key: cord-004680-u3cnsdl8 authors: lin, z.; kato, a.; kudou, y.; umeda, k.; ueda, s. title: typing of recent infectious bronchitis virus isolates causing nephritis in chicken date: 1991 journal: arch virol doi: 10.1007/bf01310957 sha: doc_id: 4680 cord_uid: u3cnsdl8 four isolates of infectious bronchitis viruses (ibv) from chickens with nephritis, were characterized by polymerase chain reaction (pcr) and restriction enzyme fragment length polymorphism (rflp), and were found to be genetically different from the other twelve strains which we previously studied. . amplification of cdna from four isolates. the strains listed at the top of the lanes were amplified by pcr in 25 cycles. ibv m 41 was also amplified as a standard strain. amplified cdnas were analyzed by electrophoresis on 1.5% agarose gel in tris-borate buffer containing 0.5 ~tg/ml of ethidium bromide. hinfi digested puc 19 dna was used as the molecular size markers (3//), and their sizes are indicated in base pairs (bp) on the left in this study, essentially the same procedure was used in an attempt to characterize four ibv isolates f-88, y-4, m-l, and ni-1 from chickens with nephritis in different endemic areas of japan between 1988 and 1989. three of them, f-88, m-l, and ni-1, were obtained from affected kidneys, while y-4 was from a gizzard, cdna fragments of the four isolates were amplified by pcr. the amplified dnas comigrated exactly in a 1.5% agarose gel, giving an identical size approximately 400 bp corresponding to the length between the two primers ( fig. 1 ). this indicates that the amplified region was well conserved without apparent deletion or insertion. to see the rflp of these amplified dnas, they were cleaved by each of 9 restriction enzymes (hpaii, maeiii, xhoii, hinfl, scai, ddei, hincii, haeiii, and psti) under the conditions recommended by the enzyme suppliers, and the digestants were run on 6% polyacrylamide gels. figure 2 shows the representative cleavage patterns of the amplified dna of m-1 and y-4 isolates. the dna of m-1 was cleaved at one site by all of the restriction enzymes, except hpaii and psti (fig. 2 a) . the dna of y-4 dna was cleaved at two sites by scai and at one site by maelii, xhoii, ddei, and hincii (fig. 2 b) . the differences in cleavage sites between the two isolates are shown in hinfi, scai, and haeiii digestion. the dna from the other two isolates, f-88 and ni-1, gave an identical cleavage pattern with that of the m-1 isolate, indicating that these three isolates are closely related to each other, and may be derived from a common origin (data not shown). to facilitate the classification of strains, a pairwise comparison of cleavage sites was performed. there are 17 cleavage sites by 9 restriction enzymes as shown in fig. 3 . for each pair of strains, the difference in restriction sites was counted (0-17). the results with four new isolates and previously defined 12 strains were schematically illustrated in fig. 4 . the four new isolates are obviously closer with each other than with the other 12 strains which previously classified (group i-v) [6] , and therefore classified into a new distinct group (vi). serological data based on the cross-neutralization test measured by plaque reduction in chicken kidney (ck) cell culture showed that the m-1 strain was not neutralized by the antiserum against any other previous strains (y. kudou, unpubl, data). this suggests that our pcr and rflp data are consistent with serological relationships. the results of the present study, indicate that the four recently obtained ibv isolates causing nephritis are, by our genetic criteria, similar to each other but different from previous isolates, hence are classified together into a new subtype (group vi). because there is no comparison between our japanese isolates and the australian t strain, it is not clear, despite their similar ne-148 z. lin et al. the grouping from i to vi is essentially according to lin et al. [6] phrotropic features, if there is any serological or genetic relationship between them. to control ibv infection, it is necessary to survey whether the prevalent ibv strains are similar to or different from current vaccine strains. several investigators have characterized the isolates obtained from ibv outbreaks, and showed that they had a strong relationship with the vaccine strain, suggesting that the prevalent strains may have originated by recombination with live vaccine strains [1, 5] . our present observation that the four new isolates used were different from the reference strains including current vaccine strains suggests that they were not derived from the live attenuated vaccine. however, it is totally u n k n o w n and remains to be clarieed how these new strains evolved. oligonucleotide fingerprinting of ribonucleic acids of infectious bronchitis virus strain infectious avian nephrosis (ureamia) in australia occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants molecular epidemiology of infectious bronchitis virus in the netherlands a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism etiology of an infectious nephritis-nephrosis syndrome of chickens key: cord-017894-8iahlshj authors: loa, chien chang; wu, ching ching; lin, tsang long title: a multiplex polymerase chain reaction for differential detection of turkey coronavirus from chicken infectious bronchitis virus and bovine coronavirus date: 2015-09-10 journal: animal coronaviruses doi: 10.1007/978-1-4939-3414-0_12 sha: doc_id: 17894 cord_uid: 8iahlshj a multiplex polymerase chain reaction (pcr) method for differential detection of turkey coronavirus (tcov), infectious bronchitis virus (ibv), and bovine coronavirus (bcov) is presented in this chapter. primers are designed from the conserved or variable regions of nucleocapsid (n) or spike (s) protein genes of tcov, ibv, and bcov and used in the same pcr reaction. reverse transcription followed by pcr reaction is used to amplify a portion of n or s gene of the corresponding coronaviruses. two pcr products, a 356-bp band corresponding to n gene and a 727-bp band corresponding to s gene, are obtained for tcov. in contrast, one pcr product of 356 bp corresponding to a fragment of n gene is obtained for ibv strains and one pcr product of 568 bp corresponding to a fragment of s gene is obtained for bcov. turkey coronavirus (tcov) contributed to signifi cant economic losses and remains as a serious threat to the turkey producers. turkey coronaviral enteritis in areas with high concentrations of turkeys on a year-round basis is not easily eliminated and is encountered frequently in turkey poults [ 1 ] . accurate and rapid method for diagnosis of tcov infection is the key to effective control of the disease. turkey coronavirus belongs to the family coronaviridae, which is a group of enveloped, positive-stranded rna viruses that infect a wide range of mammalian and avian species. the major structural proteins of coronavirus include phosphorylated nucleocapsid (n) protein, peplomeric spike (s) glycoprotein, and transmembrane or membrane (m) glycoprotein. spike protein contributes to the distinctive peplomers on the viral surface and contains neutralizing and group-specifi c epitopes. spike protein is highly variable among different coronaviruses while m and n proteins are more conserved among coronaviruses between different antigenic groups [ 2 ] . there is a close antigenic and genomic relationship between tcov and infectious bronchitis virus (ibv) according to studies of immunofl uorescent antibody assay ( ifa ), enzyme-linked immunosorbent assay ( elisa ), and sequence analysis in our and other laboratories [ 3 -8 ] . in addition, bovine coronavirus (bcov) was demonstrated to cause experimental enteric infection in turkey [ 9 ] . therefore, close relationship between tcov and bcov was previously reported and tcov was placed in an antigenic group as bcov [ 10 , 11 ] . although the sequence data revealed divergence of s genes among tcov, ibv, and bcov [ 12 -14 ] , there is still a need to detect and differentiate them accurately and quickly. polymerase chain reaction ( pcr ) assay has been an important approach for detecting many veterinary important microorganisms with the distinct advantages of high sensitivity and specifi city. this chapter describes a multiplex pcr assay to detect and differentiate tcov, ibv, and bcov in a single reaction [ 15 ] . elmer centers corp., norwalk, ct, usa). 6. ethidium bromide, 0.5 μg/ml. stomacher with fi vefold volume of pbs solution. 2. the intestinal homogenates are clarifi ed by centrifugation at 1500 × g for 10 min. the supernatants containing tcov are used as virus source for preparation of rna templates for reverse transcription (rt)-pcr reaction. 3. two hundred microliters of above supernatants are mixed with 1 ml of rnapure reagent and incubated on ice for 10 min ( see note 2 ). 4. add 180 μl of chloroform, mix the mixture, and vortex vigorously for 10 s ( see note 3 ). 5. centrifuge at 13,000 × g for 10 min at 4 °c. carefully take the upper aqueous phase into a clean tube and mix with equal volume of cold isopropanol by vortexing vigorously for 30 s. incubate on ice for 10 min. 6. centrifuge at 13,000 × g for 10 min at 4 °c. carefully discard the supernatant without disturbing rna pellet. 7. wash rna pellet with 1 ml of cold 70 % ethanol. incubate on ice for 5 min. 2. the suggested ratio of rnapure reagent to sample is 10:1. excess amount of rnapure reagent has no negative impact. the lower ratio (5:1) in this step is intended to obtain higher concentration of viral rna in the fi nal supernatants. if the upper aqueous phase after centrifugation at step 5 is more than half of the total volume, there is not enough rnapure reagent added. the appropriate reagent amount may be adjusted. chloroform is applied at 150 μl for every ml of lysate. 3. the sample mixture with chloroform at this step can be stored at −70 °c or lower than −70 °c before proceeding to the next step. 7. for routine practice in the laboratory for large number of samples, 25 μl reaction can be applied with reduced cost of reagents. tcov positive: two pcr products, a 356-bp band, and a 727bp band. ibv positive: one pcr product of 356 bp. bcov positive: one pcr product of 568 bp. negative: no pcr product. one limitation of this multiplex pcr should be noted. when both tcov and ibv are present in the sample, the ibvpositive pcr product at 356 bp is overlapping with one of the two tcov pcr products. the result of two pcr product bands at 356 and 727 bp refl ects a confi rmed diagnosis of tcov positive but does not rule out the presence of ibv. this concern is alleviated by tissue tropism of ibv. turkey enteric infection by chicken respiratory ibv has never been reported. accordingly, the presence of ibv in turkey intestines (the test sample) is not likely. any such concern should be further evaluated by a separate pcr specifi c for ibv [ 16 ] . on the other hand, ibv-positive result is able to exclude the presence of tcov. coronaviral enteritis of turkeys (blue comb disease) coronavirus immunogens antigenic characterization of a turkey coronavirus identifi ed in poult enteritis and mortality syndrome-affected turkeys phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3′ untranslated region identifi es the virus as a close relative of infectious bronchitis virus detection of antibody to turkey coronavirus by antibodycapture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus experimental bovine coronavirus in turkey poults and young chickens antigenic and genomic relationships among turkey and bovine enteric coronaviruses sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus complete sequences of 3′end coding region for structural protein genes of turkey coronavirus comparison of 3′ end encoding regions of turkey coronavirus isolates from indiana, north carolina, and minnesota complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction redesign of primers and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de072 strain of infectious bronchitis virus the protocol "a multiplex polymerase chain reaction for differential detection of turkey coronavirus from chicken infectious bronchitis virus and bovine coronavirus" outlined in this chapter had been successfully carried out in the authors' studies on molecular diagnostics and molecular virology of turkey coronavirus infection in turkeys. those studies were in part fi nancially supported by usda, north carolina poultry federation, and/or indiana department of agriculture and technically assisted by drs. tom brien and david hermes, mr. tom hooper, and ms. donna schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation. key: cord-258379-v3lceirh authors: liu, d. x.; inglis, s. c. title: association of the infectious bronchitis virus 3c protein with the virion envelope date: 1991-12-31 journal: virology doi: 10.1016/0042-6822(91)90572-s sha: doc_id: 258379 cord_uid: v3lceirh abstract a highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (ibv) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of i bv mrnas 3 and 5. one of these, 3c, a 12.4k protein encoded by the third open reading frame of the tricistronic mrna3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. these results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses. infectious bronchitis virus (ibv) is the prototype virus of the coronaviridae, a family of viruses containing a large single-stranded rna genome of positive sense. the morphology of the virion is characterised by a "corona" of widely spaced bulbous projections which consist of the virus spike glycoprotein (s) (27) . this s protein is composed of two glycopolypeptides, gp90 and gp84, which are produced by post-translational cleavage of a precursor, gp155 (7, 8, 25) . in addition to the spike protein, only two other major virion structural proteins are currently recognized. these are the nucleocapsid protein (n), which is a 5 1 k phosphoprotein found in close association with the genomic rna (26) and the membrane protein (m), an integral envelope protein which appears as a heterogeneous collection of molecules differing in their extent of glycosylation (~23, gp28, gp31, and gp36) (8, 24) . boursnell and colleagues (4) have deduced the complete sequence of ibv genomic rna through cloning of cdna. their results indicated that the ibv genome is 27.6 kb in length with at least ten open reading frames (orfs), of which only three were known certainly to encode virus proteins (the s, n, and m genes). the seven unassigned orfs were clustered in three groups on the genome-two very large orfs at the 5' end of the rna (1 a and 1 b), three small ones between the s and n genes (3a, 3b, and 3c), and two small ones between the m and the n genes (5a and 5b). recently through expression of nucleotide sequences from these unassigned orfs as bacterial fusion proteins, and production of monospecific antisera against the bacterial products, we have been able to establish posi-' to whom reprint requests should be addressed. tively the coding function of several of these orfs. it now appears that the 3a, 3b, and 3c orfs encode polypeptides in virus-infected cells (17, 23) and that the 5a and 5b orfs also represent functional genes (18) . it was therefore of interest to examine whether any of these newly identified proteins are associated with the virus particle. although their function is as yet unknown, certain of the polypeptides, particularly the 12.4k 3c and 7.5k 5a polypeptides, possess hydrophobic sequences characteristic of integral membrane proteins (2, 3) and we have shown previously that 3c can be found in association with ibv-infected cell membranes (23) . we were therefore particularly interested in the possibility that these proteins might represent previously unidentified virion envelope proteins. we report here that the 3c protein is indeed virion-associated and that it cofractionates with other virion envelope proteins on detergent disruption of virus particles. confluent monolayers of primary chicken kidney cells were prepared from 2-to 3-week-old birds as previously described (23 mm diameter) of ibv-infected cells was layered onto two tubes each containing 10 ml of 20% (w/v) tne-buffered sucrose solution (tne buffer: 50 mm tris-hci, ph 7.4, 100 mm naci, 1 rnm edta) and centrifuged at 75,000 g for 3 hr at 4'. the resulting virus pellets from the two tubes were resuspended in 10 ml of tne buffer, layered onto a 20-ml 20-55% linear sucrose gradient prepared with tne buffer, and centrifuged at 75,000 g for 18 hr at 4". the gradient was fractionated (22 samples) and the radioactivity present in each fraction determined directly by liquid scintillation. three adjacent fractions which contained most (70%) of the radioactivity were pooled, diluted three times in tne buffer, and applied to a second density gradient consisting of 20 ml of 1 o-50% renografin (meglumine diatrizoate, mw 809.1, sigma) prepared with tne buffer. this gradient was centrifuged and fractionated as before. aliquots of 13 fractions starting from the bottom of the gradient were analyzed by sds-polyacrylamide gel electrophoresis (page) (fig. 1 a) . prominent radiolabeled bands corresponding to the known structural proteins of ibv (6, 24) were observed in fractions 7-l 0 (lanes 1 o-l 4). the sedimentation rate of these proteins corresponded with that reported originally for ibv virions by stern et al. (24) whose basic protocol was used for the purification carried out here. we also observed that a polypeptide of approximately 12k, with an electrophoretic mobility similar to that of in vitrotranslated 3c (23) cofractionated exactly on the gradient with the virus major structural proteins, suggesting that it was associated with virus particles. polypeptides corresponding to in vitro-translated 5a, 5b, 3a, and 3b (17, 18) however, were not detectable. a radiolabeled protein with an apparent molecular weight of 44k, appearing in fractions l-6 (lanes 4-9), probably represents cellular actin as previously reported (19) . to confirm further the association of the putative 12k polypeptide with virions, fractions 7, 8, and 9 (lanes 10, 11, and 12) which contained most of the radioactive virions, were diluted threefold with tne buffer, pooled, and fractionated on a second 20-55% linear sucrose gradient under the same conditions as before. twenty-two fractions were collected, of which 16 (starting from the bottom of the gradient) were analyzed by sds-page ( fig. 1 b) . once again the 12k polypeptide was clearly present and cofractionated exactly with the major virion structural proteins, appearing mostly in fractions 5-9 (lanes 6-l 0). there was considerably less evidence of contaminating cellular proteins in this gradient, and the relative proportion of the 12k protein to the other virion proteins appeared similar to that observed at the previous stage in purification, strongly supporting the idea of a specific association between the 12k protein and virus particles. a single band migrating at the position expected of one of the cleaved portions of the spike protein, gp90, was observed in the upper part of the gradient, which may represent the degraded product of the spikes; morphological examination of unlabeled equivalent preparations at this stage by electron microscopy after negative staining showed that a quarter of the virions had lost their spike structures (data not shown). in order to establish the identity of the 12k polypeptide as the product of the 3c gene, and to examine the possibility that ibv virions might contain other new virus polypeptides in amounts too small to detect directly, we next carried out immunoprecipitation experiments using monospecific antisera directed against the predicted products of the 3a, 3b, 3c, 5a, and 5b orfs (17, 18, 23) . for this purpose, fractions 5, 6, and 7 from the second sucrose gradient (lanes 6, 7, and 8, fig. 1 b) were pooled, diluted three times in tne buffer, and centrifuged at 65,000 g for 3 hr at 4' to pellet the virus. the virus pellet was then resuspended in ripa buffer (50 mll/l tris-hci, ph 7.5, 150 mm naci, 1% np-40, 0.1% sds, 10% sodium deoxycholate) and aliquots were immunoprecipitated with a series of specific and control antisera as described (5) . the result of this experiment (fig. 2a) showed clearly that the 12k polypeptide was immunoprecipitated by anti-3c antiserum, but not by any of the other antisera. further evidence for the specificity of the association between 3c and purified virions comes from analysis of the relative amount of the 12k protein in virus-infected cells before virion assembly (fig. 2b) ; in this case the 3c protein is completely undetectable except by immunoprecipitation. thus the likelihood of the 3c protein appearing in virions by chance from contaminating cellular material which has survived three cycles of gradient purification is remote. these experiments indicate therefore that 3c is indeed a virion-associated polypeptide. no evidence was obtained, however, for the presence of the other small virus polypeptides. in order to assess the approximate amount of the 3c polypeptide present in the purified virion preparation, the autoradiograph shown in fig. 2a , lane "total v", was analyzed by scanning densitometry (fig. 2c) , and the relative peak areas for the virion proteins np, m, s, and 3c were measured. using these data, and taking account of the methionine content of these proteins (3c has only one aside from its initiator, and the s, np, and m proteins have 17, 4, and 4, respectively), we calculated the relative molar proportion of the four proteins to be approximately 1 .o: 1 1 .o: 10.1 :1.7 (s:np:m:3c). these relative values for the major structural proteins are broadly similar to those reported previously for ibv (6, 7) except that the proportion of envelope proteins to np is rather lower. this calculation also assumes that 2. (a) lmmunoprecipitation of radiolabeled virion proteins with specific antisera. virions were recovered from fractions 5, 6, and 7 of the sucrose density gradient shown in fig. 1 b as described in the text and immunoprecipitated with different antisera as indicated above each track. the monospecific test antisera were prepared as described elsewhere (17, 18, 23) and control anti-whole ibv serum was a gift from dr. d. cavanagh (afrc institute for animal health, houghton laboratory, uk). plbt3-radiolabeled 3a, b, and c polypeptide markers were prepared by in vitro transcription and translation as described for fig. 1 a. total 'v'unprecipitated virions. polypeptides were separated on a 22% polyacryamide gel and detected by fluorography. lanes containing anti-3a, b, and c and anti-5a and b antisera were exposed for 10 days, and other lanes for 2 days. (b) synthesis of 3c protein in ibv-infected cells. ck cells were mock-infected or infected with ibv and labeled with [?slmethionine from 6 to 7.5 hr p.i. as described before (23) . cells were harvested immediately after labeling and analyzed on a 22% sds-polyacrylamide gel the initiator methionine is not removed from any of the proteins; if one were to assume that cleavage does occur, the calculated relative proportion of 3c would increase. figures are not available for the number of molecules of each structural protein present in ibv virions, but using those reported for human coronavirus oc43 as a guide (np-726; m-726; s-88) (12) , we suggest that each virion may contain in excess of 100 molecules of 3c, as many or perhaps even more than the number of spike proteins. this relatively large proportion of 3c further supports the idea that the 3c protein is a genuine virion structural protein which may play an important role in virion assembly or infectivity. since the product of the 3c orf is known to be associated with the membranes of infected cells (23) it seemed likely that it could be located in thevirion envelope. to test this possibility, a sample of purified radiolabeled virions purified by sucrose and then renografin density gradient sedimentations as described above was pelleted bycentrifugation and disrupted according to the procedure of sturman et al. (27) by treatment with 1 ml of tne buffer containing 0.25% np-40. the ribonucleocapsid (rnp) and envelope components of the virus were then separated by centrifugation, at 180,000 g for 18 hr at 4', through a lo-ml lo-55% linear sucrose gradient containing 0.1% np-40, layered on top of a l-ml cushion of 75% renografin also containing 0.1% np-40. eighteen fractions were collected from the gradient (of which the first two correspond to the renografin cushion), and samples were analyzed by sds-page (fig. 3) . it can be seen clearly that the major virion envelope proteins sl , s2, and m cofractionate with each other, appearing maximally in fractions 15-l 8, but are well separated from the nucleocapsid protein (fractions l-l 0), indicating successful separation of the virion components. it is also evident that the 12k virion-associated protein cofractionates with the spike and membrane proteins rather than the nucleocapsids, suggesting strongly that it is indeed associated with the virion envelope. lmmunoprecipitation of the pooled rnp and envelope fractions confirmed that the 12k protein present in the envelope fraction was indeed 3c and further indicated the absence of the protein in the rnp fraction (data not shown). the function of the 3c protein is not yet clear. however, we are intrigued by an overall similarity in organization between 3c and the influenza avirus m2 protein (13, 74) . the ibv 3c protein is of a similar size and, like m2, has a stretch of hydrophobic amino acids near its n-terminus which could span the membrane. by analogy with m2, which is known to be an integral membrane protein present in small amounts in the virion (75, 31) this would leave the n-terminal 1 1 amino acids exposed on the external surface of the membrane with an internal c-terminal tail of 76 residues. recently it has been proposed that the m2 protein forms an ion channel (28) which selves to allow both uncoating of incoming virions and proper assembly of progeny virus, and this hypothesis is consistent with the observation that the m2 protein is the target for the action of the anti-viral agent amantadine (10) . m2 appears to be present in the membrane as a homotetramer composed of two disulfide-linked dimers held together by noncovalent interactions, which provides a structural basis for its proposed function in proton translocation (28) . comparison of the predicted amino acid sequence of 3c for five strains of ibv (17) indicates that the hydrophobic character of the proposed transmembrane region near the n-terminus of the protein is preserved, although the actual amino acid sequences show slight differences. there are also two conserved cysteine residues 45 and 46 amino acids from the nterminus of the predicted sequence of the 3c products in all five strains examined, which might serve the same function as cysl7 and cysl9 of the m2 protein. it will be of considerable interest to explore this possible parallel further. are 3c-like proteins a general feature of coronaviruses? on the basis of currently available information, this seems likely. messenger rna 4 of mouse hepatitis virus (mhv) is known to encode a 15k polypeptide, the predicted amino acid sequence of which contains a highly hydrophobic region from residues 8 to 41 (9, 22) , although the protein has not so far been found in virions. wesley and woods (29) have identified a 17k polypeptide in transmissible gastroenteritis virus (tgev)-infected cells, which could be a minor viral structural protein. intriguingly, sequence data deduced from the genomic rna of several species of coronaviruses have indicated that the orf immediately upstream of the membrane protein gene, in the equivalent position to that of 3c, has the potential to encode a hydrophobic protein with similar characteristics. for example the second orf (5b) of mhv mrna 5 can encode a 10.2k protein with a hydrophobic amino terminal region (16, directly or after immunoprecipitation with anti-3c antiserum. plbt3-radiolabeled 3a, b, and c polypeptide markers were prepared by in vitro transcription and translation as described for fig. 1 22 ) and the corresponding genetic location on the tgev genome has the potential to encode a 1 ok polypeptide with striking similarity to 3c (30) . furthermore, the bovine coronavirus genome at this point contains an orf which could encode a 9.5k hydrophobic protein (1). comparison of the predicted amino acid sequences of these four proteins (fig. 4 ) reveals some interesting common features. first, the amino acids flanking the proposed transmembrane region of the four proteins are negatively charged at the n-terminus and positively charged at the c-terminus. this arrangement could allow them to adopt the orientation of class iii integral membrane proteins (i i), i.e., with the n-terminus on the outside. it has also been observed that the predicted molecular weight of the 3c protein of ibv, the 5b protein of mhv, and the 9.5k protein of bcv is consistent with their migration rate in sds-page (i, 16, 17, 22) suggesting that these proteins contain uncleaved signal peptides; this is again consistent with the possibility that they are, like the m2 protein of influenza a virus, class iii integral membrane proteins (11, 20) . a further common feature is the occurrence of a cysteine residue 34 or 35 nt downstream of the charged amino acid at the n-terminal end of the proposed transmembrane region, which may play a role, as described above for the influenza m2 protein, in forming disulfide linkages. thus it may be that 3c-like proteins are conserved across the coronaviridae and may play a similar role in viral replication. proc. nat/. acad. sci. usa ce// 40 key: cord-009487-7xb4huyz authors: godwin, ir; chaffey, ga title: simple rapid method of rumen cannulation date: 2008-03-10 journal: aust vet j doi: 10.1111/j.1751-0813.1988.tb14467.x sha: doc_id: 9487 cord_uid: 7xb4huyz nan previous methods of rumen cannulation in sheep have involved 2-stage operations in which the rumen is sutured to the skin, through dissected abdominal muscles, and then several days later an incision is made through the skin and rumen wall to form a permanent fistula, and a cannula fitted (jarrett 1948 hecker (1969) adapted a method previously used for cattle (balch and cowie 1962) . a metal bar clamp is fitted to a fold of the rumen, which is exposed by laparotomy. after about 10 days the occluded fold sloughs away and a cannula may be inserted into the fistula. these methods have been found to be both time consuming and sometimes distressful to the animal. the need for a relatively large incision in the rumen wall means that leakage of rumen contents around the cannula is a frequent sequel. a siplple one-stage operation, requiring no suturing of the rumen'wall and an incision smaller than the neck of the australian veterinary journal, vol. 65, no. 7, july, 1988 cannula to ensure no leakage of rumen contents, is described here. a healthy sheep which has been fasted for 24 h is given either general anaesthesia or sedation plus local anaesthesia and placed on its right side. the left dorsal part of the abdomen is clipped of wool and the skin disinfected.* a vertical incision about 5 cm long is made about 3 cm caudal to the last rib and 3 cm ventral to the transverse process of the first lumbar vertebra. the abdominal muscles and peritoneum are gently separated by blunt dissection. the rumen is drawn through the opening and a small incision ( < 2 cm) is made in a section free of major blood vessels. a rubber cannula? (a in figure 1) is partially everted by pushing the flange through the neck of the cannula. the folded cannula (f) is then inserted through the incision and pushed inward until the neck of the cannula is tightly held by the tissue surrounding the incision. the flange of the cannula is then allowed to revert to its original shape. several swabs are placed in the cannula while the abdominal muscles (if necessary) and the skin wound closed by sutures. a pvc plate (b) is placed over the cannula neck and a cutoff 20ml syringe barrel and rubber stopper (c and d) are held in the neck of the cannula with a cable tie (e). two holes may be bored into the flanges of the syringe and wire tied over the stopper to prevent it dislodging. administering a topical antibiotic and intramuscular penicillin as prophylactic measures is advised. after several days, adhesions form between the rumen wall and the peritoneum; peritonitis has not been observed. this method has been used by several surgeons on 152 sheep. the operation can be completed in less than 20 min and most animals eat their normal ration once the effects of anaesthesia have dissipated. cannulae have remained functional for up to 3 years. occasionally the pvc plate needs to be removed and the wool clipped to reduce tightness and the possibility of blowfly attack. the authors gratefully acknowledge the funding of the the chicken is the only confirmed natural host to infectious bronchitis virus (ibv) (hofstad 1984) . we report the isolation of ibv from a flock of racing pigeons and assess its significance. during the winter of 1985, a racing pigeon fancier in north eastern victoria observed an acute illness in his loft of 150 racing pigeons. affected birds had ruffled feathers, dyspnoea and excessive mucus at the commissures of the beak. eleven birds died during the first 24 h and 11 more over the next 2 days. seven birds were submitted for necropsy. the flock was treated with antibiotics. affected birds recovered over the next 2 to 3 weeks. at post-mortem examination, all birds were in average body condition. they had recently eaten but the linings of the oesophagus and crop were ulcerated. mucoid pharyngitis and tracheitis were noted. the lower intestines contained fluid. histological examination of oesophageal tissues confirmed that the birds were infested with trichomonads. four clarified suspensions from pooled samples of 5 tracheal mucosae and from 7 cloacal swabs were each inoculated into the allantoic cavity of sets of 5 nine-day-old fertile chicken eggs, allantoic fluids, from the third passage, were collected 72 h after inoculation of each set of eggs, clarified and pelleted in an ultra centrifuge. coronaviruses were seen when the pellets were examined by electron microscopy. ten days after inoculation, all eggs were opened and the embryos examined. embryos in each set were curled and stunted, changes which are characteristic of ibv. an aliquot of a tracheal isolate was passaged 3 times in eggs at 48 h intervals. fluorescence, shown to be specific for ibv, was seen in the aliantoic cells of each passage (endo and faragher, unpublished) . after concentration and treatment with phospholipase c type 1, allantoic fluid from the third passage haemagglutinated chicken red blood cells. the haemagglutination was inhibited by specific ibv antiserum, indicating ibv of sub-type b (faragher 1987). the ibv was then sub-typed by plaque reduction serum neutralisation tests using serums specific for each of the 9 australian ibv subtypes (wadey and faragher 1981) , and shown to belong to sub-type b. twenty-six days after the onset of disease in the pigeons, samples of serum were obtained from 10 birds in the affected flock and also from 12 birds in an unaffected flock kept in a separate loft 4oom away. ibv haemagglutination inhibition (hi) antibody was detected at levels from 2 to 2'. the titres in serum from the unaffected and the affected pigeon flocks were similar. the pathogenicity of a cloacal ibv isolate was examined. four 4-week-old csiro spf chickens and four 8-week-old meat pigeons that were housed in the same cage were each inoculated by intranasal, intraocular and oral routes with allantoic fluid containing 10' eid,, of ibv. meat pigeons were chosen rather than racing pigeons to reduce the likelihood of previous exposure to ibv. a similar number of birds of both species was inoculated with phosphate buffered saline (pbs) and housed in a separate room. four days after inoculation, all the chickens inoculated with ibv had marked respiratory rales. a11 pigeons and those chickens inocuiated with pbs remained healthy for 18 d when all the birds were bled and killed. levels of ibv hi antibodies in the chickens increased from 2' before inoculation to 2' to 24 18 d after inoculation. this response is common following vaccination of chickens, whereas wild ibv stimulate higher antibody levels. no ibv hi antibody was detected in the pigeons. the ibv may have caused disease in the racing pigeons because their resistance was lowered by intercurrent disease. pigeons raced over long distances have been seen to shelter in open-sided caged-layer poultry sheds when they are attacked by raptors (j dark, personal communication). such direct contact may have been the route of transmission to the pigeons of an ibv shown to be of the same serotype as widely used australian ibv vaccine strains. a stray pigeon which the pigeon fancier had seen join his flock a week before the onset of disease may have been implicated in the transmission of the ibv. laboratory contamination as a source of the isolate was considered to be a remote possibility because no other ibv was isolated during the 5 days immediately preceding or following this case. we thank mr g beavis for submitting the pigeons and assisting with the investigation. 311 references faragher jt diseuses of poultry key: cord-257064-iafm3pcc authors: kint, joeri; maier, helena jane; jagt, erik title: quantification of infectious bronchitis coronavirus by titration in vitro and in ovo date: 2014-12-18 journal: coronaviruses doi: 10.1007/978-1-4939-2438-7_9 sha: doc_id: 257064 cord_uid: iafm3pcc quantification of the number of infectious viruses in a sample is a basic virological technique. in this chapter we provide a detailed description of three techniques to estimate the number of viable infectious avian coronaviruses in a sample. all three techniques are serial dilution assays, better known as titrations. technological advances in particle analysis have made it possible to quantify the number of virus particles in a sample with increasing accuracy. techniques such as specialized flow cytometry [1] , dynamic light scattering [2] , quantitative capillary electrophoresis [3] , and fluorescence correlation spectroscopy [4] can determine the number of particles in a sample within hours. the choice of technique depends on the sort of virus and the matrix in which it is suspended. all aforementioned techniques differentiate particles on the basis of physical properties such as size or antibody affinity. as a consequence particle analysis cannot differentiate infectious from noninfectious virus particles. there is only one technique available that can reliably quantify the number of infectious particles in a sample. this technique, developed many decades ago [5] , exploits the fact that virus can propagate in biological systems such as embryonated eggs or cell cultures. propagation of a virus is generally accompanied by changes in cell morphology (referred to as cytopathic effect or cpe), which can be visualized using a microscope, or even by eye. some viruses do not induce cpe, in which case an antibody based assay (immunofluorescence or elisa) is needed to determine presence or absence of virus. during a titration assay, tissue cultures or embryonated eggs are incubated with tenfold serial dilutions of a virus containing sample and several days later the cytopathic effect is scored. from these scores, the virus titer is calculated using the methods described by spearman and kaerber [6, 7] or reed and muench [8] . the virus titer is defined as the reciprocal of the dilution at which 50 % of the inoculated embryos or tissue cultures show cpe. in this chapter we use the method of spearman and kaerber to calculate the titer, as this calculation can cope with unequal group sizes. unequal group sizes frequently arise when eggs are lost to aspecific death of the embryo or bacterial infection. coronaviruses in general have a narrow host range and many clinical isolates only replicate in primary cells. replication of most field isolates of infectious bronchitis coronavirus is restricted to embryonated eggs or tracheal organ culture. most isolates, however, can be adapted to propagation in primary chicken kidney (ck) cells. adaptation typically requires several passages and selects for viral subpopulations and can induce mutations [9] . passaging of ibv in either embryonated eggs or primary cell cultures leads to attenuation of the virus in vivo [10] [11] [12] . the most striking example is the ibv beaudette strain, which has been passaged hundreds of times in eggs and primary chicken kidney cells [13, 14] . although ibv beaudette propagates very well on eggs, ck cells and even in vero cells, the virus is highly attenuated in vivo and vaccination using beaudette provides little protection against infection with pathogenic strains of ibv [15] . for quantification of ibv field isolates, embryonated chicken eggs are the most suitable substrate. a protocol on titration of ibv on embryonated chicken eggs is provided in the first part of this protocol. ibv strains which have been adapted to grow in cultures of primary chicken cells can be titrated on these cells using either the tcid 50 method or plaque titration. protocols for both methods are provided in this chapter. plaque-forming unit (pfu) titration yields more accurate and reproducible results then the tcid 50 method, it is however more labor intensive. both methods are presented in this chapter. 1. fertilized specific pathogen free (spf) eggs, 9-11-day-old (see note 1). 2. diluent: 2.5 % w/v tryptose, 1,000 u/ml penicillin, 1,000 μg/ml streptomycin. 3. disinfectant: 70 % alcohol in water. 4. egg shell drill or punch. 5. sterile 1 ml syringes. candle the eggs using the candling light and draw a line on the shell marking the edge of the air sac. draw an x approximately 5 mm above this line, which marks the inoculation site. 2. assign ten eggs per dilution and select those dilutions (at least three) that include the 50 % end point of the sample. 3. prepare tenfold serial dilutions in diluent. 4. disinfect the eggs by spraying them with disinfectant. 5. after the eggs have dried drill or pierce a hole in the egg shell at the marked inoculation site. 6. inoculate ten eggs per dilution each with 0.2 ml volume via the allantoic cavity by holding the syringe and needle vertically and by inserting the needle approximately 16 mm into the egg (fig. 1a, b) . 7. after inoculation, the hole in each egg is sealed with hobby glue or melted wax. 8. incubate the eggs in an egg incubator with rocker at 37.8 °c and 60-65 % humidity. 9. candle the eggs after 24 h of incubation. embryo mortality occurring up till 24 h post inoculation is considered nonspecific and therefore these eggs are discarded. 10. incubate the eggs for 6 more days in an egg incubator with rocker at 37.8 °c and 60-65 % humidity. candle the eggs at the end of the incubation period to identify embryos that have died. subsequently, macroscopically evaluate all surviving embryos for the presence of lesions characteristic for ibv infection (stunting and curling; fig. 1c ). embryos that have died and embryos that exhibit lesions characteristic for ibv infection are considered positive (see note 2). 11. any embryo with ibv specific cpe is regarded positive. virus titers in the original sample, expressed as 10 log eid 50 /ml are calculated using the method described by spearman and kaerber [6, 7] , using the following formula: in which: x 0 : logarithm of the inverse value of the lowest dilution at which all embryos are positive. d: logarithm of the dilution factor (d=1 when using tenfold serial dilutions). n: number of eggs used per dilution. r: number of positive eggs at that dilution. using the result of the titration depicted in fig. 2 , the virus titer is calculated as follows: titre eid ml 1. ck cells are seeded in 96-well plates at 7.5 × 10 4 cells/well, 1 or 2 days before the titration. one plate is needed per sample. at the time of titration, the monolayer should be nearly confluent. 3. empty the medium from the 96-well plate containing ck cells into a waste container and gently tap the plate dry on a stack of tissues. 4. fill the wells of column 1 and 12 of the 96-well plate with 100 μl/well titration medium. these are the negative control wells (fig. 3a) . 5. starting with the highest dilution, dispense 100 μl/well in row h using the multistepper pipette. proceed with filling the descending dilutions in rows g till a. 6. incubate the 96-well plates for 3-4 days at 37 °c and 5 % co 2 . 7. after 3-4 days incubation, score all wells for ibv specific cpe using a microscope. although the cpe may vary per ibv strain, it is generally characterized by clusters of rounded cells on top of the monolayer. at low dilutions the monolayer may be partly destroyed, exemplified by the ibv beaudette strain (fig. 3b ). 8. titers are calculated using the method described by spearman and kaerber (subheading 3.1, step 11) and are expressed in 10 log (tcid 50 )/ml. 10. pat plate dry and leave upside down at room temperature to fully dry. 11. plaques should be clearly visible as holes in the monolayer (fig. 4) . count the number of plaques per well at the dilution with clearly defined, individual (not overlapping) plaques (typically 10-50 plaques/well). ensure duplicate wells are counted and an average taken. 12. determine titer using the following equation: [16, 17] . dead embryos and those of which the allantoic fluid contains ibv as established by elisa are considered positive. 3. ibv titers usually do not exceed 10 8.8 tcid 50 /ml. therefore 10 −8 should be adequate as highest dilution. when the virus titer of the sample is known, select a number of tenfold dilutions (at least three) that include the 50 % end-point dilution. if the titer is unknown, select a broader range of tenfold dilutions that most likely include the 50 % end-point dilution. for a tcid 50 titration typically all the wells of the 96-well plate are used 4. alternative methods also exist for mixing media and agar. if there is concern regarding the overlay setting too quickly or risk of contamination from the water bath, hot agar can be mixed directly with cold media (4 °c). once the mixture feels warm to the touch, rather than hot, it can be added to cells. 5. the simplest method for removing agar from the cells is to hold the plate upside down with the lid removed. the small spatula is inserted between the agar and the wall of the well. once the base of the well is reached, a small amount of pressure is applied to remove the agar, being careful not to scrape off the cells. the whole agar plug should then fall out easily. evaluation of the virus counter ® for rapid baculovirus quantitation one-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles viral quantitative capillary electrophoresis for counting intact viruses kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies (evolutionary biotechnology-from theory to experiment) production of plaques in monolayer tissue cultures by single particles of an animal virus the method of 'right and wrong cases' ('constant stimuli') without gauss's formulae beitrag zur kollektiven behandlung pharmakologischer reihenversuche a simple method of estimating fifty percent endpoints plaque formation by infectious bronchitis virus in chicken embryo kidney cell cultures effect of serial embryo passage of an arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity development of attenuated vaccines from taiwanese infectious bronchitis virus strains attenuation, safety, and efficacy of an infectious bronchitis virus ga98 serotype vaccine growth kinetics of embryo-and organ-culture adapted beaudette strain of infectious bronchitis virus in embryonated chicken eggs replication of avian infectious bronchitis virus in african green monkey kidney cell line vero recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity detection and differentiation of avian infectious bronchitis viruses using a monoclonal antibodybased a monoclonal antibody-based antigen capture enzymelinked immunosorbent assay for identification of infectious bronchitis virus serotypes key: cord-022187-7c3wz6c6 authors: liu, shengwang; kong, xiangang title: a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china date: 2010-10-19 journal: avian pathol doi: 10.1080/0307945042000220697 sha: doc_id: 22187 cord_uid: 7c3wz6c6 five strains of infectious bronchitis virus (ibv) were isolated from five layer flocks that had nephropathogenic infection in four provinces in china. among them, three of the five flocks had been vaccinated against infectious bronchitis. virulence studies indicated that the five chinese ibv isolates caused 10 to 30% mortality in 15-day-old specific pathogen free chickens and gross lesions were mainly confined to the kidneys in all of the dead chickens. two oligonucleotide pairs, s1uni2 and s1oligo3′ or s1oligo5′ and s1oligo3′, were used after propagation of the isolates in embryonated eggs to amplify the s1 protein genes of the spike protein. the cdna derived by reverse transcriptase-polymerase chain reaction was cloned and sequenced. the nucleotide and amino acid sequence of s1 protein gene had a similar degree of identity (≥92%) among the five chinese ibv isolates. the nucleotide and amino acid identity of the s1 protein gene between the five chinese ibv isolates and 16 strains of other ibvs varied from 60 to 81%. this clearly showed that the five chinese ibv isolates comprised a separate genotype. these results demonstrated, for the first time, that there is a new genotype of nephropathogenic ibv circulating in vaccinated and non-vaccinated flocks in china. infectious bronchitis (ib), caused by infectious bronchitis virus (ibv), is an acute and highly contagious disease in chickens. the disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. in young chickens severe respiratory distress may occur, while in layers respiratory distress, decrease in egg production, and loss of internal and shell quality of eggs are reported. some strains of the virus cause severe kidney damage, urolithiasis and may be associated with high mortality. ib is a major health problem affecting the chicken industry in most countries of the world. through the use of attenuated live as well as inactivated virus vaccines, economic losses due to this disease have been significantly reduced. however, ibv variants may continue to circulate among vaccinated and non-vaccinated flocks and cause severe economic problems (reviewed by cavanagh & naqi, 2003) . ibv is the prototype virus of the genus coronavirus, family coronaviridae (cavanagh, 1997) . the genome of ibv contains an enveloped, single-stranded, positive-sense rna of 27.6 kb. the virion has three major virus-encoded structural proteins, namely the spike (s) glycoprotein, the membrane (m) protein, and the nucleocapsid (n) protein. the spikes of ibv are formed by posttranslational cleavage into two polypeptide components, designated s1 and s2. the molecular identification of ibv is based mainly on analysis of the s1 protein gene. in china, as in other countries, ib has occurred frequently in vaccinated and non-vaccinated flocks and has caused severe economic losses in recent years. vaccines based on massachusetts strains such as h120 and h52 and other strains such as 4-91 (nobilis ib 4-91) have been used for many years on poultry farms (farsang et al., 2002; gough et al., 2002) . however, nephropathogenic ibv strains related to the massachusetts type have been isolated in china in recent years (wang et al., 1997; wu et al., 1998; li & yang, 2001) . furthermore, other ibv strains that had partial or no relationship to massachusetts type in antigenic and immunogenic characterization have also been isolated in china (wang et al., 1997; wu et al., 1998; yu et al., 2001) . in order to investigate whether there are other genotype(s) of nephropathogenic ibv besides the massachusetts type in flocks in china, we tested five ibv isolates from layer flocks showing clinical signs of ib by sequencing and analysis of the s1 protein genes. this allowed us to evaluate the prevalence of nephropathogenic ib type(s) in vaccinated and non-vaccinated chickens in recent years in china. tissue samples of kidney were collected from layers showing clinical signs suspected to be related to ib. all flocks investigated in this study contained at least 10,000 layers. three samples were taken from chickens vaccinated with h120 massachusetts-type vaccine (nobilis ib h120) in xinjiang, shandong and heilongjiang provinces, china ( figure 1 ). the other two samples were from non-vaccinated flocks in gansu and heilongjiang provinces, china (table 1) . obvious nephropathogenic lesions were found in all the diseased layers. for virus isolation, samples of kidney were pooled and 10% w/v tissue suspensions were made in 0.1% phosphate-buffered saline containing 100 u penicillin and 100 mg streptomycin/ml. after 12 h at 48c, 200 ml supernatant from the suspensions was inoculated into the allantonic cavity of 9-day-old to 11-day-old embryos of specified pathogen free chickens (haerbin veterinary research institute, china). five eggs were used for each sample. the inoculated eggs were incubated at 378c and candled daily. two eggs were killed after 72 h incubation and five other eggs were inoculated with the harvested allantoic fluids. two to 10 blind passages were performed until the dwarfing and death of embryos were observed between 2 and 7 days after inoculation. all the allantoic fluids of inoculated eggs were harvested and tested for the presence of ibv using electron microscopy. the different passages of allantoic fluids containing ibv isolates were used in subsequent experiments (table 1) . samples of allantoic fluids after egg passages were submitted for electron microscopy. briefly, after low-speed centrifugation at 1500 )/g for 30 min (allegra tm 21r centrifuge; beckman), the supernatant of the 1.5 ml allantoic fluids were centrifuged at 12 000 )/g for 30 min. the resulting pellet was resuspended in a minimal volume of deionized water and examined by negative contrast electron microscope (jem-1200, ex). six groups each of 10 white leghorn spf chickens (haerbin veterinary research institute, china) were kept in isolators with negative pressure. at 15 days of age, groups 1 to 5 were inoculated intranasally with the five isolates (log 10 4.2 to log 10 5.0 median embryo infectious doses, per chick; table 2 ). the remaining group 6 was mock-inoculated with sterile allantoic fluid and served as a control. the chicks were examined daily for signs of infection for 30 days after inoculation. allantoic fluid (200 ml) containing virus (confirmed by electron microscopy) isolated from flocks of xinjiang, gansu, heilongjiang or shandong provinces in china, was mixed with 400 ml trizol reagent (gibco brl) and rna was isolated according to the description of the manufacturer. the rna was air-dried for 2 to 10 min and re-dissolved in 25 ml rnase-free water. the same general procedure was used for all the reverse transcriptasepolymerase chain reactions (rt-pcrs). s1oligo3? was used for the both of the rt reactions and, subsequently, the pcr with oligonucleotide s1uni2 or s1oligo5? (kwon et al ., 1993; adzhar et al ., 1997) . lyophilized oligonucleotides obtained from the manufacturer were dissolved in rnase-free water at 0.1 mmol/ml to form the stock solutions. twenty-five microlitres of viral rna was mixed with 50 ng s1oligo3? and incubated at 708c for 10 min followed by 2 min on ice. after adding a reaction mixture consisting of 8 ml 5)/first strand buffer (250 mm tris á/hcl, 375 mm kcl, 15 mm mgcl 2 ), 4 ml of 2.5 mm dntps (gibco brl), 200 u rnaseh á murine moloney leukaemia virus rt (gibco brl), 40 u rnasin (gibco brl), the mixture was incubated at 378c for 2 h. the reaction was terminated by heating at 988c for 7 min and chilling on ice. pcr, cloning and sequencing of the s1 protein genes for the pcr reaction, the following mix was made: 15 nmol oligonucleotide s1oligo3? and 15 nmol oligonucleotide s1uni2 or s1oligo5?; 1 ml cdna; 5 ml of 10)/pcr buffer (mg 2' plus; takara, japan); 4 ml of 2.5 mmol dntps; 2 u taq polymerase (takara, japan); and 34 ml water. the pcr reaction was performed using the following conditions: denaturation (948c, 1 min), annealing (508c, 1 min), and extension (728c, 2 min), 35 cycles followed by a final extension step (728c, 10 min). a product, detectable by ethidium bromide staining, of about 1700 base pairs (bp) was generated with isolates lx4 and lh2 using s1oligo3? and s1oligo5?, but no product was seen with isolates lhi10, ls2 and ld3 (table 1) . therefore, the pcr was performed for ls2, ld3 and lhi10 using s1oligo3? and s1uni2, to generate a similar size product of about 1700 bp. dna generated by pcr amplification was cloned using a t-tailed vector, pmd18-t (takara), and transformed using jm109 competent cells (takara) according to the manufacturer's instructions. three clones of each isolate were sequenced. sequence analysis of the s1 protein genes the sequences of the s1 protein gene of the five chinese ibv isolates were assembled, aligned and compared with 16 strains of other ibv with dnaman version 5.2.2 (http://www.lynnon.com/). the 16 strains of ibv and their genbank accession numbers are: beaudette (boursnell et al ., 1987 ; accession number nc_001451), m41 (niesters et al ., 1987; accession number a24863), kb8523 (sutou et al ., 1988 ; accession number m21515), b1648 (shaw et al ., 1996 ; accession number x87238), connecticut (wang et al ., 1994 ; accession number l18990), ark99 (jia et al ., 1995; accession number l10384), d274 (jordi et al ., 1989 ; accession number x15832), h120 the s1 protein gene sequences of the five chinese ibv isolates have been submitted to the genbank database and have been assigned the following accession numbers: lx4, ay189157; lh2, ay180958; ls2, ay278246; ld3, ay277632 and lhi10, ay273193. ib isolates suspected to be related to ib infection were analysed in this study. the isolates were collected from flocks in four different provinces in china, showing clinical signs of ib infection and with 10 to 30% mortality. the nephritis was observed in both the vaccinated and non-vaccinated flocks and was characterized by enlarged and pale kidneys, frequently with urate deposits in the tubules, severe dehydration and weight loss. typical signs including dwarfing and death of embryo were observed in the different passages when each of the five chinese isolates was inoculated into 9-day-old to 11-day-old chicken embryos (table 1) . diagnoses based on electron microscopy examination performed on allantoic fluids of different passages showed all five isolates had typical coronavirus morphology and were free of other agents such as newcastle disease virus (results not shown). clinical signs were observed in all of the chicks of groups 1 to 5 about 3 to 10 days after inoculation. the chicks are listless and huddled together, showed ruffled feathers and a dark, shrunken comb. some of the chicks died during the experiment (table 2 ). gross lesions of dead chicks were mainly confined to the kidneys. the kidney paryear when viruses were isolated. c different passages were performed until the dwarfing and death of embryos were observed between 2 and 7 days after inoculation. enchyma of the dead birds was pale, swollen and mottled; tubules and urethras were distended with uric acid crystals. in addition, mild respiratory signs (sneezing, rales) were also observed. the clinical signs of the inoculated birds tended to disappear gradually by 20 days of inoculation. analysis of the s1 protein gene the pair of oligonucleotides s1oligo3? and s1oligo5? was used to attempt the amplification of the s1 region of the spike gene from all five isolates. the rt-pcr amplified a â/1700 bp cdna from lx4 and lh2, but not from ls2, ld3 and lhi10. therefore, the pcr was performed for ls2, ld3 and lhi10 using s1oligo3? and s1uni2. comparison of the s1 protein gene sequences of the five chinese ibv isolates with those of 16 strains of other ibvs revealed that nucleotide and amino acid identities among the five chinese ibv isolates were between 92 and 99%, but the nucleotide identity of the five chinese ibv strains and the 16 strains of other ibv were not more than 79% and amino acid identity was not more than 78% (table 3 ). for isolation of non-egg-adapted ibv field strains, several sequential passages can be given to increase the amounts of virus before performing subsequent experiments. the extent of changes to the infected embryo that are induced by ibv vary greatly. especially for the field strains, the visible changes in the embryos in the first passage can be minimal. usually, embryo mortality and dwarfing increase as the number of serial passages increases. some field strains still caused no dwarfing on the third passage, while other methods could detect ibv using inoculated allantoic cells (reviewed by de wit, 2000) . of the five ibv isolates in this study, they showed different adaptation to embryos. all the five ibv strains did not cause visible changes in the embryos in the first passage, and four of them caused dwarfing and death of the embryos on the second to fourth passages (table 1) . for one of the ibv isolates, lhi10, obvious changes in the inoculated embryos were observed in the 10th passage. this isolate may not grow well in chicken embryo and the virus titre may be minimal in the first several passages. winterfield & hitchner (1962) first reported a nephrosis condition associated with ib in the united states. cumming (1962) reported an ib outbreak causing severe kidney lesion in chickens in australia in the same year. from this time, various nephropathogenic strains of ibv have been identified throughout the world (reviewed by meulemans & van der berg, 1998) . in china, nephropathogenic ibv strains of the massachusetts genotype have isolated in rural chicken farms of beijing (li & yang, 2001 ). in the present study, we isolated three ibv strains from h120-vaccinated flocks and two from non-vaccinated flocks of layer chickens that experienced nephropathogenic infection in four provinces of north china. virulence studies showed that severe kidney lesions were observed in the dead chicks that had been inoculated with each of the five chinese ibv isolates. it was reported that nephropathogenic ib occurred in pennsylvania from 1997 to 2000 in commercial broiler-type and layer-type chicken flocks, with mortality as high as 20% (ziegler et al., 2002) . similar mortality was also found in this study by inoculating 15-day-old specific pathogen free chickens with each of the five chinese ibv isolates (table 2) . although the results of this experiment were not a true reflection of the situation in the flocks that the five ibv strains came from, they confirmed that the five ibv isolates are nephropathogenic. the s1 protein genes of the five ibv isolates, ld3, ls2, lx4, lh2, and lhi10, isolated from 1999 to 2003 in china, shared 95 to 99% of nucleotide identity and 92 to 98% amino acid identity. this strikingly high identity implied a close genetic relationship and possibly indicated a common origin. not more than 76% nucleotide and amino acid identity was shared between the five ibv isolates and three strains, q1, t3, and j2, which were also isolated in china before 1999 (yu et al., 2001) . isolates q1, t3, and j2 were isolated from the proventricular tissues of infected chickens with a syndrome associated with lesions of alimentary and respiratory tracts. unlike the ibv isolates in this study, nephropathogenic infections were not observed from chickens infected with those three ibv strains (yu et al., 2001) . in addition, nephropathogenic ibv strains that showed more than 99.1 and 97.8% nucleotide and amino acid identity with the s1 protein of standard ibv strain m41 were isolated in beijing (li & yang, 2001) . the s1 protein genes were also compared between the ibv strains isolated in this study and three massachusetts-type ibv strains, m41, h52 and h120. the results showed that they had only 74 to 77% nucleotide and amino acid identity at the level of s1 protein gene and indicated that they belonged to different genotypes. on the basis of the genotype and pathogenicity, we can speculate that there were at least three different genotypes of ibv strains circulating in china. the spreading of a virus from one area or country to another could be due, at least in part, to its improper introduction by the trading of birds or by the use of attenuated vaccines. with the exception of the massachusetts strain, a very interesting aspect of ibv epidemiology, as far as it is possible to know, is the presence and the spreading of the various ibv serotypes in different continents. about 20 emergent serotypes in north america did not spread to other continents. similarly, the european, australian, and asiatic serotypes apparently did not spread elsewhere. in this study, the s1 protein genes of the five chinese isolates were compared with two ibv strains, a1211 and a1171, which were isolated in taiwan; one japanese isolate, kb8523; three european isolates, uk/7/93, d274, and b1648; four american isolates, ga/2787/98, connecticut, beaudette, and ark99. these ibv strains shared only 48 to 79% nucleotide and amino acid identity with the s1 protein of the five chinese isolates. obviously, they belonged to different genotypes. however, we cannot conclude that other ibv types are not present in china because the research on the matter is rather poor and it would be worthwhile to conduct more studies. molecular studies have shown that a new serotype can emerge as a result of only a few changes in the amino acid composition in the s1 part of the virus spike protein, with the majority of the virus genome remaining unchanged (cavanagh et al., 1992) . this could be due to immunologic pressure caused by the widespread use of vaccines, to recombination as a consequence of mixed infections, or to the decrease of dominant serotypes as a result of vaccination, allowing other field strains to emerge. further to this study, more epidemiological investigations are needed to better understand the origin, diffusion and persistence of the new genotype. the sharing of antigens between the ibv isolates and vaccinal viruses might suggest that currently available vaccines should be able to provide protection against challenge from viruses belonging to different serotypes from the vaccine. the low nucleotide and amino acid similarities between the h120 massachusetts vaccine and the new genotype viruses (table 3 ) may account for the occurrence of the disease caused by the new genotype viruses in h120 vaccinated layer flocks. molecular analysis of the 793/b serotype of infectious bronchitis virus in great britain completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus infectious bronchitis infectious bronchitis virus: evidence for recombination within the massachusetts serotype nidovirales: a new order comparing coronaviridae and arteriviridae. archives of virolgy the aetiology of 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sequence of the m41 strain of coronavirus ibv and its comparison with beaudette strains sequence of the spike protein of the belgian b1648 isolate of nephropathogenic infectious bronchitis virus cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus isolation, pathogenicity, and h120 protection efficacy of infectious bronchitis viruses isolated in taiwan isolation and identification of infectious bronchitis virus from chickens in sichuan evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus aetiology of an infectious nephritis */nephrosis syndrome of chickens antigenic and immunogenic characterization of infectious bronchitis virus strains isolated in china between 1986 and 1995 molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia nephropathogenic infection bronchitis in pennsylvania chickens á 77 77 77 77 77 78 78 78 75 76 77 77 77 79 78 75 75 73 78 52 kb8523 76 77 77 77 77 76 76 76 81 81 97 99 99 78 81 77 95 87 76 53 beau 76 77 77 76 76 76 76 76 81 81 98 97 97 78 81 78 97 86 76 53 c o n n 7 4 7 4 7 4 7 4 7 3 7 4 7 4 7 4 7 8 7 8 9 1 9 1 9 0 7 5 7 6 7 4 9 0 9 0 7 3 5 1 ark99 75 75 76 75 75 76 76 76 77 78 78 78 77 77 78 78 78 78 75 51 ga/98 60 60 60 60 60 61 61 61 61 62 62 62 62 60 61 61 62 62 61 61 nucleotide identity (%)top right, amino acid identity (%); bottom left, nucleotide identity (%). a the first 1605 nucleotides, starting at the aug translation start codon, of the s1 protein genes were compared. nephropathogenic ibv 325 key: cord-277804-ujabzic4 authors: yuk, seong-su; kwon, jung-hoon; noh, jin-yong; hong, woo-tack; gwon, gyeong-bin; jeong, jei-hyun; jeong, sol; youn, ha-na; heo, yong-hwan; lee, joong-bok; park, seung-yong; choi, in-soo; song, chang-seon title: comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs date: 2016-01-21 journal: j virol methods doi: 10.1016/j.jviromet.2016.01.008 sha: doc_id: 277804 cord_uid: ujabzic4 a sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (ibv) is important to commercial manufacturers for improving vaccine quality. typically, ibv is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. in this study, we used a dot-immunoblotting assay (dia) to measure the titers of ibv vaccines that originated from different pathogenic strains or attenuation methods in embryonated eggs, and we compared this assay to the currently used method, clinical sign evaluation. to compare the two methods, we used real-time reverse transcription-pcr, which had the lowest limit of detection for propagated ibv. as a clinical sign of infection, dwarfism of the embryo was quantified using the embryo: egg (ee) index. the dia showed 9.41% higher sensitivity and 15.5% higher specificity than the clinical sign determination method. the dia was particularly useful for measuring the titer of ibv vaccine that did not cause apparent stunting but propagated in embryonated chicken eggs such as a heat-adapted vaccine strain. the results of this study indicate that the dia is a rapid, sensitive, reliable method for determining ibv vaccine titer in embryonated eggs at a relatively low cost. infectious bronchitis virus (ibv) is a gamma coronavirus that causes a highly contagious disease in chickens. the virus causes severe economic losses to the poultry industry worldwide because it can affect the upper respiratory and reproductive tracts, and some strains can cause nephritis in chickens (jackwood, 2012) . despite intensive vaccination using both live attenuated and killed vaccines to prevent the disease, the emergence of new variant strains that do not serologically cross-react has complicated disease control and demonstrates the importance of vaccinating chickens with the disease-causing ibv types (cavanagh, 2005 (cavanagh, , 2007 . vaccination is considered the most cost-effective approach for controlling ibv infection (meeusen et al., 2007) . to prevent the economic losses caused by ibv, live attenuated vaccines and inactivated oil-emulsion vaccines containing both the km91 and massachusetts 41 (m41) strains are widely used in korea. more recently, vaccine strains providing broad cross-protection have been developed, including the k2 and k40/09 strains (kim et al., 2013; lim et al., 2012 lim et al., , 2015 . in korea, the viral content of a vaccine preparation is quantified by ibv titration according to standard procedure approved by the animal and plant quarantine agency. for this procedure, the chicken embryos are inoculated with serial dilutions of the virus preparation, and then the embryos are examined for the presence of specific lesions caused by the virus, i.e., dwarfing, curling, and stunting (doherty, 1967) . however, as a variety of live-attenuated vaccines have been developed for variant field strains, it is unclear whether the clinical sign determination method reflects viral propagation in ibv-infected embryos. furthermore, as there are no specific standards for measuring clinical signs, the measurements of specific lesions can differ among observers. our previous study revealed that a novel dot-immunoblotting assay (dia) using monoclonal antibodies against several ibvs could detect viruses propagated in embryonated chicken eggs (song et al., 1998) . to accurately, reproducibly, and efficiently measure vaccine titers, we used the dia to detect ibv propagated in inoculated http://dx.doi.org/10.1016/j.jviromet.2016.01.008 0166-0934/© 2016 elsevier b.v. all rights reserved. embryonated eggs. the aim of this study was to evaluate and compare the sensitivity and specificity of the dia to the clinical sign determination method for detecting ibv in inoculated embryonated eggs during titration of ibv vaccines. a respiratory strain belonging to the mass group (m41), a nephropathogenic strain belonging to the km91-like subgroup (km91), and a recombinant nephropathogenic strain belonging to korean new cluster 1 (k40/09) were used to evaluate the titer of ibvs used in killed vaccines as described by kim et al. (2013) . two nephropathogenic strains that were attenuated by 170 serial passages (k2p170) or heat-adapted passages (k40/09hp40) in chicken embryos were used to evaluate the live-attenuated vaccine strains (lee et al., 2010) . all viruses were propagated in 10-day-old specific-pathogen-free embryonated chicken eggs (ece; hy-vac, adel, ia) at 37 • c for 72 h. allantoic fluid from each egg was harvested, aliquoted, and frozen at −70 • c until use. all animal protocols used in this study were reviewed, approved, and supervised by the institutional animal care and use committee of konkuk university. to confirm the detection limit of the dia, each vaccine strain was serially diluted 2-fold in phosphatebuffered saline (pbs; invitrogen, carlsbad, ca) and analyzed by dia and real-time reverse transcription (rt)-pcr. concurrently, triplicate 10-fold serially diluted samples were used to determine the detection limit of real-time rt-pcr. to measure vaccine titer based on the infectivity of chicken embryos, 10-fold serial dilutions of a 10 0 virus solution (10 −1 -10 −9 ) were generated by mixing 1 ml of the virus with 9 ml of pbs containing 50 g/ml gentamicin sulfate (sigma-aldrich, st. louis, mo); all dilutions were kept on ice. next, 0.1 ml of the 10 −4 through 10 −9 dilutions were inoculated into five 10-day-old spf ece (hy-vac), and the eggs were incubated at 37 • c. no eggs had to be discarded due to non-specific death of embryos within one day of inoculation. after three days of incubation, 500 l of allantoic fluid was extracted from the inoculated eggs using a 1 ml syringe and was used to detect propagated ibv by the dia and real-time rt-pcr. eggs were then resealed with paraffin for further incubation. seven days after inoculation, the embryo:egg (ee) index was calculated for all eggs. propagation of the inoculated virus was determined using real-time rt-pcr, dia, and the ee index method, and the 50% egg infectious dose (eid 50 ) of the five vaccines was calculated based on the method of reed and muench (1938) . rna was extracted from each inoculated allantoic fluid sample using the exiprep viral rna/dna extraction kit (bioneer co., daejeon, korea) according to the manufacturer's instructions, and eluted in a 50 l volume. real-time rt-pcr analysis of the extracted rna samples was conducted as previously described (callison et al., 2006) . the primers and taqman dual-labeled probe were synthesized by macrogen (seoul, korea). the primers and probe were utilized in a 25 l reaction containing 12.5 l of quantitect probe rt-pcr 2× mix (qiagen, hilden, germany), 0.25 l of rt enzyme (qiagen), 0.5 mol of primers, 0.1 mol of probe, and 5 l of the rna sample from 50 l of extracted rna. amplification was performed in an abi prism 7500 real-time pcr system using the following program: 50 • c for 30 min, 95 • c for 15 min, and 45 cycles of 94 • c for 1 s and 60 • c for 60 s, with emitted fluorescence measurement. cycle threshold (ct) values below the detection limit were considered positive for ibv. the dia was conducted as described previously with slight modifications (song et al., 1998) . allantoic fluids from eces inoculated with the serially diluted vaccines were centrifuged at 1000 × g for 1 min. next, 150 l of the supernatant was dotted onto a nitrocellulose membrane (0.45 m pore size) using a hybri-dot 96-well filtration manifold (bio-rad laboratories, hercules, ca). uninfected normal allantoic fluid was used as a negative control. the membrane was then blocked with 3% bovine serum albumin in tris-buffered saline (tbs; 100 mm tris, 0.9% sodium chloride, ph 7.5) at 37 • c overnight. the primary monoclonal antibodies (3f5) were diluted 1:1000 with tris tween-buffered saline (ttbs; 100 mm tris, 0.2% tween 20, 0.9% sodium chloride, ph 7.5) and incubated with the membrane for 30 min at 37 • c. after washing the membrane in ttbs three times for 15 min each with gentle agitation, the membrane was incubated for 30 min at 37 • c with biotinylated anti-mouse immunoglobulin g (vectastain abc kit; vector laboratories inc., burlingame, ca) diluted in ttbs. following washing, the membrane was incubated for 30 min at 37 • c with biotin and avidin-conjugated peroxidase complex (vectastain abc kit) diluted in ttbs. after the final washing, the membrane was developed using 6 mg of diaminobenzidine (pierce, rockford, il) in 10 ml of 50 mm tris and 10 l h 2 o 2 for 1 min. the reaction was then stopped by rinsing with distilled water (three times) and the membrane was allowed to air-dry. dark brown-colored dots were considered positive for ibv. dwarfing of the infected chicken embryos was detected by determining the ee ratio. the weights of the eggs were measured 7 days after inoculation of the serially diluted viruses. the respective eggs and embryos were weighed using an electronic balance (ohaus, florham park, nj). the ee ratio was defined as the weight of embryo divided by the weight of respective egg. a total of 120 spf eggs (hy-vac) were used to determine the ee ratio of control ece (mock-inoculated). briefly, we inoculated 10-day-old eggs with 100 l of sterile pbs as a mock-inoculated control. the ee ratio of 20 eggs was determined at 7 days post-inoculation. the average ee ratio and the standard deviation of the mock control were calculated. the ee index was determined by dividing the ee ratio of inoculated eggs by the mean ee ratio of mock-inoculated eggs (dhinakar raj et al., 2004) . to assess the receiver operating characteristics (roc) using medcalc ® version 15.8 statistical software (mariakerke, belgium), positive or negative results confirmed by real-time rt-pcr were assigned as "true-positive" or "true-negative," respectively. the area under the roc-curve (auc index) was then calculated for both the dia and clinical sign detection methods. high values (close to 1) indicate a highly accurate test (greiner et al., 2000) . the concordance of both assays to the properly classified samples as positive or negative was estimated by calculating the weighted kappa statistic (ä test). ä test values of 0.41-0.60 indicate moderate agreement, values of 0.61-0.80 indicate substantial agreement, and values of 0.81-0.99 indicate nearly perfect agreement (viera and garrett, 2005) . the 2-fold diluted virus was detected concurrently by dia and real-time rt-pcr (supplementary material fig. 1 ). the highest dilution that showed dark-brown dots in the dia was 2 −7 for k2p170 and k40/09 and 2 −6 for m41, km91, and k40/09hp40. the 10-fold diluted viruses were also examined by real-time rt-pcr (supplementary material fig. 2 ). for the tested pathogenic or attenuated ibvs, there were no detectable ct values at the 10 −7 dilution. therefore, the detection limit of the real-time rt-pcr assay was set to the ct value of the 10 −6 dilution. the real-time rt-pcr method was found to be approximately 2 15 -fold more sensitive than the dia method. thus, real-time rt-pcr is suitable for detecting propagated viruses and can be used to compare the dia and ee ratio detection methods for propagated ibv during titration. because the dia does not distinguish between infectious and noninfectious virus, we had to confirm that the assay does not detect inoculated virus in the eggs. as shown by the detection limit of the dia, we observed that the input virus was not detected at dilutions higher than 2 −6 -2 −7 . thus, none of the dilutions used in the titering (10 −4 -10 −9 ) would give a positive result without viral replication, especially after further dilution in the allantoic fluid. after 7 days of incubation, the mean ee ratio and standard deviation of mock-inoculated eggs was 0.375 ± 0.036. to detect dwarfism, the ee index was defined as the ee ratio of ibv-inoculated ece divided by the mean ee ratio of mock-inoculated ece. embryos with ee indices below the lowest individual ee index for mock-inoculated ece (0.880) were classified as positive for dwarfism. supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2016.01.008. based on the results of real-time rt-pcr, the titer of respiratory the type ibv m41 strain was 10 8.4 eid 50 /ml ( table 1) . four of the five eggs inoculated with the 10 −7 dilution showed positive results. the dia results were identical to the real-time rt-pcr results, even the individual egg results. in contrast, only three of the five eggs inoculated with the 10 −6 dilution were positive by the ee index. the weight of the other two eggs exceeded the lowest value of the individual ee index of mock-inoculated ece (0.885, 0.890). because one embryo inoculated with the 10 −8 dilution died 3 days after inoculation, we excluded this egg from the ee index measurements. one egg inoculated with the 10 −9 dilution showed a positive result for the ee index (0.800). the titer of m41 obtained using the ee index was lower than that obtained using the other assays (10 8.3 eid 50 /ml). according to real-time rt-pcr analysis, the titer of the korean nephrogenic type ibv (km91) and its attenuated form (k2p170) were both 10 7.0 eid 50 /ml. based on the individual values, four of the five eggs inoculated with the 10 −5 dilution and three of the five eggs inoculated with the 10 −6 dilution showed positive results for both viruses. the dia results were identical to the real-time rt-pcr results, including the individual egg results. unexpectedly, in the km91 titration, one egg at the 10 −4 dilution showed a negative result (0.933) for the ee index, despite showing positive results in the other assays. because one embryo inoculated with the 10 −5 dilution died 3 days after inoculation, we excluded this egg from the ee index measurements. ee indices of two of the eggs inoculated with the 10 −7 virus dilution (0.878, 0.833) were slightly below the lowest individual ee index of the mock-inoculated ee index. the titer of km91 determined using the ee index was higher than that determined using the other assays (10 7.3 eid 50 /ml). in the k2p170 titration, the results according to the ee indices were identical to those obtained by real-time rt-pcr, except for one egg inoculated with the 10 −7 dilution (0.742). accordingly, the titer of k2p170 determined using the ee index was higher than that determined using other assays (10 7.2 eid 50 /ml). based on the real-time rt-pcr results, the titers of the recombinant nephrogenic type ibv k40/09 and its heat-attenuated form k40/09hp40 were 10 7.8 and 10 8.2 eid 50 /ml, respectively ( table 1) . two of the five eggs inoculated with the 10 −7 dilution of the k40/09 were positive. however, the ct value of one egg inoculated with the 10 −7 dilution of k40/09hp40 (27.96) was higher than the detection limit of the dia (21.07) but lower than that of real-time rt-pcr (35.05). thus, the egg was negative according to the dia. in the k40/09 titration, two embryos inoculated with the 10 −7 and 10 −8 dilutions died at four and five days after inoculation, respectively; therefore, we excluded these eggs from the ee index measurement. the ee index of one egg inoculated with the 10 −8 dilution (0.747) was positive. in the k40/09hp40 titration, one embryo inoculated with the 10 −6 dilution died at two days after inoculation and was excluded from the ee index measurement. the ee indices of two eggs inoculated with the 10 −5 dilution (0.881, 0.907) and one egg inoculated with the 10 −6 dilution (1.159) were negative. two eggs inoculated with the 10 −7 dilution (0.984, 0.919) and one egg inoculated with the 10 −8 dilution (0.938), which were positive according to real-time rt-pcr, did not show clear dwarfism. consequently, the three assays showed three different titers. when compared to the titer measured by real-time rt-pcr (10 8.2 eid 50 /ml), the titers measured by dia (10 7.9 eid 50 /ml) and ee index (10 7.2 eid 50 /ml) were 2-fold and 10-fold lower, respectively. the performance of the two methods was also compared ( table 2) . dia detected propagated ibv in 77 (97.25%) of 79 positive samples, with perfect specificity (100%) and an auc index of 98.7%. the value for this test was 0.973, indicating that the values were highly consistent with the real-time rt-pcr results. of the 79 true-positive samples, two samples in k40/09 and k40/09hp40 were false-negatives by dia, which was also observed using the clinical signs method. the results of the ee index, which was defined by the cut-off value of the lowest ee index of the mockinoculated ece (0.880), showed propagated ibv in 65 (87.84%) of 74 positive samples, with relatively low specificity (84.50%) and an auc index of 86.2%. the ä value for this test was 0.724, indicating moderate agreement with the real-time rt-pcr results. of the nine false-negative results obtained using the clinical sign method, two samples were m41, four samples were km91, two samples were k2p170, and one sample was k40/09. of the 11 false-positive results obtained using the clinical sign method, three samples were m41, two samples were km91, one sample was k2p170, and five samples were k40/09hp40. for the ee index results, which were obtained using the cut-off value of the lowest individual ee index of the mock-inoculated eces, we compared the cut-off value to the optimum value calculated based on the ee index distribution. the optimum cut-off (0.890) calculated based on the ee indices distribution suggested that the cut-off value of the lowest individual ee index of the mock-inoculated eces (0.880) was reliable and could be used to classify clinical signs by weight. using this new cut-off, only one false-negative sample was corrected to a positive result, and the assay showed 89.19% sensitivity and 85.92% specificity (fig. 1) . we compared the clinical signs and dia results to the real-time rt-pcr results and found that the dia results were highly consistent with the real-time rt-pcr results. among the 150 eggs inoculated with serially diluted ibv samples, only two samples showed false-negative results. however, 20 results obtained using the clinical signs method did not match the real-time rt-pcr results. for the m41 respiratory strain, the titer calculated by the clinical signs method were lower than the titer calculated by real-time rt-pcr. the nephropathogenic strain km91 and its attenuated form k2p170 showed higher titer when measured by the clinical signs method. compared to km91, k2p170 showed clearer signs of stunting. this demonstration that egg-adapted strains induce clearer embryo dwarfism than wild strains is in agreement with those of dhinakar raj et al. (2004) . however, the recombinant nephrogenic strain k40/09 and its attenuated counterpart k40/09hp40, which was egg-passaged with heat adaptation, showed opposite results. the overall ee indices were higher for k40/09hp40 than for k40/09. moreover, the k40/09hp40 titer measured by the clinical signs method was approximately 10-fold lower than the titer measured by real-time rt-pcr. these results suggest that the clinical signs induced by this heat-adapted virus might not be sufficient to produce dwarfism in eces. the extensive genetic diversity and high mutation rate of ibv generate many different virus types that the existing ibv vaccine strains do not protect against. thus, there is great demand for a multiple serotype ibv vaccine that provides broad protection. in addition, several research groups have attempted to develop a multivalent vaccine against not only multiple ibv serotypes but also other avian infectious diseases such as newcastle disease, avian influenza, and infectious laryngotracheitis, as well as nonrespiratory diseases such as marek's disease and fowl pox (ndegwa et al., 2015; sharma et al., 2002; vagnozzi et al., 2010; winterfield, 1968) . for such multivalent vaccines, which must be mixed accurately to induce a protective immune response, it is thought that the higher or lower titers observed by the clinical sign method (e.g., for the nephropathogenic strain or heat-adapted strains) could prompt manufacturers to add less or more antigen than required, respectively. this is especially important for live vaccines, because the virus first infects the host and then induces an immune response via innate and acquired immunity, the virus quantitation must represent the true propagating ability within the host. in korea, the currently approved methods for quantifying freeze-dried live ibv vaccine or killed ibv vaccine before virus inactivation state that quantification should be determined by observing infectious bronchitis-induced lesions at seven days after inoculation of serially diluted vaccine into 10-day-old ece (qia, 2015) . generally, the disease-induced lesions are clinical signs, including stunting, curling, and clubbed down. in the us, the method for titrating ibv vaccines also states that on the seventh day after virus inoculation, all eggs should be examined for ibv lesions (usda, 2014) . in this method, specific lesions, such as bile stasis and kidney urates, are listed as general clinical signs. however, assessing specific lesions is laborious and time consuming. moreover, well-trained observers who can recognize the clinical signs and can skillfully approach without damaging them are required. in this study, we choose stunting as a typical clinical sign because it is the most objective and quantifiable clinical sign, and it can be easily distinguished without observer error. real-time rt-pcr is a reliable method because its detection limit is lower than that of other methods. however, the fluorescence thermal cycler requires continuous maintenance and trained operators who can perform the experiments without introducing cross-contamination between samples, which is very costly. accurate measurement of vaccine titer could help manufacturers increase vaccine production or prevent vaccine failure resulting from antigen content that is insufficient to induce an immune response. the dia method could be used to measure the titer of ibv vaccines that do not elicit clear stunting signs but propagate in eces, such as the heat-adapted vaccine strain. in addition, this method is faster than clinical signs method, and could reduce inter-observer differences. the results indicate that the dia presented in this study is a relatively low cost, timesaving, sensitive, and reliable method for detecting ibv during ibv vaccine titration in embryonated eggs. development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus egg:embryo weight ratio as an indicator of dwarfism induced by infectious bronchitis virus titration of avian infectious bronchitis virus in the tissues of experimentally infected chickens principles and practical application of the receiver-operating characteristic analysis for diagnostic tests review of infectious bronchitis virus around the world cross-protective immune responses elicited by a korean variant of infectious bronchitis virus characterization of a novel live attenuated infectious bronchitis virus vaccine candidate derived from a korean nephropathogenic strain live attenuated nephropathogenic infectious bronchitis virus vaccine provides broad cross protection against new variant strains successful cross-protective efficacy induced by heat-adapted live attenuated nephropathogenic infectious bronchitis virus derived from a natural recombinant strain current status of veterinary vaccines combined infectious bronchitis virus arkansas and massachusetts serotype vaccination suppresses replication of arkansas vaccine virus korean standard assay of veterinary biological products, chicken infectious bronchitis freeze-dried live vaccine standard examination a simple method of estimating fifty per cent endpoints field trial in commercial broilers with a multivalent in ovo vaccine comprising a mixture of live viral vaccines against marek's disease, infectious bursal disease, newcastle disease, and fowl pox detection and classification of infectious bronchitis viruses isolated in korea by dot-immunoblotting assay using monoclonal antibodies united states department of agriculture center for veterinary biologics testing protocol. in: supplemental assay method for the titration of newcastle disease vaccine, infectious bronchitis vaccine, and combination newcastle disease-infectious bronchitis vaccine in chicken embryos. united states department of agriculture animal and plant health inspection service protection induced by infectious laryngotracheitis virus vaccines alone and combined with newcastle disease virus and/or infectious bronchitis virus vaccines understanding interobserver agreement: the kappa statistic respiratory signs, immunity response, and interference from vaccination with monovalent and multivalent infectious bronchitis vaccines this work was supported by a grant from the agricultural biotechnology development program, ministry of agriculture, food and rural affairs, republic of korea (no. 314017-03). key: cord-263785-0iift8zy authors: zhang, xiaorong; liao, kai; chen, shuqin; yan, kun; du, xubin; zhang, chengcheng; guo, mengjiao; wu, yantao title: evaluation of the reproductive system development and egg-laying performance of hens infected with tw i-type infectious bronchitis virus date: 2020-07-31 journal: vet res doi: 10.1186/s13567-020-00819-4 sha: doc_id: 263785 cord_uid: 0iift8zy the prevalence of tw i-type infectious bronchitis virus (ibv) has been increasing rapidly, and it has become the second most common genotype of ibv in china threatening the poultry industry. in this study, 1-day-old specific-pathogen-free (spf) chickens infected with tw i-type ibv were continuously observed for 200 days. tw i-type ibv affected the respiratory, urinary, and female reproductive systems, resulting in a mortality rate of 10% as well as a decrease in egg quantity and an increase in inferior eggs. during the monitoring period, serious lesions occurred in the female reproductive system, such as yolk peritonitis, a shortened oviduct, and cysts of different sizes with effusion in the degenerated right oviduct. the infective viruses persisted in vivo for a long time, and due to the stress of laying, virus shedding was detected again after the onset of egg production. our findings suggest that tw i-type ibv is deadly to chickens and could cause permanent damage to the oviduct, resulting in the poor laying performance of female survivors and decreasing the breeding value and welfare of the infected flock. avian infectious bronchitis is a highly contagious acute viral disease in chickens caused by infectious bronchitis virus (ibv) [1] . as a member of the genus gammacoronavirus, ibv has a remarkably high mutation and recombination rate, leading to numerous types and variants that differ from each other in pathogenicity [2, 3] . although the site of entry of ibv is the upper respiratory tract, where the initial infection occurs, the virus can spread systemically, replicating in the epithelial cells of many organs and causing injuries of the kidneys and female reproductive tract [4] . the kidney, trachea, caecal tonsil, and cloaca have been demonstrated to be tissues in which the long-term persistence of ibv is observed [5] . it has been reported that the virus can even be re-excreted from the faeces of h52-infected and h120-infected chickens at 227 days post-infection [6] . the mechanisms involved in the long-term persistence of virulent ibv in convalescent chickens are assumed to be related to viral pathogenicity, and further study is needed [7] . tw i-type ibv was first discovered in 1992 in taiwan and subsequently identified as a new genotype. since the first report in chinese mainland in 2009, the prevalence of this ibv type has increased rapidly nationwide [8] . it [9] . due to differences in antigenicity, the protection provided by commercial vaccines in chickens infected with tw i-type ibv is not complete, and the incidence of immune failure caused by this strain type has increased in recent years [10] . the majority of tw i-type ibv strains exhibit widespread tissue tropism, and they can affect the respiratory, urinary and reproductive systems, and lead to the death of young chickens [11] . there is evidence suggesting that tw i-type viruses have undergone extensive evolution, with diverse strains circulating in chicken flocks, and the need to comprehensively evaluate the pathogenicity of this type of strain has become even more urgent [12, 13] . in this study, the pathogenicity of tw i-type ibv was evaluated by examining clinical symptoms, mortality rates, virus shedding, lesions, and laying performance in terms of egg quantity and quality in infected chickens. the aim of the study was to comprehensively reveal the pathogenicity of tw i-type ibv, particularly regarding the long-term impact on egg production. strain ck/ch/ah/2011/3 (abbreviation: ah1103) of tw i-type ibv used in this study was isolated in 2011 from the trachea and kidney of chickens in a broiler flock exhibiting respiratory signs and death [14, 15] . ah1103 was serially diluted, and five replicate samples of 10 −3 , 10 −4 , 10 −5 , 10 −6 , and 10 −7 dilutions were inoculated into 10-day-old embryonated spf chicken eggs, the 50% egg infections dose (eid 50 ) of this strain was calculated by the reed-muench method [16] . spf chicken eggs were purchased from the beijing boehringer ingelheim merial vital laboratory animal technology co., ltd, china. one-day-old spf white leghorn chickens were purchased from the jinan sipai furui livestock technology co., ltd. the operation and treatment of the animals were approved by the institutional animal care and use committee of yangzhou university (yzudwll-201902-001). one-day-old spf chickens (n = 140) were randomly divided into two groups, each group contained 70 chickens. at 30 days of age, all cockerels were picked out and discarded. the chickens were housed in separate negative-pressure isolators in biosafety level 2 facilities and supplied with feed and water ad libitum. at 1 day of age, the challenge group was infected via oculo-nasal route, with 100 μl of pbs diluent containing 10 5.5 eid 50 of the ah1103; and the control group was administered pbs instead. after challenge, the chickens were observed daily. the full trial period lasted 200 days. the pathogenicity of tw i-type ibv in the early stage post-infection was evaluated via the clinical symptoms, pathological lesions, and virus shedding from trachea and cloaca. dyspnea, gasping, tracheal rales, diarrhoea, depression, anorexia, and death in the infected chickens were recorded as clinical symptoms. at 5 days post-infection (dpi), five chickens in each group were euthanized by cervical dislocation for microscopic lesion examination. at 7 dpi and 14 dpi, the oral and cloaca swabs were collected from 10 chickens in each group for the detection of virus shedding by reverse transcription-quantitative polymerase chain reaction (rt-qpcr). at 140 days of age, the effects of tw i-type ibv infection on the female reproductive system were evaluated. nine hens of the challenge group and three hens of the control group were euthanized by cervical dislocation and necropsied. the developmental status of oviducts and ovaries were checked and documented. then, fourteen chickens in each group were randomly chosen for comparison of egg production performance and egg quality after the onset of laying. in the laying period, the eggs were collected daily, and quality traits were evaluated for each egg in the next morning [17] . eggs were weighed individually, and their egg weight recorded to the nearest 0.1 g. egg length and width was measured to the nearest 0.1 cm using calipers. the width was divided by the length and multiplied by 100 to obtain the shape index. eggs were broken onto a flat surface and the height of the albumen was measured in millimeters using a tripod micrometer halfway between the edge of the yolk and thick albumen. the thickness of the eggshell was measured in 3 places around the midline to the nearest 0.01 mm, and averaged. at 156 days of age, the oral and cloaca swabs were collected from 6 hens in each group for the detection of virus re-excretion by virus isolation and rt-qpcr. at 200 days of age, all of the hens were euthanized by cervical dislocation and necropsied. the samples of trachea, lungs, kidneys, oviduct, heart, liver, spleen, cecal tonsils, duodenum, and glandular stomach of 6 chickens were collected, and tissues from every two chickens were pooled for viral rna detection by rt-qpcr. the oral and cloaca swabs were washed in pbs, and suspensions of tissues were also prepared in pbs (20% w/v). samples of 156-day-old hens were blind passaged three times in the allantoic cavity of 10-day-old embryonated spf chicken eggs. total rna of the samples were extracted using the ultrapure rna kit (cowinbio, beijing, china), and reverse transcription was performed with the easyscript ® reverse transcriptase [m-mlv, rnaseh-] kit (transgen biotech, beijing, china). the primers and probe used for rt-qpcr were described in a previous study [18] . the reaction mixture and the thermal profile employed for rt-qpcr were as specified in the aceq qpcr probe master mix kit (vazyme biotech, nanjing, china). for haematoxylin-eosin staining, the collected samples of the trachea, lungs, kidneys, and oviduct were fixed in 10% neutral formalin for 48 h at room temperature. the fixed samples were processed, embedded in paraffin wax, and cut into 5 μm sections and examined by light microscopy for the presence of lesions. for the examination of ultrastructural alterations, the cylindrical trachea were cut crosswise with two sharp blades into small strips of 1 mm wide and 3-4 mm long, and then the strips were cut into 1 mm 2 small squares. the squares were then fixed in 2.5% glutaraldehyde in 0.1 m pbs for 12 h at 4 °c. the samples were then washed with pbs and dehydrated via an alcohol gradient, followed by drying (cpd-30d) and conductive treatment (scd 500). finally, the ultrastructure of the tracheal cilia was observed by scanning electron microscopy (gemin-isem 300) with a magnification of 5000×. all statistical analyses were performed with spss 26.0 software. non-parametric mann-whitney u test was used to analyze the significant difference, and p < 0.05 was considered as significant. the clinical manifestations in the early infection stage caused by tw i-type ibv were identified as respiratory symptoms. nearly half of the chickens in the challenge group exhibited gasping, tracheal rales or dyspnea. seven chickens died from 9 to 30 dpi, accounting for 10% of the infected flock. gross lesions such as light hyperaemia and serious catarrhal exudation in the tracheal mucosa and urate deposition in the tubules of the pale kidney, appeared in the infected flock from 5 to 30 dpi. the development of oviducts and ovaries was assessed at 140 days of age. the reproductive system of chickens in the control group was well developed. however, there were 4/9 chickens in the challenge group that showed delayed development of oviducts and ovaries ( table 1 ). the right oviducts of all chickens in both control and challenge groups were completely degenerated. right cystic dilatations attached to the cloaca with a watery content were present in 5/9 of the chickens from the challenge group, and another 1/9 chicken had a cyst on the left immature oviduct wall (table 1, figure 1 ). the pathogenicity of tw i-type ibv in the laying period was characterized by a decrease in the quantity of eggs and an increase in the quantity of inferior eggs (table 2, figure 2 ). during the 21.5-to 27.5-week-old in which laying was monitored, egg production in the challenge group was lower than that in the control group; the decrease of egg production was 19.3% in the challenge group; and the peak egg production per week (66.3%) was lower than that in the control group (85.7%). the mean albumen height in the challenge group was 0.82 mm thinner than that in the control group, representing a decrease of 10.39% (p < 0.05). these results indicate that tw i-type ibv has a significant effect on subsequent laying performance when the infection occurs at a very young age in chickens. at the age of 200 days, various lesions were observed in the reproductive systems of the hens (table 3, figure 5 ). in the challenge group, lesions of yolk peritonitis were found in 7 infected females, which were assumed to have been caused by the mature follicles or eggs falling into the abdomen and failing to be captured (table 3) . almost all of the females exhibited a large or small oviduct cyst, three of which presented a diameter greater than 3 cm (figure 1) . a fibrin clot (yolk-like) in the lumen of the oviduct was observed in 3 females (figure 3 ). none of these lesions were found in the control group. the rt-qpcr results for the oral and cloaca swabs collected at 7 dpi and 14 dpi indicated virus shedding via the respiratory tract and cloaca at 7 dpi (figure 3 ). at 156 days of age, the viruses were re-excreted from the cloaca (2/6 in the challenge group), about 10 2.2 copies/μl, and two positive samples were blind passaged three times in spf chicken eggs, 10 6.7 and 10 8.3 copies/ μl were detected. at 200 dpi, one of the pooled caecal tonsil samples was detected containing 10 4.1 copies/μl ( figure 4 ). the ultrastructural examination showed the presence of catarrhal exudates and the adhesion, lodging, and shedding of cilia on the tracheal surface of the infected flock at 5 dpi (see additional file 1). the tracheae were collected at 5 dpi; the lesions of the blurred boundary between the cilia, congestion, inflammatory cell infiltration, and necrosis of ciliated epithelial cells were widespread. in the laying period, the lesions of the congestion, inflammatory cell infiltration, broadening of the interstitial region, and desquamation of epithelial cells in the oviducts were common in the infected flock ( figure 5 ). no significant lesions were observed in the control group. the prevalence of tw i-type ibv has increased greatly in china in recent years, which has caused tremendous economic losses to the poultry industry [9] . some reports have illustrated that the majority of tw i-type ibv show tissue tropism to the kidneys and trachea and result in a moderate mortality rate [19, 20] . however, there are no long-term reports on the effects of tw i-type ibv on the quantity and quality of eggs when infection occurs at a very young age in chickens, although the poor laying performance and occurrence of "false layers" caused by qx-type ibv have attracted widespread attention [21, 22] . in this study, a 200-day pathogenicity study of the tw i-type ibv strain in chickens was performed. the results suggest that this strain can result in death and has consecutive adverse effects on the female reproductive system, which decrease the breeding value and welfare of the infected flock. in the early infection stage, the pathogenicity of tw i-type ibv in chickens is similar to that of other prevalent types of strains regarding, for example, the symptoms of tracheal rales, and dyspnea or lesions of urate deposition in the kidneys [22, 23] . in terms of the ultrastructural and microstructural examination of the trachea, the lesions of adhesion and lodging and the shedding of tracheal cilia caused by tw i-type ibv are assumed to result directly from inflammatory cell infiltration and the presence of serious catarrhal exudates [24] . respiratory lesions, which are the most common pathogenic characteristic of ibv, subsequently recover and do not lead to the death of infected chickens [25, 26] . similar to qx-type strains, the renal lesions of urate deposition may be responsible for the death of chickens, although the mortality rate caused by tw i-type ibv was relatively low [27, 28] . during the laying period, the impacts caused by tw i-type ibv in females can be summarized into four categories: a high incidence of oviduct cysts, consecutive lesions in the female reproductive system, decreases in egg production, and poor quality of eggs. the mechanisms involved may contribute to the lesions in the reproductive system that emerged throughout the monitoring period and were induced by the virus reserves in the caecal tonsil. some previous studies have illustrated that ibv exhibits reproductive tissue tropism and that the virus induces an immune response involving an influx of cytotoxic cells and upregulation of inflammatory cytokines in the oviduct and ovary [29, 30] . according to a previous report, the two main candidate sites for ibv persistence are cecal tonsils and the kidneys [31] . however, in this study, we found that the cecal tonsil was the only site of virus replication at 200 days of age. in addition, the exacerbated inflammation and physiological disorders in the oviduct caused by reinfection would be further worsened due to the effect of oestrogen [32] . as a result, during the laying period, the congestion and inflammatory cell infiltration in the oviducts were more severe than at any earlier time. these adverse impacts were subsequently reflected in a series of lesions in the female reproductive system, such as shortening of the length of the oviduct and a decrease in the number of hierarchical follicles in the ovary, which were directly responsible for a portion of the decrease in egg production. however, the pathogenicity of ibv in the female reproductive system is irregular, and the factors involved include the strains of the virus, the age of the infected chickens, and the protection afforded by antibodies [21, 33] . some qx-type strains can also induce oviduct cysts with fluid accumulation in chickens of different ages, but some 4/91-type strains fail to do so [34, 35] . not all mass-type strains can induce cystic oviducts or the occurrence of false layers, which differ in different strains [26, 35] . some recombinant strains can also induce oviduct cysts even when chickens have been inoculated with commercial vaccines [33] . the long-term decrease in egg production and the shortened oviduct observed at 200 dpi suggest that the damage to the reproductive system caused by tw i-type ibv might be a permanent impairment rather than just a developmental delay [36] . in conclusion, tw i-type ibv is characterized by respiratory symptoms, urate deposition lesions in the kidneys, and the continuous disturbance of the female reproductive system, resulting in a high incidence of oviduct cysts and a decline in the quantity and quality of eggs. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-00819 -4. abbreviations ibv: infectious bronchitis virus; spf: specific-pathogen-free; dpi: days post infection; rt-qpcr: reverse transcription-quantitative polymerase chain reaction. coronavirus avian infectious bronchitis virus evolution of infectious bronchitis virus in china over the past two decades recombination in avian gamma-coronavirus infectious bronchitis virus induction of ibv strain-specific neutralizing antibodies and broad spectrum protection in layer pullets primed with ibv massachusetts (mass) and 793b vaccines prior to injection of inactivated vaccine containing mass antigen the long view: 40 years of infectious bronchitis research a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus re-excretion of infectious bronchitis virus in chickens induced by cyclosporin phylogenetic analysis of the s1 glycoprotein gene of infectious bronchitis viruses isolated in china during epidemiology and characterization of avian infectious bronchitis virus strains circulating in southern china during the period from serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china altered pathogenicity of a tl/ch/ldt3/03 genotype infectious bronchitis coronavirus due to natural recombination in the 5′-17 kb region of the genome genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems characterization and analysis of an infectious bronchitis virus strain isolated from southern china in 2013 molecular epidemiology of infectious bronchitis virus in china between 2009 and 2011, and development of recombinant vaccine using marek's disease virus as vector development and efficacy of an attenuated tw-i like infectious bronchitis virus strain as a candidate live vaccine for chickens in china coronaviruses: methods and protocols. in: walker jm (ed) methods in molecular biology egg quality traits differ in hens selected for high as compared with low antibody response to sheep red blood cells development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/ch/ljl/140901-like backbone and an s2 fragment from a 4/91-like virus induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d388 (genotype qx) by maternally derived antibodies and by early vaccination comparative histopathology and immunohistochemistry of qx-like, massachusetts and 793/b serotypes of infectious bronchitis virus infection in chickens pathogenicity differences between a newly emerged tw-like strain and a prevalent qx-like strain of infectious bronchitis virus safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine genetic and pathologic characterization of a novel recombinant tc07-2-type avian infectious bronchitis virus pathogenicity differences between qx-like and mass-type infectious bronchitis viruses preparation and protective efficacy of a chicken embryo kidney cell-attenuation gi-19/qx-like avian infectious bronchitis virus vaccine a multi-omics study of chicken infected by nephropathogenic infectious bronchitis virus effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct effects of the routine multiple vaccinations on the expression of innate immune molecules and induction of histone modification in ovarian cells of layer chicks the effects of oestrogen and progesterone on re-excretion of infectious bronchitis virus strain g (correction of straing) in spf chickens the effect of estrogen on the early cytotoxic response to ib virus infection in hen oviduct new insights on infectious bronchitis virus pathogenesis: characterization of italy 02 serotype in chicks and adult hens comparative pathogenicity of malaysian qx-like and variant infectious bronchitis virus strains in chickens at different age of exposure to the viruses comparison of the pathogenicity of qx-like, m41 and • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc /b infectious bronchitis strains from different pathological conditions pathogenicity of virulent infectious bronchitis virus isolate yn on hen ovary and oviduct publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. xiaobo wang of yangzhou university for providing help during histopathologic examination. the datasets analyzed during the current study are available upon request from the corresponding authors. the authors declare that they have no competing interests.received: 3 february 2020 accepted: 16 july 2020 key: cord-022378-ovxmy1as authors: cook, jane k.a. title: coronaviridae date: 2009-05-15 journal: poultry diseases doi: 10.1016/b978-0-7020-2862-5.50033-7 sha: doc_id: 22378 cord_uid: ovxmy1as nan infectious bronchitis was fi rst reported in north dakota, usa, in 1931 as an acute, highly infectious respiratory disease of chickens. it now has worldwide distribution. th e primary target organ is the respiratory tract, where initial infection and disease occurs. ibv also aff ects egg-laying performance, and renal damage associated with infectious bronchitis has become increasingly important, particularly in broilers. economically, the most important aspects are the eff ects on egg production and quality in laying hens and production performance in broilers, where the initial respiratory infection is frequently exacerbated by secondary infections. th e ibv contains four structural proteins: a surface projection, known as the spike, a nucleoprotein, a membrane protein and a small membrane protein. th e spike (s) glycoprotein is responsible for attachment to and fusion of the virus membrane with the host cell membrane, as well as for inducing protective immune responses. molecular studies have shown that the s gene of ibv is also responsible for determining the serotype or genotype of an ib virus or variant. it is now known that only a small number of amino acid changes in the s1 part of the spike can result in what is defi ned by laboratory tests as a new variant. since it is possible for concurrent infection with more than one type of ibv to occur, new variants may emerge as a result of both recombination between two diff erent ibvs, as well as by mutations of the genome. many diff erent ibv variants are recognized both on the basis of antigenic variation, determined by virus neutralization (vn) tests, and increasingly by molecular analysis of the genome (genotyping). variants of economic importance include: massachusetts, which is the serotype most commonly found worldwide; connecticut, arkansas and delaware 072 from the usa; t from australia; d274; d1466; 793b (4/91); b1648; italian 02 from europe, and so on. ibvs in australia have evolved in their own distinct lineages and other areas, for example parts of south-east asia, have their own unique ibv variants, as well as ones found in other parts of the world. th e number of new ibv variants continues to increase as simpler methods for their detection become available. while these diff erent variants are important epidemiologically, their signifi cance in terms of control of infection is less clear. th is is because new variants are defi ned by a very small number of amino acid changes, so that the majority of the genome is conserved. in addition to antigenic variation within ibvs, variations in virulence are now reported. th ere have been reports from the usa and europe of ibv strains with increased virulence compared with previous isolates of the same serotype. th ere may also be variations in pathogenesis. one such example of this is the 793b (4/91) variant, which has been associated with both muscle myopathy and scouring in broilers, as well as with mortality in adult breeding hens. more recently a variant ibv isolated in europe, designated d388 (qx), has been associated with 'false layers'. although domestic fowl have usually been regarded as the exclusive host of ibv, there are reports of ibv isolations from other avian species. in turkeys, coronaviruses (tcov, also called bluecomb disease virus and turkey enteric coronavirus) are known to be associated with enteric disease, mortality and underperformance and to aff ect egg-laying performance in older birds. in pheasants, coronaviruses have been associated with respiratory disease and renal problems. sequence data for coronaviruses from turkeys and pheasants show them to be at least as closely related to ibv as diff erent ibv strains may be to each other and it is clear that the host range of ibv itself extends beyond the chicken. in the last few years, possibly as a result of renewed interest in coronaviruses with the emergence of the severe acute respiratory syndrome (sars) virus, coronaviruses have been reported in other gallinaceous birds, including partridge, guinea fowl and peafowl, as well as in nongallinaceous birds such as teal (in this case, the virus was possibly an ibv that had spread from nearby chickens). in some cases, analysis of the genome of these coronaviruses has shown them to be ibv, and examples exist to indicate both reisolation of a vaccine strain and isolation of a virulent fi eld strain. however, some recent coronavirus isolates from gallinaceous birds, for example mallard duck and a pigeon, have two small extra genes near the 3ј end of the genome, indicating that they cannot be classifi ed as ibv and may represent new species. th is is a new area of investigation and, while the coronaviruses detected in some gallinaceous and nongallinaceous birds have not so far been associated with disease, these species are potential carriers of ibv and other coronaviruses and could therefore play a role in global transmission of infection. avian coronaviruses are not known to pose any human health risk. ibv is highly infectious and only a few virus particles may initiate an infection. after a short incubation period of 1-3 days, bird-to-bird transmission occurs rapidly and signs are seen in most birds in a susceptible fl ock. virus is shed via both the respiratory tract and the faeces and a high standard of biosecurity is essential to minimize both entry of infection to a fl ock and spread between fl ocks. th e virus may be shed for several weeks after clinical recovery and can persist in the intestinal tract for several months. th e virus is rapidly killed by common disinfectants but its survival in the environment has not been adequately studied. direct airborne transmission of virus from the respiratory tract to susceptible birds is probably the most common method of spread, particularly when respiratory signs are present. however, transmission through infected faeces is also important and spread by fomites certainly occurs. th e greatest source of infection is birds in which virus is rapidly replicating. while true egg transmission is not believed to be signifi cant, surface contamination of eggs is likely to occur. because the virus may persist in the bird for many weeks, carrier birds may exist. th ere is no evidence for the involvement of vectors in ibv transmission. respiratory signs are the fi rst and most common clinical manifestation in birds of all ages and include tracheal rales, gasping, sneezing, watery nasal discharge, sometimes accompanied by lacrimation, and facial swelling. generally birds appear huddled and depressed; food conversion and weight gain are usually aff ected. mortality is generally negligible in the uncomplicated disease, except in very young chicks and in the absence of maternal antibody. however, signifi cant morbidity and mortality may occur as a result of secondary bacterial infection. th e uncomplicated disease may last 10-14 days but secondary infection may increase the duration. renal problems caused by ibv often follow an initial respiratory infection in broilers. aff ected birds, commonly at 3-6 weeks of age, show depression, scouring and wet litter, associated with increased water intake. in the mild form there may be little or no mortality, but mortality can reach 30%. th e kidneys are often pale, swollen and blotchy and the distended tubules are white, because of the presence of urates ( fig. 28 .2). in ibv infection of commercial layers or broiler breeders, respiratory signs may or may not be observed and the most common manifestation is the eff ect on egg production and egg quality. flocks infected prior to or during lay are aff ected. in unprotected fl ocks, the drop in egg production may exceed 50%. however, in birds that have received a complete vaccination programme but are challenged with fi eld strains against which those vaccines do not provide complete protection, the disease may present as failure to lay at the full potential or production falls of up to approximately 10% ( fig. 28 .3). production may take 4-6 weeks to return to normal, or this may never be achieved. as production begins to increase, deterioration in external and internal egg quality is seen. eggs may be smaller than normal; they may be misshapen or show ridging. th e shells may be depigmented, some becoming almost completely white ( fig. 28.4) , or have calcareous deposits. eggs are often very thin-shelled or completely shell-less. internally, the albumen loses its viscosity ('watery whites') and the chalazae are often broken so that the yolk fl oats free. small haemorrhages may be seen in the albumen or yolk. some birds in an infected fl ock may lay normally, despite showing respiratory signs. it has been known for many years that infection of very young, susceptible chicks with a particularly virulent ibv strain may be followed by aberrant oviduct development. th ere may be partial, or almost complete absence of the duct, or vestiges that are nonpatent or cystic. at maturity aff ected birds may ovulate normally, with the ova then being shed into the body cavity. such birds go through the motions of oviposition but fail to lay ('blind or false layers'). sometimes ova pass along a patent but abnormal oviduct, giving eggs of reduced shell and albumen quality. until recently, there was little evidence to suggest that this phenomenon was a signifi cant problem under normal commercial conditions. however, since 2004, there have been reports from parts of europe of poor egg production in otherwise apparently healthy fl ocks, but in which some hens may present with a pendulous abdomen. in such cases, ovaries may appear to be normal and % egg production functional, whereas the oviduct is thin-walled and frequently contains large, watery cysts. a novel ibv variant (designated d388 or qx) has been detected in a number of such cases; however, experimental reproduction of the condition with this isolate has yet to be reported. th ese include excess mucus in the trachea, nasal cavity and sinuses, accompanied by infl ammation and catarrhal exudate that may become caseous. th is may lead to formation of mucoid plugs of pus in the primary or secondary bronchi, frequently causing asphyxia. lungs may be congested and air sac walls may be cloudy and thickened, often with yellow, caseous exudate. in infected layers, the oviduct may appear normal. however, deposits of yolk may accumulate in the abdomen: so called 'egg peritonitis'. within about 18 h of infection, the trachea and bronchi show loss of cilia with epithelial hyperplasia and metaplasia, often with sloughing of the surface cells. subepithelial thickening is marked, with oedema and massive monocyte and lymphocyte infi ltration of the lamina propria and loss of mucous glands. disease of the mature oviduct results in regression in size with metaplasia of the epithelium, glandular dilatation, infi ltration of subepithelial tissues with monocytes and proliferation of lymphoid follicles and later fi broplasia. when aberrant oviduct development follows infection with a highly virulent strain at a very young age, there is hypoplasia of the epithelium and tubular glands and the lumen may be obliterated. in renal infections, interstitial lymphocytic infi ltration occurs, with granular degeneration, vacuolation and necrosis of the tubular epithelium, together with accumulation of urates and necrotic material in the lumen. in the ureters there is metaplasia and necrosis of the epithelium with sloughing into the lumen. visceral gout is sometimes seen. some innate increase in resistance to infectious bronchitis infection occurs with increasing age. th e immune status of the host infl uences protection, and both maternal immunity and active immunity, resulting from natural infection or vaccination, may prevent or reduce disease and limit virus excretion. experimental data suggest that there are diff erences between inbred lines of chicken in their susceptibility to ibv infection, but there are no data to suggest that this extends to commercial breeds. th ere is evidence that onset of lay (probably associated with hormonal changes) can cause re-excretion of virus that has been latent in the host following an earlier infection. in young chickens, particularly broilers where stocking density is highest, infection with other pathogens may result in more severe and prolonged respiratory disease. such pathogens include newcastle disease virus, infectious laryngotracheitis virus, avian pneumoviruses, bacteria such as haemophilus paragallinarum or escherichia coli, and mycoplasma gallisepticum or mycoplasma synoviae. chilling in brooding chicks can exacerbate the disease, as can poor ventilation and build-up of ammonia levels in the shed. immunosuppressive agents, such as infectious bursal disease virus, may reduce the protective immune response to vaccination or fi eld challenge. in the nephritic form of the disease, some breeds appear to be more severely aff ected and a highprotein diet is an exacerbating factor. th e clinical features and gross and histological lesions are not diagnostic. th e respiratory infection may resemble diseases caused by other pathogens, either alone or as part of a multifactorial disease syndrome. poor egg production and quality may be caused by many infectious and noninfectious factors, including poor management. th ere are also other causes of abnormal oviduct development, and kidney disease may be associated with nutritional defi ciencies or be of unknown cause. th erefore, proof of ibv infection depends on detecting either the virus itself or an increase in specifi c antibody levels in serum. th e detection of ibv in respiratory tract tissues is easiest in the early stages of infection, when virus is replicating most rapidly. beyond about 7-10 days after infection virus is diffi cult to isolate. at later stages, or when cases of aberrant egg production are being investigated, faeces, intestinal tract tissue (possibly caecal tonsil) or kidney are the material of choice for virus isolation attempts. oviduct is not a rewarding site from which to attempt ibv isolation. sentinel birds have been used successfully to aid the isolation of ibv. specifi c-pathogen-free (spf) chickens, possibly vaccinated against the ibv serotypes common to the area, are introduced into a fl ock and sampled at weekly intervals thereafter. for attempted virus isolation from fi eld material, the most successful systems are embryonated chicken eggs inoculated via the allantoic route or chick embryo tracheal organ cultures (tocs). material is homogenized with broth containing antibiotics and centrifuged or fi ltered to remove debris and contaminants. th e material is then inoculated into the allantoic cavity of 9-11-day embryonated chicken eggs or into tocs. following embryo inoculation, ibv if present, will cause characteristic embryo dwarfi ng and curling by the 18th day of incubation, but several 'blind' passages, to allow embryo adaptation of the virus, may be required. th ree or four passages are therefore usually given before a sample is discarded as negative, making it an expensive and time-consuming procedure. in tocs, ibv causes ciliostasis within 2-3 days of inoculation. th is is easily observed by low-power microscopy. as with embryo inoculation, two or three passages at 1-2-day intervals may be necessary. th e identity of the virus isolated in either system should be confi rmed. th is may be achieved by examining centrifuged allantoic fl uid or toc supernatant by electron microscopy, immunochemical assays, antigen-detecting enzyme-linked immunosorbent assays (elisas) or increasingly by using molecular methods. by incorporating serotypespecifi c monoclonal antibodies into an assay it is possible to identify the ibv serotype involved. in situations where it is not essential to actually obtain an isolate of the virus, ibv may be detected by immunochemical assays (immunofl uorescence or immunoperoxidase using an anti-ibv serum) and increasingly by molecular methods. th ese involve the reverse-transcription polymerase chain reaction (rt-pcr) together with restriction enzyme fragment length polymorphism (rflp) analysis, dna probes or nucleic acid sequencing. th ey are increasingly used for the detection of ibvs generally, as well as for the diff erentiation of specifi c variants. th ese techniques have also facilitated molecular epidemiological studies to monitor the distribution of specifi c variants. while direct sequencing of the genome is the most accurate method for identifying new variants, rflp, which identifi es sites unique to a particular serotype, is acceptable and has provided a rapid method for variant identifi cation. th e rna required for these techniques can be extracted directly from either swab material or appropriate tissues of infected birds, or from allantoic fl uid or toc supernatant of samples from which virus isolation is being attempted. since viral nucleic acid is what is being detected, it is not necessary for live virus to be present and rt-pcr probably detects the presence of ibv for longer than is possible by virus isolation methods. while, for many purposes, the lack of a live virus is not a problem, for epidemiological studies and for further research, the availability of an isolate could be necessary. for use in genome analysis, universal oligonucleotide primer sets, designed to detect all known ibvs, as well as ones designed to detect only specifi c serotypes, have been developed. initially these were based on sequence data from parts of the spike protein gene. however, as this is the part of the genome showing the most variation, sequence data from parts of the membrane and nucleoprotein genes have also been used. more recently, eff ort has concentrated on the use of the 3ј end of the genome, an area known to be conserved in coronaviruses. by so doing, it is hoped to develop assays that will detect and diff erentiate not only ibv strains but also novel coronaviruses of other avian species. molecular techniques give results more quickly than virus isolation methods. however, their sensitivity means that great care is needed to avoid contamination of reagents and the tests should be performed only in a dedicated laboratory where a high level of technical expertise is available. th e methods available to detect ibv antibodies include agar gel precipitation (agp), haemagglutination inhibition (hi), immunofl uorescence, elisa and vn. th e vn test is the most reliable test for identifying and diff erentiating ibv variants but is time-consuming and expensive and is only performed when it is important to identify the particular serotype involved in an outbreak, or for epidemiological studies. it can be performed in indicator systems such as chicken embryos, tocs, or avian cell culture using a cell-culture-adapted virus. th e hi test is a simple and reliable test but the virus must fi rst be treated with an appropriate enzyme. when performed carefully with appropriate controls, the hi test can diff erentiate between responses to particular serotypes. however, following exposure to more than one ibv serotype, diff erentiation by hi becomes unreliable. th e other assays all detect group-specifi c antibodies. th e agp test is simple to perform but relatively insensitive. some birds never develop precipitins while in others they are present for only a few weeks. for this reason, the only advantage of this test is that a positive result indicates recent exposure to the virus. th e elisa detects all known ibv serotypes and therefore cannot be used to identify particular variants. it is the most commonly used test for monitoring responses to vaccination and to indicate possible fi eld challenge. because of the availability of reliable commercial kits, it is widely used for fl ock screening. attention to management factors, such as temperature and ventilation, are essential, particularly in controlling the eff ects of secondary bacterial infection. th e use of antimicrobials may also be benefi cial against secondary infections. however, drug therapy is of no value in controlling the virus. while strict biosecurity is an important part of disease control, because ibv is ubiquitous and spreads rapidly it is impossible to exclude it completely by hygienic means. th us control depends on increasing the resistance of the bird by vaccination. live attenuated and inactivated (oil-adjuvanted) vaccines are highly eff ective and widely used. th e live attenuated vaccines are used to prevent and control infection in young birds and to 'prime' future breeders and layers prior to administration of inactivated vaccines. it is important to remember that, for inactivated vaccines to be eff ective, chickens must have been 'primed' with a live vaccine. to achieve optimal benefi t from the inactivated vaccine, at least 4-6 weeks should elapse between the last application of live vaccine and administration of the inactivated vaccine. ideally the live vaccine should contain ibv serotypes that will stimulate protection against the variants existing in a particular area. th e massachusetts serotype is the one most commonly included but, depending on local circumstances, other serotypes, where this is permitted by the regulatory authorities, may be included in order to optimize protection. frequently, two or more applications of live attenuated vaccine are given, often varying the serotype of ibv in the later vaccinations. despite the apparent protective eff ect of maternally derived immunity in very young chicks, live vaccines can be administered successfully from 1 day of age by coarse spray, beak dipping or nasal or eye drop. older birds may be vaccinated via the drinking water, by eye drop or coarse spray. diff erent vaccination protocols are available, designed for diff erent types of bird (see ch. 5) but the most important point is to ensure that the vaccine is carefully and correctly administered so that each bird receives the required dose. whatever protocol is followed, a highly attenuated live virus vaccine is given initially. for broilers this is likely to be given in the hatchery by coarse spray. revaccination of broilers, possibly at 2-3 weeks of age, is now common practice in some areas. in order that protection against both infectious bronchitis and newcastle disease may be achieved by one application of vaccine, the two vaccines may be combined. because of the risk that, if it is present in excess, the infectious bronchitis vaccine may interfere with the response to the newcastle disease vaccine, the use of a combined product is preferable to the use of two separate vaccines given together. for future breeders or layers, the fi rst vaccination is usually given at about 3 weeks of age, in the drinking water or by spray, and may be followed by one or more further applications of live attenuated vaccine. before onset of lay an inactivated vaccine is given intramuscularly or subcutaneously. increasingly, the inactivated vaccine is likely to be a multivalent one containing possibly two diff erent ibv antigens, as well as antigens to other important poultry pathogens. excellent protection against homologous challenge is obtained, provided that vaccination is carried out carefully. however, there is now increasing evidence that licensed vaccines provide heterologous protection against challenge with some diff erent ibv serotypes or variants, although it is not currently possible to predict when this might occur based on either serotyping or genotyping. recent data suggest that, if two antigenically distinct live-attenuated ibv vaccines are applied, preferably separated by at least 2 weeks, good protection may be achieved against challenge with a range of heterologous ibv serotypes. th is is probably because of the very small number of amino acid diff erences between diff erent serotypes or variants in the immunity-inducing part of the virus. although a high antibody titre following vaccination or natural infection is indicative of protection, particularly against the homologous strain of ibv, a low titre, even against homologous strains, may not indicate poor protection. th is is because local and cell-mediated immunity are very important in protecting against ibv challenge but are diffi cult to measure by currently available methods. locally produced antibody in the upper respiratory tract is the important fi rst line of defence against ibv challenges. it is therefore very important that the live attenuated infectious bronchitis vaccines are applied very carefully to ensure good, even stimulation of local antibody in every bird. development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine. a review coronaviruses in poultry and other birds infectious bronchitis epidemiology of infectious bronchitis virus detection of infectious bronchitis diseases of poultry avian infectious bronchitis virus proceedings of the international symposium on avain coronaand pneumoviruses and complicating pathogens key: cord-285052-aql0vrzv authors: kamble, nitin machindra; pillai, aravind s.; gaikwad, satish s.; shukla, sanjeev kumar; khulape, sagar aashok; dey, sohini; mohan, c. madhan title: evolutionary and bioinformatic analysis of the spike glycoprotein gene of h120 vaccine strain protectotype of infectious bronchitis virus from india date: 2016-02-12 journal: biotechnol appl biochem doi: 10.1002/bab.1298 sha: doc_id: 285052 cord_uid: aql0vrzv the infectious bronchitis virus is a causative agent of avian infectious bronchitis (aib), and is is an important disease that produces severe economic losses to the poultry industry worldwide. recent aib outbreaks in india have been associated with poor growth in broilers, drop in egg production, and thin egg shells in layers. the complete spike gene of indian aib vaccine strain was amplified and sequenced using a conventional reverse transcription polymerase chain reaction and is submitted to the genbank (accession no kf188436). phylogenetic analysis revealed that the vaccine strain currently used belongs to h120 genotype, an attenuated strain of massachusetts (mass) serotype. nucleotide and amino acid sequence comparisons have shown that the reported spike gene from indian isolates have 71.8%–99% and 71.4%–96.9% genetic similarity with the sequenced h120 strain. the study identifies live attenuated ibv vaccine strain, which is routinely used for vaccination, for the first time. based on nucleotide and amino acid relatedness studies of the vaccine strain with reported ibv sequences from india, it is shown that the current vaccine strain is efficient in controlling the ibv infection. continuous monitoring of ibv outbreaks by sequencing for genotyping and in vivo cross protection studies for serotyping is not only important for epidemiological investigation but also for evaluation of efficacy of the current vaccine. avian infectious bronchitis (aib) is an acute respiratory disease mainly found in young chickens in commercial poultry farms, causing significant economic losses. the prevalence of infectious bronchitis virus (ibv) is pandemic in a number of countries including india and assumes a variety of clinical forms, ranging from a respiratory disease to infection of the oviduct leading to permanent damage to immature birds and in hens, leading to reduced egg production and a nephropathogenic form of ibv causing acute nephritis, urolithiasis, and mortality [1] . ibv, a causative agent of aib disease, belongs to the genus gammacoronavirus, subfamily coronavirinae, family coronaviridae, and order nidovirales (international committee on taxonomy of viruses (ictv) 2011). the genome of ibv is a positive-sense single-stranded rna of about 27.6 kb with the following genomic organization: 5 -pol-s-3a-3b-e-m-5a-5b-n-untranslated region-3 [2] . all coronaviruses maintain a set of essential genes, including those that encode the polymerase (pol), spike (s), small membrane (e), membrane (m), and nucleocapsid (n) proteins [3] . the spike gene is translated as a precursor spike s o , which is posttranslationally cleaved by host serine protease into s1 and s2 at cleavage sequence motif [4] . the s1 protein is the primary cause of antigenic variations in ibv and contains the serotype-specific neutralization epitopes, whereas the envelope and membrane proteins are conserved. antigenic variation among ibv strains is common [1, [5] [6] [7] [8] ; the major prevalent form of ibv in the indian subcontinent is respiratory and nephropathogenic forms [9] . the ibv classification of different serotypes is based largely on virus-neutralization tests, a gold standard test for serodiagnosis of infectious bronchitis in infected birds. serotype determination is of trivial importance as different serotypes do not always cross protect. genotyping of ibv strains can also be done by genetic characterization of the spike glycoprotein gene by reverse transcription polymerase chain reaction (rt-pcr), restriction fragment length polymorphism, and nucleotide sequencing, which for the most part correlates with the viral serotype [10, 11] . nucleotide sequencing of the s1 gene is the foremost technique used for differentiation of ibv strains into various genotypes. the emergence of antigenic variants is mostly attributed to variation in the spike gene by recombination [12, 13] and is investigated by nucleotide sequencing of the s1 portion of the s gene coding for the s1 subunit [14, 15] , where most of the epitopes to which neutralizing antibodies bind are found [16] . the impact of regular emergence of antigenic variants of the disease and subsequent vaccines used to control varies depending on different geographical locations. genetic assessment of field isolates of viruses from outbreaks is essential for evaluation of vaccine efficiency on antigenic variants that arise. a critical advantage of sequencing s1 is for comparison and analysis of sequences of unknown field isolates and variants with reference vaccine strains for establishing potential relatedness. ibv vaccines currently used are either live attenuated or killed showing varying degrees of efficacy. at present, many countries only permit live vaccines of the massachusetts type, such as the h120 strain. some countries have also licensed other live strain vaccine such as connecticut, arkansas, or delaware 072 in the united states or the 4/91 strain in the united kingdom. in india, verma, [17] for the first time reported the prevalence of ibv infections in chickens. the emergence of a nephropathogenic ibv with a novel genotype in india has also been reported [9] . recently, an outbreak of nephropathogenic aib in broiler flocks was reported in the chhattisgarh region of india [18] . in this study, we amplified and sequenced the spike gene of the live infectious bronchitis attenuated vaccine virus, which is routinely used in india. bioinformatics was applied to evaluation of the spike gene for characterization and spike gene's relatedness with previously reported indian ibv isolates. viral rna was isolated from the aib vaccine, living bp vet (mass type strain; ventri biologicals, pune, india). a virus sample was obtained in a freeze-dried form that was dissolved in 1 ml normal saline; 0.1 ml of the inoculum was inoculated in five 10-day-old specific pathogen free chicken eggs (venky's hatcheries, pune, india), and the eggs were candled daily for 96 h. the allantoic fluid was collected and centrifuged at 1700 g for 10 min at room temperature, and a supernatant was stored at -20 • c until use. total rna from the infected allantoic fluid was extracted by a trizol reagent (sigma, st. louis, mo, usa) as per the manufacturer's protocol. the extracted rna was first reverse-transcribed with gene-specific primers and a thermoscript rt kit (invitrogen, carlsbad, ca, usa) to synthesize the first-strand cdna. synthesized cdna was screened for the presence of ibv genome by a polymerase chain reaction (pcr) with a set of primers specific to the spike gene of ibv. primers were used to amplify the complete coding sequence (cds) of the s gene including forward 5 -cccgaattcatgttggtaacacctcttttactag-3 (ecori) (nucleotide position 20374-20398) and reverse primer 5 -gcggagctcttaaacagactttttaggtc-3 (saci) (nucleotide position 20843-23862), designed from massachusetts m41 serotype (fj904723). pcr amplification was carried out using pfu dna polymerase (thermo scientific, waltham, ma, usa) with the following cycling conditions: 94 • c for 5 min followed by 30 cycles of 94 • c for 1 min, 60 • c for 1 min, 72 • c for 1.5 min, with a final extension of 72 • c for 10 min. the pcr-amplified gene fragment was cloned with a cloning vector clonejet tm (thermo scientific). the size of amplicon was 3489 bp. the pcr product was visualized by agar gel electrophoresis and confirmed by restriction enzyme digestion and sequencing. sequences were analyzed using chromas lite 2.1.1. the nucleotide sequence of the s gene of indian ibv vaccine strain was assembled and aligned by clustalw [19] after basic local alignment and search tool (blast) search with published ibv sequences deposited to the genbank database. sequence identities by blast analysis were included in the alignment and phylogenetic tree construction. the multiple sequence alignments and phylogenetic tree construction with the neighbor joining method were performed using mega version 5 [20] . the bootstrap values were determined from 1,000 replicates of the original data. phylogenetic analysis of nucleic acid and deduced amino acid sequences was carried out with the neighbor joining method using the jukes-cantor model and pairwise deletion. the s gene sequences from the genbank database, which were used for comparison or phylogenetic analysis in this study, are enlisted in tables 1 and 2. n-glycosylation sites were predicted by using the facility available at http://www.cbs.dtu.dk/services/netnglyc. leucinerich repeat regions (lrr) were determined by lrrfinder, which is available at http://www.lrrfinder.com/result.php. the spike gene was amplified using a cdna template prepared from isolated rna of commercial ibv vaccine available in india. on agarose gel electrophoresis, a positive amplification of 3.4kb band was seen (fig. 1) , which was gel eluted and cloned. the resulting full-length cds of spike gene was submitted to the genbank (accession number kf188436). the partial cds of the s gene was compared with relevant available ibv sequences in the genbank (tables 1 and 2 phylogenetic characteristics and molecular epidemiology of the indian vaccine strain. no complete spike gene sequences of the indian ibv isolates genotype were available in the genbank; therefore partial cds of kf188436 and other previously reported indian isolates were analyzed. the phylogenetic analysis based on the partial s1 gene sequence of the indian vaccine strain showed that it belongs to the h120 strain of ibv, which is a type of massachusetts strain routinely used as a vaccine strain worldwide (fig. 2) . some indian isolates were clustered together to form a distinct cluster, whereas ay091551 and hm748585 were clustered with the chinese nephropathological strain (dq973112 and dq973114) and 6/41 of uk (af093794 and jn192154), respectively. interestingly, one indian isolate hm163471 was clustered with the egyptian isolate dq487085, which is a nephropathogenic strain of aib virus found in egypt. to determine the relatedness among indian isolates, a comparative analysis of the nucleotide sequences of the spike gene (kf188436.1) with previously reported sequences of the s gene from indian isolates was carried out. a higher nucleotide sequence homology was shown by indian isolates gu967405, gq219712, and gu967392 with kf188436, which is around 98.4%, 99.0%, and 99.0%, respectively. the isolates gu967387, hq291841, hq291842, and hm179146 showed around 90.6%-93.7% nucleotide sequence homology to kf188436. the lowest sequence homology to kf188436 was shown by isolates hm748585 and hq291840, which was about 71.8% and 87.7%, respectively. the nucleotide sequence divergence of reported indian isolates from kf188438 was 0.1%-3.5% with the exception of hm748585. the spike gene of infectious bronchitis harbors recombinational hotspots; no crossover was observed between the vaccine strain and field isolates. the deduced amino acid sequences of the spike glycoprotein from the indian ibv vaccine strain and previously reported indian isolates exhibited 71.4%-96.9% homology. indian isolates gq219712 (96.9%), gu967392 (96.6%), gu967401 (96%), a deduced amino acid sequence of indian isolates has shown a cleavage sequence motif arg-arg-phe-arg-arg in a sequenced kf188436 h120 vaccine strain and other reported indian isolates at the amino acid position 533 [21] (table 3 ). n-glycosylation sites were predicted based on the presence of conserved motif asn-xaa-ser/thr in a sequence where an asn residue is n-glycosylated. twenty-six n-glycosylation sites were found in the kf188436 strain. in other indian isolates, most of the sites were found to be conserved with some exception (table 4) the lrr region of 84 amino acids in length was found in the infectious bronchitis vaccine strain kf188436 starting at position 1079 (leklsilktyi). a signal peptide mlvtplllvtllcalcsa was found in the vaccine strain h120 with a signal peptide probability of 0.729. in this study, we carried out propagation, amplification, sequencing, and bioinformatic analysis of the complete spike gene from the indian vaccine strain, routinely used for vaccination of poultry birds. sequencing result showed that the open reading frame (orf) of the indian vaccine strain was 3,489 bp with a cleavage site for spike located at 1,596 bp, which is equivalent to 532 amino acids. this is the first report on a complete cds spike gene of an indian ibv vaccine strain. universal primers are available for cloning and sequencing of the spike gene; self-designed primers were used in this study. in phylogenetic analysis, the spike sequence was clustered together with the h120 vaccine strain of ibv. the h120 strain is a live attenuated vaccine strain of massachusetts (mass) serotype, originally prepared after 120th serial passage of strain h, isolated from embryonated chicken eggs in the year 1956 in the netherlands [22] . the h120 strain is considered to be one of the safest vaccine strains and is used worldwide as a primary vaccine in broilers, breeders, and future layers. it has been reported that the differences in s1 probably contribute to failure of cross protection. however, common epitopes exist between isolates, which are of importance in cross-immunity; some of these are likely to be on proteins other than s1. the degree of cross protection induced by uk/6/82 against homologous and heterologous ibv isolates diminished as the similarity of the s1 proteins diminished [12] . in a study involving vaccination of birds with single vaccination with 6/82 failed to protect all chicks against isolates with extremely similar (>98% identity) s1 sequences, indicating that a slight change in the s1 sequence may have led to the failure of vaccine [12] . the indian ibv vaccine strain h120 showed 71.4%-96.9% homology at an amino acid level and 71.8-99% homology at a nucleotide level. so the degree of protection may be higher with homologous genotype compared to heterologous ones. it is presumed that the degree of cross protection between different serotypes is less, but some strains of the ibv viruses do induce cross protection against other serotypes and are known to as protectotypes. the h120 strain of ibv is a well-known protectotype and has been extensively used for vaccination. to overcome the problem associated with different serotypes and genotypes, it has been reported that vaccinating with two different types of ibv vaccine can provide broad protection against different ibv types [5] . a different combination of protectotypes can be tested to achieve a high level of cross protection and to overcome a problem of vaccination failure. the study involving the indian vaccine strain ma41 with 94.8%-98.8% homology to field isolates showed good cross protection [23] . other than spike, enhancement of humoral immunity against ibv by a bicistronic dna vaccine plasmid encoding nucleocapsid protein and interleukin-2 has shown up to 80% of protection [24] . twenty-six n-glycosylation sites were found in the vaccine strain, out of which 17 sites are present in the s1 region, which is an ectodomain of the spike gene. the reported indian ibv isolates have lost some of the n-glycosylation sites ( table 4 ). as n-glycosylation is a regular event in posttranslational modification of the protein, changes in a glycosylation pattern can modulate antigenic properties of the protein as antibodies raised against glycoprotein may be specific for a carbohydrate moiety of the glycoprotein [25] . diversification of the glycosylation pattern may influence a survival pattern and a transmission property of virus, in lactate dehydrogenase elevating virus, a member of the coronaviridae family; determinants of changes in virulence and cellular tropism of virus are loss or acquisition of n-glycosylation sites of the protein [26] . the precursor spike glycoprotein (s) of ibv is posttranslationally cleaved by a host serine protease at the cleavage site motif into two subunits (s1 and s2). the cleavage recognition site consists of five basic amino acid residues, and variation in a sequence and composition does not correlate with serotype or pathogenicity of ibv as different serotypes as well as at-tenuated and pathogenic isolates contain the same cleavage recognition site that correlate with viruses of different geographic regions [21] . the indian vaccine strain kf188436, which belongs to massachusetts (mass) serotype, is shown to have an arg-arg-phe-arg-arg cleavage recognition site, which correlates with isolates tha241251, tha280252, tha290252, and tha320352 of thailand [21] ; fj888351, gq154655, and fj888351 of china; and gu393335 of the united states. it shows that the h120 strain mostly has a rrfrr cleavage recognition site. lrr is a structural motif of protein like alpha/beta horseshoe fold and is responsible for protein==protein interactions [27, 28] . in kf188436, lrr is present at position 1079 that is a part of the s2 protein and may be responsible for an interaction with other proteins of ibv. in conclusion, the results of the current study show that the current vaccine strain, which is routinely used for field vaccination of poultry birds, belongs to the h120 strain. the percent identity of the spike gene sequence of the h120 strain with the indian isolates shows varying degrees of similarity. the vaccine strain used is sufficiently protective based on a similarity study but further in vivo cross protection studies should be carried out to check the cross protection potential of the current vaccine against field isolates. diseases of poultry location of publisher the authors are thankful to the csir-ugc, new delhi, india, and the director, indian veterinary research institute, bareilly, india, for providing necessary grants and facilities to carry out this research work. the authors hereby declare that there is no conflict of interest. key: cord-268788-jcu3pasy authors: thor, sharmi w.; hilt, deborah a.; kissinger, jessica c.; paterson, andrew h.; jackwood, mark w. title: recombination in avian gamma-coronavirus infectious bronchitis virus date: 2011-09-23 journal: viruses doi: 10.3390/v3091777 sha: doc_id: 268788 cord_uid: jcu3pasy recombination in the family coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. however, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. in this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (ibv) isolates were sequenced and along with other full-length ibv genomes available from genbank were analyzed for recombination. evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. the extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within ibv. these data indicate that reticulate evolutionary change due to recombination in ibv, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. avian infectious bronchitis virus (ibv) is a gamma-coronavirus in the family coronaviridae, the order nidovirales, and the genus coronavirus that causes a highly contagious upper-respiratory disease of domestic chickens. in layer type birds it can cause a drop in egg production and some strains are nephropathogenic. infectious bronchitis remains one of the most widely reported respiratory diseases of chickens worldwide despite the routine usage of attenuated live vaccines to control the disease. control of ibv is difficult because there is little to no cross-protection between the numerous different serotypes of the virus. infectious bronchitis virus is an enveloped, single-stranded, positive-sense rna virus with a genome length of approximately 27 kb. the 3' end of the genome encodes four structural proteins; spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as several non-structural proteins [1] . the s glycoprotein of ibv forms projections on the surface of the virion. spike is post-translationally cleaved into s1 and s2 subunits with the s1 subunit forming the outermost portion and s2 forming a stalk-like structure that is embedded in the viral membrane. the s1 subunit contains hypervariable regions that play a role in attachment to host cell receptors, and it contains conformationally-dependent virus-neutralizing and serotype-specific epitopes [2, 3] . spike is also involved in membrane fusion and viral entry into the host cell. the e and m proteins are integral membrane proteins involved in assembly of the virus. the n protein is closely associated with the viral genome and plays a role in replication. the 5' two-thirds of the genome, approximately 21 kb, encodes two polyproteins 1a and 1ab. a minus one frame-shift mechanism is used to translate the 1ab polyprotein. the polyproteins are post-translationally cleaved into 15 non-structural proteins (nsps), nsp 2-16 (ibv does not have an nsp1) that make up the replication complex. key nsps encoded, include a papain-like protease 2 (plp2) within nsp 3, a main protease (mpro) within nsp 5, and the rna-dependent rna-polymerase (rdrp) within nsps 11 and 12. genetic diversity in coronaviruses is due to adaptive evolution driven by high mutation rates and genetic recombination [4] . high mutation rates are attributed to minimal proof reading capabilities associated with the rdrp. recombination is thought to be due to a unique template switching "copy-choice" mechanism during rna replication [5] . evidence of recombination among strains of ibv has been observed both experimentally and in the field [6] [7] [8] [9] [10] [11] . the emergence of several alpha-and beta-coronaviruses has been attributed to recombination [12, 13] but only recently was recombination shown to be the mechanism behind the emergence of a novel gamma-coronavirus, turkey coronavirus (tcov) [14] . although "hot spots" of recombination in the genome of ibv have been reported [9, 15] , a thorough study of recombination using multiple different strains across the entire genome has not been conducted. in this study we sequenced and analyzed the entire genome of eight ibv strains that represent different serotypes that have not been previously sequenced, and we compared these sequences with other gamma-coronavirus full-length genome sequences available in genbank for evidence of recombination [16] . different serotypes of field viruses and vaccine type viruses were selected to provide a wide variety of sequences potentially capable of contributing gene fragments to recombinants. the full-length genomes of eight isolates of ibv were sequenced at 5× to 10× coverage, and the consensus sequences were assembled. the genome size (see the end of the 3'utr in table 1 ), organization of the genome, and the location and size of the open reading frames (orfs) are listed in table 1 for each of the viruses. the gene order is the same for all the viruses examined; 5'utr-1a/ab-spike-3a-3b-envelope-membrane-4b-4c-5a-5b-nucleocapsid-3'utr. in addition, the genomes for cav/cav56b/91, de/de072/92, fl/fl18288/71, mass/h120, iowa/iowa97/56 and jmk/jmk/64 contain orf 6b between nucleocapsid and the 3'utr. the full-length genomes were aligned and phylogenetic trees were constructed using the neighbor-joining, minimum evolution, maximum parsimony and upgma programs in mega4 [17] . the trees all had similar topology and bootstrap support, and a representative tree is shown in figure 1 . the feline coronavirus fcov/fipv/wsu-79-1146 and the beluga whale virus belugawhalecov/sw1/08 were included as out-groups. the wild bird viruses isolated from a munia (muniacov/hkuy13/09), thrush (thrushcov/hku12/09) and bulbul (bulbulcov/hku11/09) formed a unique clade, which is not surprising as this group might represent a new coronavirus genus provisionally designated deltacoronavirus [18] . the remaining viruses separated into clades consisting of ibv isolates from the us and vaccine viruses, tcov isolates, an ibv isolate from west africa and ibv isolates from china and taiwan. vaccines for ibv used in commercial poultry include the serotypes mass, conn, de and ark. the peafowlccv/gd/kq6/03, ck/ch/lsd/051/06 and ck/ch/zj971/97 strains from china grouped with mass type viruses indicating that they are closely related, which is not surprising since mass type vaccines are used in china. the overall percent similarities between the various strains are listed in supplemental table 1 . all ibv genomes examined are greater than 80% similar at the nucleotide level. genomes available in the database for ck/ch/ep3, ck/ch/p65, and mass/beaudette were excluded from the analysis because they are viruses not found in the field. the recombination programs can be used to detect recombination without reference sequences, and our analysis was conducted without regard to date of isolation because that information was not available for some of the viruses. although the programs attempt to identify major and minor parent sequences contributing to each recombinant, the data reported herein only represents sequences in other viruses that are most closely related to the sequence surrounding the transferred fragment (major sequence) and the sequence closely related to the transferred fragment (minor sequences) and doesn't imply origin or source of the transferred fragment. in many cases, the transferred fragment has undergone mutations making it difficult to identify all the endpoints for the major and minor sequences. in addition, some of the transferred fragments overlap suggesting that recombinations have occurred between recombinant viruses. twenty-five ibv strains were examined and the viruses with the most transferred fragments in table 2 are cav/56b/91 and mass/h52 both with 8 fragments, and ck/ch/lsd/051/06 and ga98/0470/98 both with 7 fragments. the strains with the fewest transferred fragments are iowa/iowa97/56 and tw/2575/98 with only 2 transferred fragments and the ck/ch/bj/97, holte/holte/54, and nga/a116e7/06 strains with only 1 transferred fragment. the ark/ark-dpi-p11/81 and ark/ark-dpi-p101/81 strains are the same virus that was passaged 11 and 101 times in embryonated eggs, respectively. both viruses share identical transferred fragments indicating that they have identical recombination history. in addition, conn/conn46/66 and conn/conn46/91 share the same recombination history (4 identical transferred fragments). the conn/conn46/66 field virus was used to produce an attenuated live vaccine, which is currently used in commercial poultry. viruses that share the same recombination history are likely derived from the same parent virus suggesting that conn/conn46/91 is reisolated conn vaccine derived from the conn/conn46/66 virus. the fl/fl18288/71 virus also shares all 4 transferred fragments with the conn viruses, however; fl/fl18288/71 and conn viruses are different serotypes suggesting that fl/fl18288/71 is a field virus that emerged due to point mutations accumulating in spike over time rather than from recombination. all 6 of the transferred fragments in ck/ch/zj971/97 are identical to all 6 of the transferred fragments in vaccine strain mass/h120, providing compelling evidence that ck/ch/zj971/97 is reisolated mass/h120 vaccine. that observation was also reported by zhang et al. [23] . it is interesting that mass/h52 (8 transferred fragments) and mass/h120 (6 transferred fragments) share only 5 identical transferred fragments. the mass/h52 and mass/h120 viruses were isolated circa 1955 in the netherlands and it is widely accepted that h stands for holland, but it actually stands for houben, the owner of the broiler farm where the viruses were isolated [24] . it is thought that mass/h120 was derived from mass/h52 but the actual relationship between the viruses is not certain. our data indicates that they are not necessarily parent and progeny but they are closely related. the gray/gray/60 and jmk/jmk/64 viruses share 99.7% nucleotide similarity across the entire genome and have 4 identical transferred fragments with jmk/jmk/64 having one additional fragment located in the 5'utr, which is not found in gray/gray/60. it is well known that the gray/gray/60 virus is nephropathogenic, whereas the jmk/jmk/64 virus is strictly respirotropic. perhaps sequence differences in the 5'utr, which is involved in replication of the viral genome, play a role in the different pathobiologies observed for these viruses. there is evidence that some transferred fragments in field viruses come from vaccines. as an example, ck/ch/lsd/051/06 has 3 of 7 and 2 of 7 transferred fragments in common with vaccine strains mass/h52 and mass/h120, respectively. in addition, the only fragments that usa viruses have in common with the viruses from china and taiwan are fragments also associated with mass type vaccines, which are used in both regions, providing further evidence that some of the fragments in field viruses come from vaccines. that result and the observation in figure 1 that the viruses separated into clades based on geographic location also supports the conclusion that usa viruses have not recombined with asian viruses. a difference in the order of taxa in phylogenetic trees constructed from different regions of the genome is further evidence of recombination [25] . the ordering of taxa in sequential trees [26, 27] was conducted and inconsistent phylogenetic relationships were observed for all of the examined virus strains across the entire genome, indicating a substantial amount of recombination (data not shown). there is a high number of breakpoints in the 1a region of the genome and immediately upstream of the s gene, which has been previously shown to be a 'hot spot' for recombination [9] . a phylogenetic compatibility matrix constructed at the 70% bootstrap level for 250 bp sequence fragments at 100 bp intervals also showed that recombination breakpoints were distributed throughout the ibv genomes (data not shown). to determine recombination hot and cold spots, a recombination breakpoint distribution plot (figure 3 ) was generated in rdp4 using a 200 nt window and 1,000 permutations [21] . no global hot-spot regions were observed in the 95% and 99% confidence thresholds (dotted lines at the top of the graph). the detectable recombination breakpoint positions are shown at the top of the figure and were distributed throughout the genome with a relatively high number clustered just upstream of the s gene. that region also had the highest breakpoint count within the 99% local hot/cold-spot confidence interval. a high number of breakpoints were also observed in the 1a region of the genome; nsp 2, nsp 3, and nsp 16, in the envelope and matrix protein genes and in a small area near the 3'utr. table 3 shows that nsp2, nsp3, nsp16 and spike genes were associated with the greatest number of transferred fragments, which is consistent with the location and number of breakpoints in figure 3 . recombination in the 1ab orf area, which encodes the nonstructural proteins involved in the viral replication complex, has the potential to alter the pathogenicity of the virus [28] . the nsp 2 contains hydrophobic residues that likely anchor the replication complex to the golgi [29] . the nsp 3 encodes the protease plp2 which cleaves nsps 2, 3, and 4 and an area with adp-ribose 1'-phosphatase (adrp) activity. the protease plp2 has been shown to have deubiquinating-like activity [30] and also to be a type i interferon (ifn) antagonist [31] . changes in the amino acid composition of this area could affect the ability of the virus to replicate in a variety of cell types. the adrp region of nsp 3 is conserved among coronaviruses [32, 33] , and a recent study suggested a biological role for the coronavirus adrp in modulating the expression of pro-inflammatory immune modulators such as tumor necrosis factor alpha and interleukin-6 [34] . recombination in this area could alter the pathogenicity of the virus by modulating host cytokine expression. the nsp16 is reported to be an s-adenosyl-l-methionine (adomet)-dependent rna (nucleoside-2'o)-methyltransferase (2'o-mtase) responsible for capping the viral mrna nascent transcripts [32] . an alteration in the efficiency of this protein could profoundly decrease not only viral replication but also pathogenicity. the spike glycoprotein of ibv on the surface of the virus plays a role in attachment to host cell receptors, membrane fusion and entry in this study, evidence was obtained that recombination is occurring among avian coronavirus ibv isolates across their entire genome. every sequence included in the analysis was recognized as a potential recipient of horizontally acquired sequences at some point in its viral evolutionary past. the nsp2, nsp3, nsp16 were associated with the greatest number of transferred fragments. in addition, the area immediately upstream of the spike gene had the highest number of recombination breakpoints. breakpoints in the 1ab polyprotein gene have the potential to alter pathogenicity of the virus, and breakpoints near or in spike have the potential to lead to the emergence of new serotypes of ibv or new coronaviruses. although the spike region determines the serotype of the virus, the remainder of the genome may be a mosaic of sequence fragments from a variety of gamma-coronaviruses. the only evidence of a gamma-coronavirus possibly recombining with an alpha or beta-coronavirus was the the pcr reaction was run on the same machine as the rt step and included a one-time initial denaturation step of 94 °c for 2 min, followed by 30 cycles of 94 °c for 30 s, 60 °c for 30 s and 72 °c for 3 min. the pcr products were agarose gel purified using the qiaquick gel extraction kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. the pcr products were cloned into the topoxl vector using the topoxl cloning kit (invitrogen, carlsbad, ca, usa) according to manufacturer's protocol to prepare cdna libraries for sequencing. plasmid dna from the libraries of the cloned cdna fragments for each virus was isolated using an alkaline lysis method modified for the 96-well format and incorporating both hydra and tomtek robots. sequencing reactions were performed using the bigdye™ terminator® cycle sequencing kit version 3.1 (applied biosystems, foster city, ca, usa) and mj research (watertown, ma, usa) thermocyclers. sephadex filter plates were used to filter each reaction into perkin-elmer microamp optical 96-well plates. a 1/12-strength sequencing reaction on an abi 3730 was used to sequence each clone from both the 5' and 3' ends. primers for one-step rt-pcr were specifically designed for each virus (supplemental table 2 ). viral rna was amplified using the titan one tube rt-pcr kit (roche diagnostics, indianapolis, in, usa) following manufacturer's instructions. a dna engine peltier thermocycler (bio-rad laboratories, inc., hercules, ca, usa) was used for the rt-pcr reaction, which had the following steps: one cycle of 42 °c for 60 min and 95 °c for 5 min, followed by 10 cycles of 94 °c for 30 s, 50 °c for 30 s, and 68 °c for 1 min 30 s, and then 25 cycles of 94 °c for 30 s, 50 °c for 30 s, 68 °c for 1 min and 30 s adding 5 s with each cycle. the resulting pcr products were agarose gel purified using the qiaquick gel extraction kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. the resulting cdna was sequenced using abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca, usa) following the manufacturer's protocol. the reactions were prepared for sequencing by centrifugation through either a centri-sep column (applied biosystems, foster city, ca, usa) or using the edge system (edgebio, gaithersburg, md, usa) plate. the samples were sequenced at the georgia genomics facility (university of georgia, athens, ga, usa). chromatogram files and trace data were read and assembled using seqman pro, and genome annotation was conducted with seqbuilder (dnastar, inc., madison, wi, usa). each sequence was aligned to a representative genome; mass/mass41/41 (genbank accession #ay851295), or cal99/cal99/99 (genbank accession #ay514485) as a backbone for genome assembly. whole genome analyses were generated and phylogenetic trees constructed with the neighbor-joining method with 1000 bootstrap replicates as well as with minimum evolution, maximum parsimony and upgma methods [17] . ibv strains in phylogenetic trees generated from sequential genome fragments using treeorder scan (version 1.6, simmonics, university of warwick, coventry, uk) [26, 27] . changes in the tree position of taxa supported at the 70% or greater bootstrap level for a 250 bp sequence window were examined at 100 bp intervals. in addition, a phylogenetic compatibility matrix was constructed and used to examine the frequency and location of recombinations across the entire genome. potential recombination sites were identified using the rdp4 software [22] and a breakpoint map was constructed. a breakpoint density plot was then created from this map by moving a 200 nt window 1 nt at a time along the length of the map. the number of breakpoints falling within a window was plotted at the central window position. a 99% (upper) and 95% (lower) confidence threshold for globally significant breakpoint clusters (defined as windows with more breakpoint positions than the maximum found in >95% of the 1,000 permuted plots) was calculated. in addition, 99% and 95% confidence intervals were calculated for local breakpoint clusters (defined as windows with more breakpoint positions than the maximum found in >99% of the windows at that location in 1,000 permuted plots). genbank accession numbers virus genome sequences generated in this study were submitted to genbank and assigned the following accession numbers: cav/cav56b/91 (gu393331 gray/gray/60 (gu393334) mass/beaudette (nc_001451), nga/a116e7/06 (fn430415), ita/90254/05 (fn430414) references and notes the viruses and their replication does ibv change slowly despite the capacity of the spike protein to vary greatly? the neutralization epitopes on the spike protein of infectious bronchitis virus and their antigenic variation the evolution and emergence of rna viruses rna recombination in animal and plant viruses sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains origin and evolution of georgia 98 (ga98), a new serotype of avian infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv a recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adaptedvaccine strain naturally occurring recombination between distant strains of infectious bronchitis virus coronavirus diversity, phylogeny and interspecies jumping recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs emergence of a group 3 coronavirus through recombination identification and analysis of the georgia 98 serotype, a new serotype of infectious bronchitis virus molecular evolutionary genetics analysis (mega) software version 4.0 coronavirus genomics and bioinformatics analysis evolutionary aspects of recombination in rna viruses a simple and robust statistical test for detecting the presence of recombination recombination patterns in aphthoviruses mirror those found in other picornaviruses recombination detection and analysis using rdp3 complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h120 comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 frequency and dynamics of recombination within different species of human enteroviruses recombination in the genesis and evolution of hepatitis b virus genotypes the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity dynamics of coronavirus replication-transcription complexes the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses mouse hepatitis virus liver pathology is dependent on adp-ribose-1''-phosphatase, a viral function conserved in the alphalike supergroup testing the hypothesis of a recombinant origin of the sars-associated coronavirus analysis of recombination and natural selection in human enterovirus 71 infectious bronchitis application of phylogenetic networks in evolutionary studies neighbor-net: an agglomerative method for the construction of phylogenetic networks a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints possible emergence of new geminiviruses by frequent recombination analyzing the mosaic structure of genes evaluation of methods for detecting recombination from dna sequences: computer simulations sister-scanning: a monte carlo procedure for assessing signals in recombinant sequences an exact nonparametric method for inferring mosaic structure in sequence triplets evaluation of methods for detecting recombination from dna sequences: empirical data detecting and characterising individual recombination events: practice this work was supported by usda, csrees award number 2007-35600-17786. the authors would like to thank the technical help of jon s. robertson and cornelia lemke with sequencing. discovery of the mosaic nature of the sars-coronavirus genome [35] . although this type of recombination is possible it appears to be rare in nature.in this study, we characterized recombination in the full-length genomes of avian gammacoronavirus ibv strains from around the world. our bioinformatic analysis was similar to a previous study on enteroviruses [36] and we found that recombination in ibv is more extensive than formerly thought, involving regions across the entire genome. our data suggests that reticulate evolution due to a high frequency of recombination in ibv, likely plays a major role in the generation of new serotypes of the virus. the characterization, distribution and frequency of recombination breakpoints are important information that will further our understanding of the mechanisms behind the diversity and evolution of these viruses so that better control methods can be developed. all of the viruses sequenced in this study (table 4 ), were propagated in 9-11 day-old specific-pathogen-free (spf) embryonated eggs as described [37] . total rna was isolated from 200 μl of allantoic fluid collected from the infected eggs using the high pure rna isolation kit (roche applied science, mannheim, germany) following the manufacturer's instructions. the amplification reactions were carried out using strand displacement rt-pcr or one step rt-pcr. strand displacement rt-pcr uses a random (at the 3' end) primer and an amplification primer. the sequence of the random primer was (agcgggggttgtcgaatgtttgannnn) and the sequence of the amplification primer was (agcgggggttgtcgaatgtttga). the rt-pcr reaction was carried out using the takara rna la pcr kit (takara bio. inc., otsu, shiga, japan) according to the manufacturer's protocol. a dna engine peltier thermocycler (bio-rad laboratories inc., hercules, ca, usa) was used for the rt reaction, which included an rna denaturing step at 65 °c for 10 min followed by 30 °c for 10 min, 42 °c for 60 min, 99 °c for 5 min, and 5 °c for 5 min. we used neighbor-net analysis to examine the ibv genomes for evidence of networked relationships and the pairwise homoplasy index (phi) in splitstree (version 4, simmonics, university of warwick, coventry, uk) [20, 38, 39] to statistically determine the likelihood of recombination. in addition, the ibv genomes were examined for recombination breakpoints using the recombination detection program (rdp4, version 4, simmonics, university of warwick, coventry, uk) [21, 22] . unless otherwise stated, default settings were used in all of the programs. the specific algorithms used were rdp [40] , geneconv [41] , bootscan/rescan [40] , maximum chi square [42] , chimaera [43] , siscan [44] , and 3seq [45] . we used more than one method to analyze the data because evaluation of these recombination detection methods using both simulated and empirical data showed that the results from only a single method were not very reliable [46] . automasking was used for optimal recombination detection. the rdp analysis was run without a reference and a window size of 60, bootscan window size was increased to 500, maxchi and chimaera number of variable sites per window was increased to 120, and the window size and step size for siscan was increased to 500 and 20, respectively. the window sizes were increased from their default settings because ibv has a high mutation rate, which can mask recombination signals. increasing the window size was shown to increase the ratio of recombination signals relative to mutational "noise" [47] . inconsistent phylogenetic relationships between different regions of the viral genome provide further evidence of genetic recombination. herein, we examined the order of avian gamma-coronavirus key: cord-007648-tm0hn0hz authors: mockett, a.p.adrian title: envelope proteins of avian infectious bronchitis virus: purification and biological properties date: 2002-12-20 journal: j virol methods doi: 10.1016/0166-0934(85)90138-7 sha: doc_id: 7648 cord_uid: tm0hn0hz immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (ibv). the purified proteins were inoculated into rabbits to produce antisera. the rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. ibv-infected chickens produced antibodies to both the spike and membrane proteins. both these antibodies were at their highest concentration about 9–11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. neutralizing antibodies were at their highest concentration 21 days after inoculation. a second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. the neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7–11 days after this inoculation. the advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed. avian infectious bronchitis virus (ibv) is a coronavirus whose principal site of replication is the ciliated epithelial cells of the respiratory tract mucosa of chickens. viral replication occurs in the cytoplasm of the cell and virions are formed by budding from the endoplasmic reticulum. there are three viral structural proteins: spike (s; peplomers), membrane (m) and nucleocapsid (n). s and m proteins are both glycosylated and parts of them are exposed at the surface of the virion. the spike protein consists of two glycopolypeptides, s 1 and $2, which have molecular weights of 90 kda and 84 kda, respectively (cavanagh, 1981) . the membrane protein is present as a number of distinct species which have molecular weights ranging from 23 kda to 34 kda; the molecular weight differences are associated with the various degrees of glycosylation. n protein (54 kda) is associated with the viral rna. the ibv spike protein, associated with the outer projections, plays an important part in the infection of cells. chicken antisera to this protein and spike-specific monoclonal antibodies (mockett et al.. 1984) can neutralize the infectivity of the virus. a similar function has been found for the spike protein of murine coronavirus mhv-4 (collins et al., 1982; fleming et al., 1983) and the porcine coronavirus tgev (garwes et al., 1978) . the spike protein of ibv also contains strain-specific determinants (mockett et al., 1984) . the membrane protein appears to be a more highly conserved antigen and it is possible that only a small amount (approx. 1 kda) is exposed at the viral surface (boursnell et al., 1984) . the nucleocapsid protcin interacts with the viral rna to form a helical nucleocapsid. the objectives of this work were to produce immunoadsorbents using monoclonal antibodies which have been prepared previously (mockett et al., 1984) and to purify the virus-coded proteins of the viral envelope in a single step in relatively large amounts. this allowed hyperimmune rabbit antisera to the proteins to be produced and tested for neutralizing antibodies. in addition the sequential humoral antibody response of chickens after ibv infection has been studied using the purified viral proteins and whole virus in elisas and compared to the results using the neutralization test. the massachusetts m41 strain of ibv was grown in the allantoic cavities of i 1-day-old embryonated chicken eggs and purified on isopycnic sucrose gradients as described by cavanagh (1981) . purified virus was pelleted in a 6 x 14 ml rotor at 70,000 x g for 3 h at 4°c and resuspended in phosphate-buffered saline (pbs). an equal volume of pbs containing 4% (wt./vol.) np40 was added, mixed using a dounce homogeniser and incubated for 2 h at 25°c. the material was centrifuged for 5 min in an eppendorf microcentrifuge and the resulting supernatant, containing soluble viral components, was used for the affinity chromatography purification. monoclonal antibodies (designated a38 and c!24) to the spike and membrane proteins respectively of ibv strain m41 were prepared (mockett et al., 1984) . the gammaglobulin fraction of ascitic fluids containing either anti-spike or anti-membrane monoclonal antibodies was isolated by salt precipitation using a final concentration of 18% (wt./vol.) na2so4. for the spike immunoadsorbent 5.6 mg of gammaglobulin was coupled to 0.75 mg of cnbr-sepharose 4b (pharmacia) according to the manufacturers' instructions and for the membranc immunoadsorbent 5.5 mg was coupled to the same amount of gel. unreactive groups on the gel were blocked using 1 m ethanolamine, pit 8.0, and any non-covalently bound proteins wcre removed by repeated washings with 0.1 m nahco~ buffer, ph 8.3, containing0.5 m naci and 0.1 m acetic acid buffer, ph 4.4, containing0.5 m naci. the immunoadsorbent was stored in pbs containing 0.2% nan~ at 4°c until used. it was washed twice with 3 m nh4scn in pbs containing 0.1% octylglucoside, four times with pbs and twice with pbs containing 2% np40 before use. all wash volumes were 10 ml. the solubilised virus preparation was mixed with the immunoadsorbent for 16 h at 4°c using a rotary stirrer. the gel was poured into a chromatography column and washed with pbs containing 0.1% np40 (40 ml) and pbs containing 0.1% octylglucoside (10 ml). 3 m nh,scn in pbs containing 0.1% octylglucoside was added and 10 fractions of 1 ml collected. the absorbance at 280 nm of each of the fractions was read using a sp1800 pyeunicam spectrophotometer. the fractions in the absorbance peak were dialysed against pbs. a sample of each fraction was then subjected to electrophoresis in a polyacrylamide gel. those fractions containing detectable viral protein were pooled and constituted the purified protein prepar~,tion. ten per cent polyacrylamide slab gels containing sds were used (laemmli, 1970) and after electrophoresis samples were stained first with coomassie brilliant blue r-250 and then silver (morrisey, 1981) . new zealand white rabbits were used. samples of purified proteins were mixed with an equal volume of freund's complete adjuvant and inoculated intramuscularly into the rabbits. a similar inoculation was given 1 wk later. after 5 wk the same antigen in incomplete freund's adjuvant was inoculated subcutaneously. five months later blood was collected from the ear vein and the resulting serum stored at -20°c until used. the houghton poultry research station line of rhode island red chickens was used. ibv (m41) was inoculated intratracheally (500 ciliostatic dose fifty (cd 50); darbyshire et al., 1979) into 8 chickens and sequential blood samples were taken from the wing vein (see fig. 2 for times after inoculation). sera from the blood samples were stored at -20°c until used. a serum pool for each time of sampling was made by mixing an equal volume of serum from each of the eight samples. three different antigens were used for the ei,isas: spike protein, membrane protein and ib virus. the purified spike and membrane proteins were used at a dilution of 1 : 20 in carbonate buffer whilst the purified ib virus was used at a 1 : 100 dilution. antigens were adsorbed for i h at 37°c. in the second step chicken sera were serially diluted in 0.5 m nacl containing 0.5~7c np40 (saline/np40) from an initial 6.64 iog2 dilution. any specifically bound antibody was detected using a rabbit anti-chicken igg serum ( 1 : 300) and a goat anti-rabbit igg alkaline phosphatase conjugate ( 1 : 1,000) (sigma), both diluted in saline/np40. the substrate was p-nitrophenyl phosphate (1 mg/ml)in diethanolamine buffer and the reaction stopped using 3 m naoh. each step was for 30 min at 37°c, the reaction volumes 50 pl and the plate was washed three times with pbs containing 0.1c~ tween 20 between each step. titres were calculated graphically (mockett and darbyshire, 1981) using an absorbance value of 0.1 as the cut off. the chicken antisera were tested for neutralizing antibody to ibv as described by darbyshire et al. (1979) . rabbit sera were precipitated with 18°~ (wt./vol.) nazso4 and the resulting gammaglobulin tested because rabbit sera have high concentrations of non-specific inhibitors of ib virus replication. the viral proteins purified by affinity chromatography are shown in fig. 1 . the spike protein, which is composed of two polypeptides, was the only protein detected in the two fractions shown using the sensitive silver staining procedure. similarly the membrane protein was not contaminated with other proteins, although this protein did not stain as well as the spike. there were other stained bands present, but these are artifacts sometimes observed, even in the absence of protein, with this staining procedure. the purification was highly reproducible. the purified viral proteins were inoculated into rabbits and the gammaglobulin fraction of the antisera tested for neutralizing activity. only the anti-spike gammaglobulin neutralized the virus. however the membrane protein was a good immunogen because the rabbit anti-membrane sera tested in the elisa had high activity against the whole virus: in fact the titres were higher than those of the rabbit anti-spike sera (see table 1 ). the results of testing sera from ibv-infected chickens for antibody to spike and membrane proteins showed that both anti-spike and anti-membrane antibodies were produced early after infection (see fig. 2 ). peak titres were between 9 and 11 days after infection. the antibody response to the whole virus had a similar profile. however, the the second inoculation of virus induced an anamnestic antibody response. the elisa detected a similar increase in antibody titres using spike, membrane and whole virus -the peak was at 10-11 days after infection. the neutralizing antibody response was also similar and the peak titres were at 11 days after infection which contrasted to the slow rise to the peak titres after the primary inoculation. this paper describes the application of affinity chromatography using monoclonal antibodies for the purification of the two viral structural proteins present at the surface of the ib virion -spike and membrane. a previous report has described procedures for the purification of these viral proteins and also nucleocapsid protein, the only other major structural protein (cavanagh, 1983) . ibv was solubilised in np40 detergent and centrifuged in a sucrose gradient containing this detergent in order to purify the nucleocapsid protein. the addition of 1 m naci to the sucrose solutions was required for the purification of the spike and membrane proteins, as they co-migrated in gradients containing low salt concentrations. however, the nucleocapsid protein could not be purified in gradients containing high salt concentrations. the yield of material from these gradients was relatively low, due to the limited number of fractions which contained purified viral components. in other studies purified spike material contained some nucleocapsid protein and the membrane preparation contained other proteins which were thought to be of cellular origin. there are a number of advantages in using affinity chromatography. by making use of the specificity of the antibody pure material can be isolated, even from a crude mixture of proteins. the method is very quick and easy and the immunoadsorbent can be used several times. thus, relatively large amounts of purificd material can be obtained. the availability of spike and membrane proteins in a highly purified form will allow more biochemical, structural and immunological studies to be done. the conditions used to solubilise the virus did not dissociate the two spike polypeptides, therefore, both s i and $2 were detected in thc material eluted from the anti-spike immunoadsorbent. the results of experiments using the rabbit antisera to the viral proteins confirmed the biological importance of the spike protein as only antibodies to this protein neutralized the infectivity of the virus. thc chicken, about 10 days after an ibv infection, has antibodies to both the spike and membrane proteins in its serum but only very low concentrations of neutralizing antibodies. thc profile of neutralizing antibodies shown in this paper agrees with previous published findings (holmes, 1977; mockett and darbyshire, 1981; hawkes et al., 1983) . the results show that anti-spike antibodies produced early after infection are non-neutralizing, as assessed by our in vitro technique. this raises the question as to the function of these antibodies in thc chicken. previous evidence has shown only the spike protein to be capable ofeliciting neutralizing antibodies. there is a possibility that the anti-spike antibodies could be neutralizing in vivo and the function of the anti-membrane antibodies could be similar. the possible role of these antibodies in protection remains to be resolved. purified viral proteins can be used to determine which is responsible for protecting thc chicken from infection. protection tcsts such as thc ciliostasis (darbyshire, 1980) and the mixed infcction (escherichia coil and ibv) (smith et al., 1985) tcsts are available. it is only by using methods such as affinity chromatography that stflficientl~, large amounts of pure viral proteins can be made available to enable such tests to bc done. 1984. \.'iru~ rc~ i 11. pov, cll virolog, 119, 35~. darbyshire artwrlght, lg":'bl vet ph. i). i hcsis, i. tnl,ct~,it', oi ]'he author wishes to thank ms. ,i.k.a. ('ook for her help with the neutralilatton tests, ms. debra southee for her excellent technical assistance and i)r. i'.i).k. br()wn for useful discussions. key: cord-263193-paeosfiu authors: zhu, jinyan; xu, shuang; li, xueyan; wang, jue; jiang, yueqi; hu, weichen; ruan, wenke title: infectious bronchitis virus inhibits activation of the tlr7 pathway, but not the tlr3 pathway date: 2020-06-10 journal: arch virol doi: 10.1007/s00705-020-04690-8 sha: doc_id: 263193 cord_uid: paeosfiu various strains of infectious bronchitis virus (ibv) cause different forms of infectious bronchitis with different clinical signs. here, primary chicken embryo kidney (cek) cells and specific-pathogen-free (spf) chickens were infected with three pathogenic ibv strains, and it was observed that the tlr7-myd88 pathway was inhibited but the tlr3-tirf pathway was activated. after treatment with poly(i:c)-lmw, poly (i:c)-lmw/lyovec, and imiquimod, the replication of ibv was significantly suppressed after 24 h. however, treatment with tlr3 pathway inhibitors such as pepinh-trif, celastrol, chloroquine, and bx795 resulted in increased replication of ibv after 36 h. these results also showed that chloroquine and celastrol were most effective inhibitors of the antiviral response at 48 hpi. infectious bronchitis is a contagious disease of poultry caused by infectious bronchitis virus (ibv), a member of the genus gammacoronavirus [16] . different strains of ibv cause various pathologies [10, 16] . avian toll-like receptor (tlr) 3 and tlr7 recognize viral rna in endosomes and induce immune responses [20, 24] . avian melanoma differentiation associated gene 5 (mda5) is a rig-i-like receptor (rlr) that recognizes long viral rna in the cytoplasm [28] . previous studies showed that tlr3 contributes to the host defense against severe acute respiratory syndrome coronavirus (sars-cov) and porcine epidemic diarrhea virus (pedv) [2, 23] . stimulation of tlr3 hindered murine coronavirus infection, but stimulation of tlr7 did not affect murine coronavirus production [18] . it was also shown that mda5 is critical for host defense during infection with murine coronavirus [29] . ibv strains may different in their pathogenicity and induce different immune response. here, three ibv strains associated with three classic types of pathogenicity were chosen to explore potential differences in the innate immune responses they induce. the ah and tm strains of ibv were isolated from sick chickens with respiratory signs, kidney changes, and proventriculus changes from chicken farms. ibv strain h52 was obtained from qyh biotech company limited, beijing, china. chicken embryo kidney (cek) cells were prepared from the kidneys of 17-day-old specific-pathogen-free (spf) chicken embryos and were cultured in six-well plates (5 × 10 5 cells/well) at 37 °c. for viral infection, cek cells were inoculated with ibv strain ah, tm, or h52 at a multiplicity of infection (moi) of 1 for 1 h at 37 °c. mock-treated cek cells were incubated with sterile saline solution without virus and then cultured under the same conditions. the cells were collected at 12, 24, 36, or 48 h post-inoculation (hpi), and total rna was extracted from the tissue samples and cells using trizol reagent (invitrogen, carlsbad, ca). all experiments were performed in triplicate. the extracted rna was treated with dnase i (1u/μg rna) for 20 min and then reverse transcribed into cdna. the 20-μl reaction mixture contained 2 μg of total rna, 0.5 μg of anchored oligo (dt18), and 2 μl of easyscript rt/ri enzymemix in 1× es reaction mix (transgen biotech, beijing, china). the reaction mixture was incubated at 42 °c for 30 min and then at 85 °c for 5 min. the initial rna and dna concentrations were determined using a spectrophotometer (nanodrop technologies, wilmington, de). β-actin (actb) was selected as a control because it is a stably expressed and frequently used reference gene. the qpcr reaction mixture contained 0.5 μl (10 μmol) of each primer, 10 . these proteins were subjected to sds-page on a 12% acrylamide gel, and the separated protein bands were transferred to a pvdf membrane using a trans-blot (bio-rad, ca, usa). the membrane strips were tested for their reactivity with an anti-myd88 antibody, an anti-trif antibody, an anti-nf-κb antibody, or a β-actin antibody (cell signaling technology, ca, usa): for in vivo experiments, 40 fifteen-day-old spf chickens were randomly divided into four groups (animal ethics approval no. 2017-02-13). they were challenged with 0.2 ml of a virus suspension containing10 6 eid 50 of ibv strain ah, tm, or h52 or with 0.2 ml of sterile saline solution. the kidneys of these chickens were collected at 5 days post-inoculation (dpi), since the virus titer in the kidney has been shown to reach a peak after 5-7 days [3, 19, 27] . the tissues were stored in liquid nitrogen and used later for rna extraction and qpcr assays. to investigate the effect of innate immune molecules, agonists of tlr3, tlr7, and mda5 and inhibitors of key molecules of the tlr3 pathway were added to cek cells that had grown to 80-90% as instructed by the reagent manufacturer (invivogen, ca, usa) as follows: 5 μg of imiquimod (a tlr7 agonist) per ml for 12 h, 10 μg of low-molecular-weight (lmw) polyinosinic-polycytidylic acid (poly(i:c)) (a tlr3 agonist) per ml for 12 h, 0.5 μg of poly (i:c)-lmw/lyovec (an mda5 agonist) per ml for 12 h, 10 μm pepinh-trif (a trif inhibitor) for 6 h, 5 μm celastrol (an nf-κb inhibitor) for 1 h, 50 μm chloroquine (an inhibitor of endosomal acidification) for 0.5 h, and 5 μm bx795 (a tbk1 inhibitor) for 6 h. the cells were then washed with pbs, and their viability was tested using trypan blue staining. the treated cells were incubated at an moi of 1 with ibv strain ah, tm, or h52 for 1 h at 37 °c and washed again with pbs. control cek cells were infected with ibv without pretreatment and cultured under the same conditions. the cells were collected at 12, 24, 36, or 48 hpi for qpcr using a pair of primers specific for the ibv n (fig. 1a-f) . the differences in mrna and protein expression suggested that transcription or translation of ifnβ could be regulated by the virus. expression of the myd88, trif, and nf-κb proteins was detected by western blot. compared with the levels in uninfected cells, the expression of myd88 was significantly downregulated in all ibv-infected cells from 12 to 48 hpi. however, the expression of trif was significantly upregulated in all ibv-infected cells from 12 to 48 hpi. moreover, at 48 hpi, the expression of both myd88 and nf-κb was significantly higher in cells infected with ibv strain h52 than in those infected with strain ah or tm (fig. 1g-i) . importantly, we found significant activation of the tlr3-trif pathway and inhibition of the tlr7-myd88 pathway at 48 hpi by different ibv strains in cek cells. previous studies have shown the expression of innate immune molecules in chickens [3, 19, 27] . we found that tlr7 and ifnβ mrna expression in the kidney was significantly downregulated in chickens infected with ah and tm ( fig. 1k and m) . however, there was no significant difference in mrna expression of tlr3 and mda5 in any the experimental groups ( fig. 1j and l) . the observed differences might be explained by the fact that more kinds of molecules are involved in the antiviral response in vivo than in vitro. the replication of strain ah was significantly inhibited compared to the control from 24 to 36 hpi, whereas the replication of strain h52 was significantly inhibited after 24 hpi, and the replication of strain tm was significantly inhibited from 12 to 36 hpi (fig. 2) . all of the inhibitors that were tested promoted ibv replication after 36 hpi. after treatment with pepinh-trif, bx795, or chloroquine, the rate of replication of ibv in ah-infected cek cells was significantly higher than in control cells at 36 hpi, but the replication of ah in celastrol-treated cells was significantly higher at 48 hpi. after treatment with pepinh-trif and bx795, the replication rate of ibv strain tm was significantly higher at 36 hpi. the rate of replication of tm was significantly higher at 48 hpi after treatment with all four inhibitors. after treatment with chloroquine, the rate of replication of strain h52 was significantly higher at 36 hpi, and the rate of replication of h52 in pepinh-trif-, celastrol-, and chloroquine-treated cells was significantly higher at 48 hpi. at 36 hpi, the most effective inhibitor of replication of ibv strain ah was chloroquine, but at 48 hpi, it was celastrol. at 36 hpi, the most effective inhibitor of tm was bx795, but at 48 hpi, it was chloroquine. from 36 to 48 hpi, the most effective inhibitor of h52 replication was chloroquine (fig. 2) . previous research has suggested that ibv strains differ in their pathogenic mechanisms [16] . tlr3, tlr7, and mda5 play critical roles in the activation of the innate immune response to ibv infection [13, 24] . for example, tlr3 levels were significantly downregulated in the respiratory epithelial cells and lungs of 6-day-old chickens infected with the ibv connecticut strain [11] . however, another report showed that tlr3 and tlr7 levels in the trachea and kidneys were upregulated from days 3 to 8 in 2-week-old spf chickens infected with ibv strain m41 [8] . several studies have shown that the expression of tlr3 and tlr7 after ibv infection decreases after the passage of the infectious virus, which demonstrates the ability of ibv to adapt to the host environment [25, 30] . mda5 has been shown to be upregulated in the trachea but downregulated in the kidneys [8] . further research is required to clarify the complex regulatory mechanism in the kidneys after ibv infection [19, 27] . ibv has a single-stranded rna genome, and tlr7 recognizes single-stranded viral rna, subsequently activating an immune response [9, 17] . our results suggest that ibv inhibits the expression of tlr7, but the mechanism by which this occurs is unclear. innate immune receptors are competitive determinants of cell fate [5] . in the case of ibv, in addition to the structural proteins, more than 20 nonstructural proteins and secretory proteins have important biological functions that still need to be elucidated. tlr3 activates an immune response after it recognizes viral double-stranded rna [1] . we found that tlr3 was upregulated by ibv in cek cells. viral double-stranded rna is synthesized in cek cells late in ibv infection, so tlr3 probably plays an important role in the recognition of ibv during the late period of ibv infection. myd88 and trif were the adaptor molecules of tlr7 and tlr3, respectively [17, 26] . here, we found that myd88 protein expression was significantly suppressed, but trif was significantly upregulated. there is an evident correlation between activation and inhibition of immune recognition receptors and signal pathway molecules. nf-κb protein expression was upregulated at 48 hpi in cells infected with ibv strain h52, which is an efficient vaccine strain. this result might therefore identify another signaling pathway by which the immune response is activated after infection with h52, such as the tlr3 pathway [1] . in contrast to earlier reports, we found that ibv 6 eid 50 of ibv, and the kidneys of these chickens were collected at 5 dpi. the expression of chicken tlr3, tlr7, mda5, and ifnβ mrna was detected using qpcr. data were analyzed using unpaired t-tests or anova, followed by dunnett's multiple comparison test, using graphpad prism (graph-pad software, san diego, ca). the significance of the differences between the infected and mock-infected groups is indicated by the p-value strains with different pathogenicity induced different levels of mda5 mrna [14] , indicating that tlr3 is probably the main pattern recognition receptor (prr) recognizing the ibv genome, rather than tlr7 or mda5. imiquimod (also known as r837) is a ligand of tlr7 [15] . poly(i:c) is recognized by tlr3 [12] . poly(i:c)-lmw/lyovec is a preformed complex consisting of poly(i:c)-lmw and the transfection reagent lyovec. naked poly(i:c) is recognized by tlr3, whereas transfected poly(i:c) is sensed by mda5 [6] . both showed a strong effect on immune activation in this study. we suggest that the tlr3 pathway is the major immune pathway that inhibits early replication of ibv. trif and tbk1 are key molecules in tlr3 signaling pathway. pepinh-trif contains a 14-aa sequence that corresponds to the sequence of trif [22] . bx795 inhibits the catalytic activity of tbk1/ ikkε by blocking its phosphorylation [4] . celastrol is an effective inhibitor of nf-κb [21] . chloroquine is an inhibitor of endosomal acidification [7] . al of these compounds enhanced ibv replication after 36 hpi in this study, consistent with the signal transmission by these downstream molecules. celastrol inhibited all of the ibv strains at 48 fig. 2 the effect of agonists and inhibitors on the replication of different ibv strains. agonists of tlr3, tlr7, mda5 and inhibitors of key molecules of the tlr3 pathway were added to the cek cells, which were infected with ibv strain ah, tm, or h52 at an moi of 1. the cells were collected at 12, 24, 36, or 48 hpi, and ibv n and β-actin mrna expression was measured using qpcr. data were analyzed using unpaired t-tests or anova, followed by dunnett's multiple comparison test, using graphpad prism (graphpad software, san diego, ca). the significance of the differences between the treatment and control groups is indicated by the p-value hpi, since nf-κb is the downstream molecule of the immune pathway. in addition to celastrol, chloroquine also showed a strong ability to promote replication of ibv strains tm and h52. this suggests that endosomal acidification and rna degradation are important for immune recognition of ibv. the three ibv strains tested showed different sensitivity to inhibitors. this study provides preliminary data on the immune responses induced by different ibv strains and their pathogenicity. future studies can use these data to analyze the mechanisms underlying the variations in ibv pathology and immune responses. these data also can be helpful for future ibv vaccine research. recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 porcine epidemic diarrhea virus infection induces nf-kappab activation through the tlr2, tlr3 and tlr9 pathways in porcine intestinal epithelial cells differential innate immune responses induced by classical and variant infectious bronchitis viruses in specific pathogen free chicks use of the pharmacological inhibitor bx795 to study the regulation and physiological roles of tbk1 and ikappab kinase epsilon: a distinct upstream kinase mediates ser-172 phosphorylation and activation innate immune receptors as competitive determinants of cell fate essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr7/8-mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production responses of the toll-like receptor and melanoma differentiation-associated protein 5 signaling pathways to avian infectious bronchitis virus infection in chicks species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 vaccination against infectious bronchitis virus: a continuous challenge induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens toll-like receptor and rig-i-like receptor signaling rig-i in rna virus recognition activation of the chicken type i interferon response by infectious bronchitis coronavirus molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor 7 infectious bronchitis virus variants: molecular analysis and pathogenicity investigation recognition of singlestranded rna viruses by toll-like receptor 7 protective role of toll-like receptor 3-induced type i interferon in murine coronavirus infection of macrophages early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles polymorphisms of chicken tlr3 and 7 in different breeds celastrol, a novel triterpene, potentiates tnf-induced apoptosis and suppresses invasion of tumor cells by inhibiting nf-kappab-regulated gene products and tak1-mediated nf-kappab activation differential involvement of bb loops of toll-il-1 resistance (tir) domain-containing adapter proteins in tlr4-versus tlr2-mediated signal transduction toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection toll-like receptors and innate antiviral responses differential modulation of avian beta-defensin and tolllike receptor expression in chickens infected with infectious bronchitis virus role of adaptor trif in the myd88-independent tolllike receptor signaling pathway induction of innate immune response following introduction of infectious bronchitis virus (ibv) in the trachea and renal tissues of chickens avian infectious bronchitis virus disrupts the melanoma differentiation associated gene 5 (mda5) signaling pathway by cleavage of the adaptor protein mavs mda5 is critical to host defense during infection with murine coronavirus the establishment and characteristics of cell-adapted ibv strain h120 we would like to thank the national natural science foundation of china (#31572499 to wenke ruan) for financial support. conflict of interest the authors declare that they have no conflict of interest.ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. key: cord-280442-jtvez46y authors: wu, xuan; song, zengxu; zhai, xiwen; zuo, lei; mei, xueran; xiang, rong; kang, zhuangzhuang; zhou, long; wang, hongning title: simultaneous and visual detection of infectious bronchitis virus and newcastle disease virus by multiple lamp and lateral flow dipstick date: 2019-11-01 journal: poultry science doi: 10.3382/ps/pez372 sha: doc_id: 280442 cord_uid: jtvez46y abstract infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are both important viruses seriously affecting poultry industry worldwide. in this study, reverse-transcription lamp (rt-lamp) was combined with lateral flow dipstick (lfd) forming a novel detection tool which could simultaneously detect ibv and ndv visually. primers targeted the 5′-untranslated region (5′-utr) of ibv genome and the conserved region of ndv large polymerase gene (lp). the specificity and sensitivity of this multiple reverse transcription-lamp-lfd (mrt-lamp-lfd) assay were compared with those of conventional rt-pcr, nested rt-pcr (nrt-pcr), quantification rt-pcr (qrt-pcr), and rt-lamp monitored by electrophoresis. no non-specific amplifications were observed when the assays were tested with unrelated viruses. according to the sensitivity study, when detecting ibv or ndv alone, the lowest detection limits of mrt-lamp-lfd were 100.8 ibv rna copies/reaction and 100.7 ndv rna copies/reaction. furthermore, when detecting ibv and ndv simultaneously, the lowest detection limit was the same as that of the single detection assays. in the clinical sample study, mrt-lamp-lfd performed the best among these assays. when tested with ibv or ndv single infected samples, the mean detection rates were 98.65% and 97.25%, respectively. in the ibv and ndv co-infected sample study, the mean detection rates of ibv and ndv were both 95%. this study showed that mrt-lamp-lfd was a promising qualitative detection tool suitable for field single or multiple ibv and ndv detection. infectious bronchitis virus (ibv) and newcastle disease virus (ndv) are 2 of the most important viruses seriously affecting the poultry industry and causing huge economic losses worldwide (bande et al., 2017; brown and bevins, 2017) . ibv and ndv belong to the gammacoronavirus of the coronaviridae family and the avulavirus of the paramyxoviridae family, respectively (http://www.ictv.global). the genome of ibv is about 27.6 kb in length. it encodes 15 non-structural proteins, and 4 structural proteins: spike glycoprotein (s), small membrane protein (e), membrane glycoprotein (m), and phosphorylated nucleocapsid protein (n). at the 5 and 3 ends of the genome, there is an untranslated region (utr) each (armesto et al., 2009 ). ndv possesses a 15 kb long genome comprising 6 genes c 2019 poultry science association inc. received april 18, 2019. accepted june 9, 2019. 1 corresponding author: whongning@163.com which individually encode the nucleocapsid (n), matrix protein (m), phosphoprotein (p), fusion protein (f), hemagglutinin-neuraminidase protein (hn), and large polymerase protein (lp) (de leeuw and peeters, 1999) . ibv and ndv both have high mutation rates, making their prevention and control difficult. quick and accurate detection of ibv and ndv is important for preventing the viruses from spreading. a wide variety of diagnostic assays for ibv and ndv have been developed, including virus isolation, and serological and molecular assays (bande et al., 2016; brown and bevins, 2017) . costs, requirements of stringent techniques, and time required limit the use of virus isolation as a routine virus detection assay (bande et al., 2016) . serological assays, such as hemagglutination inhibition and elisa, are faster and simpler than virus isolation, but tend to lack specificity and sensitivity, especially in the case of ibv, and poor crossreactions between serotypes makes serological tests less applicable (cavanagh, 2007; miller et al., 2010) . in view of their high sensitivity, specificity, and reduced flow time, molecular assays are the most commonly used 5401 methods for ibv and ndv monitoring. according to previous studies, both ibv and ndv quantification rt-pcr (qrt-pcr) detection methods were highly specific, and the lowest detection limits were 10 2 -10 4 genome copies indicating that these qrt-pcr methods were highly sensitive (callison et al., 2006; farkas et al., 2009; wise et al., 2004) . another highly specific and sensitive molecular method is nested rt-pcr (nrt-pcr) which involves 2 rounds of pcr amplifications. as previously reported, the lowest detection limits of ibv and ndv nrt-pcr assays were 10 1.9 and 10 4.0 eid 50 /ml, respectively (nguyen et al., 2013) . while pcr assays are widely applied in pathogen detection, the conduct of pcr requires sophisticated laboratory equipment and observation of pcr product requires electrophoresis, making pcr assays unsuitable for point-of-care and visible detections, especially in some low-resource regions. loop-mediated isothermal amplification (lamp) amplifies dna under isothermal conditions by the bst dna polymerase large fragment (notomi et al., 2000) . numerous studies have demonstrated that the amplification efficiency of lamp is quite high (khan et al., 2017; zhang et al., 2014) . moreover, the specificity of lamp is also satisfactory as there are 4 specially designed primers recognizing 6 distinct regions on the target dna (asiello and baeumner, 2011; zhang et al., 2014) . furthermore, unlike conventional pcr assays, only simple devices are needed during lamp, such as a water bath or a heat block. lamp is thought to revolutionize molecular biology not only because of its excellent performance on dna amplification but also due to its diverse, simple, and intuitional reaction monitoring methods. several naked eye monitoring approaches have been applied, such as adding color indicators into reactions and combining with immunochromatographic techniques (parida et al., 2008; zhang et al., 2014) . lateral flow dipstick (lfd), an immunochromatographic technique, utilizes antibody capture followed by secondary antibody labeling (chen et al., 2016; zhang et al., 2014) . lamp combined with lfd (lamp-lfd) could be used for highly sensitive, simple, visual, and multiple pathogen detections (chen et al., 2016) . lamp products can be labeled by employing biotin/fitc modified fip/bip primers, and subsequently, these biotin-fitc double labeled lamp products can be captured by biotin-antibodies and immobilized at specific locations on lfd strips (test line). subsequently, fitc at the other end of the products can specifically combine with gold particles labeled with fitc-antibodies, thus making the results readable using the naked eye (nimitphak et al., 2008) . however, no previous studies have reported multiple detection of avian pathogens using lamp-lfd. both ibv and ndv are pathogens that cause avian respiratory diseases, and single or multiple infection by them may cause similar clinical signs. studies have shown that multiple conventional rt-pcr could be used for detecting and differentiating respiratory dis-ease pathogens in poultry diseases (pang et al., 2002; rashid et al., 2009) . however, the sensitivity of multiple conventional rt-pcr is not satisfactory. multiple nested rt-pcr is much more sensitive than multiple conventional rt-pcr but time-consuming (nguyen et al., 2013) . furthermore, these assays are not suitable for on-site pathogen detection, because products of rt-pcr need to be monitored by electrophoresis and qrt-pcr need to be conducted with highly accurate instruments. here, we developed a visual multiple rt-lamp-lfd (mrt-lamp-lfd) assay which could simultaneously detect ibv and ndv and be easily carried out and monitored by the naked eye. to evaluate this novel detection method, pcr assays (including conventional rt-pcr, qrt-pcr and nrt-pcr) and reverse-transcription lamp (rt-lamp) monitored by electrophoresis were also conducted and the specificity and sensitivity of the assays were compared with those of the mrt-lamp-lfd assay. a total of 13 ibv strains, 7 ndv strains, and the pcr and lamp target sequences of 6 ndv and 1 turkey coronavirus strains (tcov) synthesized by sangon biotech (shanghai, china) co, as well as 6 other avian virus strains, were used for the determination of the specificities of rt-pcr and rt-lamp assays. the genbank numbers of ibv and ndv strains were labeled in figure s1 (supplementary information); the tcov strain and the 6 other avian virus strains used in the specificity study were listed in table s1 (supplementary information). tissue samples used in this study were stored at −80 • c. rna in the samples was extracted with trizol (invitrogen, carlsbad, ca). subsequently, cdna was synthesized using primescript rt reagent kit (takara, beijing, china) following the manufacturer's instructions. briefly, the reverse transcription reaction mixture consisted of 2 μl 5 × primescript buffer, 0.5 μl primescript rt enzyme mix i, 0.5 μl oligo dt primer, 0.5 μl random 6 mers, the rna of the virus, and rnase free dh 2 o, thus creating a final volume of 10 μl. complete genome sequences of 224 ibv strains and 331 ndv strains available in genbank were aligned using mega 6 software. subsequently, to determine conserved regions in the ibv and ndv genomes, aligned results were used for similarity plotting analysis with the simplot program 3.5.1. primers for rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays were designed tgcatgtgccacatgagact nd-b3 ctttcctctgtattctctctcc on base of ibv and ndv genome conservative regions: ibv primers targeted the 5 -utr region, and ndv primers located in the conserved region of lp gene. primers for rt-pcr, nrt-pcr, and qrt-pcr were designed by primer premier 6 software (premier inc., palm desert, ca); primers for rt-lamp assays were designed by primerexplorer v4 software (fujitsu, tokyo, japan). in mrt-lamp-lfd reactions, modified fip and bip primers were used: bio-ib-fip was modified by biotin on the 5 -end, dig-nd-fip was modified by digoxigenin on the 5 -end, and fitc-ib-bip and fitc-nd-bip were modified by fitc on the 5 -end. the sequences of the primers are listed in table 1 . the most appropriate annealing temperatures for each pair of primers were determined using gradient pcr and listed in table 1 . conventional rt-pcr reaction mixture consisted of 12.5 μl 2 × m5 pfu pcr mas-termix (mei5, beijing, china), 10 pmol of each primer, 1 μl template and double distilled water (ddh 2 o) creating a final volume of 25 μl. the pcr parameters included an initial denaturation step for 5 min at 94 • c followed by 32 cycles of denaturation at 94 • c for 50 s, annealing for 50 s, extension at 72 • c for 10 to 30 s depending on the sizes of the products and a final extension step at 72 • c for 10 min. nrt-pcr involved 2 rounds of amplifications. the reaction mixture and parameters of each round of amplification were the same as that of conventional rt-pcr. the products of conventional rt-pcr and nrt-pcr were monitored by electrophoresis in 1% agarose gels. qrt-pcr assays were conducted with 10 μl 2 × sso-fast evagreen supermix (bio-rad, hercules, ca), 10 pmol of each primer, 1 μl template and ddh 2 o making the final volume to 20 μl. the parameters were: initial denaturation for 30 s at 95 • c followed by 39 cycles of denaturation at 95 • c for 5 s, annealing/extension for 5 s, and a final melting curve at 65 to 95 • c with increment 0.5 • c/5 s. data was analyzed by bio-rad cfx maestro 1.1 software (bio-rad, hercules, ca). plate read was added during the extension and melting curve steps. to establish ibv and ndv qrt-pcr standard curves, fragments amplified using qib-f/r and qnd-f/r were individually cloned into peasy-t1 vector (transgen, beijing, china). plasmids were extracted using tianprep mini plasmid kit (tiangen, beijing, china). subsequently, ibv plasmids were 10-fold serial diluted from 5.09 × 10 9 to 5.09 × 10 4 copies/μl and ndv plasmids were 10-fold serial diluted from 3.97 × 10 9 to 3.97 × 10 4 copies/μl. diluted plasmids were used as standard samples during the establishment of figure 1 . principle of mrt-lamp-lfd. ibv and ndv lamp amplify products were labeled with fitc/biotin and fitc/digoxigenin, respectively. gold particles modified with fitc-antibodies can combine with the labeled ibv and ndv products. subsequently, ibv products will be captured by biotin-antibodies immobilized on the test line 1, ndv products will be captured by digoxigenin-antibodies immobilized on the test line 2, and the free gold particles will be immobilized on the control line. thus, the products are visualized. ibv and ndv qrt-pcr standard curves. slopes and intercepts of standard curves, amplification efficiency (e) and corresponding correlation coefficients (r 2 ) were generated using bio-rad cfx maestro 1.1 software (bio-rad, hercules, ca). the rt-lamp reactions were conducted under gradient temperatures (50 to 68 • c) to determine optimal reaction temperatures. the concentration of mgso 4 (4 to 10 mm) and dosage of bst 2.0 warmstart dna polymerase (new england biolabs, ipswich, ma) (1 to 8 u) were also optimized. the optimal reaction mixture contains 2.5 μl 10 × isothermal amplification buffer to evaluate the specificities of the assays, the phylogenetic analyses on pcr and lamp target sequences of 224 ibv and 331 ndv strains were conducted. according to the phylogenetic trees, 224 ibv strains were grouped into 4 clades, and 331 ndv strains were grouped into 6 clades. a total of 13 ibv strains were used to determine the specificities of the assays distribute in all 4 clades, likewise, 13 ndv strains were used to determine the specificities distribute in all 6 clades ( figure s1 , supplementary information) . in addition, cdna of avian reovirus (arv), infectious bursal disease virus (ibdv), and avian influenza virus (aiv), dna of gallid alphaherpesvirus 2 (gahv-2) and fowl adenovirus (fadv), as well as synthesized sequence of 1 tcov strain were used as templates for further evaluation of the specificities of the assays (table s1, supplementary information) . to determine the lowest detection limits of the assays in terms of rna copy numbers, 10-fold serial diluted in vitro-transcribed rna of target regions was used as templates. briefly, fragments containing ibv 5 -utr and ndv lp target regions were separately amplified using primer pairs 5 -atcacactagccttgc gctaga-3 /5 -gcaaaagcatcagcgtaatcc-3 and 5 -aatctgtattacatgtctagg-3 /5 -aga gagaatatatcctttcgc-3 . subsequently, the fragments were separately ligated downstream t7 promoter. in vitro-transcriptions were conducted using hiscribe t7 high yield rna synthesis kit (neb, beijing, china). the concentrations of the rna transcripts were measured using nano drop 2000 (thermo, shanghai, china) , and the copy numbers of ibv and ndv rna molecules were calculated following the formula reported previously (fronhoffs et al., 2002) . the copy numbers of ibv and ndv rna molecules were 10 6.8 and 10 5.7 copies/μl, respectively. ibv rna was 10-fold serial diluted into 10 6.8 to 10 0.8 copies/μl; ndv rna was 10-fold serial diluted into 10 5.7 to 10 0.7 copies/μl. whereafter, 1 μl rna from each concentration was used in reverse transcription reaction (10 μl reaction volume). after reverse transcription, 1 μl cdna was used in the pcr and lamp assays. thus, the concentrations of serial diluted ibv and ndv rna, which was finally used as templates, were separately 10 5.8 to 10 −0.2 copies/reaction and 10 4.7 to 10 −0.3 copies/reaction. furthermore, serial diluted ibv and ndv rna was mixed to test the lowest detection limit of mrt-lamp-lfd when simultaneously detecting ibv and ndv. negative control reactions in specificity and sensitivity studies were conducted with total rna extracted from allantoic fluid of health specific pathogen-free (spf) chick embryo as templates. ibv-positive samples, including 144 tissue samples (78 tracheas and 66 lungs), 124 swabs (82 oral swabs and 42 cloacal swabs), ndv-positive samples, including 87 tissues (52 tracheas and 35 lungs), and 76 swabs (40 oral swabs and 36 cloacal swabs) were used to examine the performance of the assays in detecting ibv and ndv in clinical samples. in addition, to further investigate the specificities of the assays when detecting clinical samples, 33 negative tissues (including 10 tracheas, 10 lungs, and 13 kidneys), 10 fadv positive livers, and 7 aiv h9n2 positive lungs were also tested by the assays. all these samples were collected from 32 chicken farms distributed in 7 provinces, china, during our routine monitor on avian diseases. to investigate whether the mrt-lamp-lfd assay could detect ibv and ndv in co-infected samples, both accurately and simultaneously, ten 4-wk-old spf chickens were inoculated with 10 2.7 eid 50 ibv m41 and 10 2.4 eid 50 ndv f48e9 by the nasal route to mimic ibv-ndv co-infected chickens. oral and cloacal swabs were collected on 3 and 6 days-post-infection (dpi) from each bird. on 6 dpi, all the chickens were sacrificed, and lungs and tracheas were collected. the animal experiment in this study was approved by the animal ethics committee of the college of life sciences, sichuan university (license: syxk-chuan-2013-185). all experimental procedures and animal welfare standards strictly followed the guidelines of animal management at sichuan university. statistical significance differences in the mean detection rates of the assays, when detecting different kinds of samples, were evaluated by one-way anova using graphpad prism version 6 (graphpad software inc., san diego, ca). differences were considered to be significant at * p < 0.05. under optimal annealing temperatures, rt-pcr and nrt-pcr amplified specific products of the expected lengths, and there were no non-specific bands observed in the negative controls ( figure 2a ). both in ibv and ndv qrt-pcr assays, fluorescent signals were detected with the target ibv or ndv templates, while no fluorescent signals were detected in negative control reactions ( figure 2b ). according to the standard curves, c t values (y), and log of copy numbers (x) were linearly correlated (ibv: y = −3.323x + 41.249, e = 99.9%, r 2 = 0.999; ndv: y = −3.369x + 41.444, e = 98.1%, r 2 = 0.999). the melting curve showed a single peak indicating no primer dimer formed ( figure 2c ). after ibv and ndv rt-lamp amplification, symbolic ladder-like bands were observed in a 2% agarose gel ( figure 3a ). as for mrt-lamp-lfd assays, ibvpositive reactions generated test line 1 and the control line; ndv-positive reactions generated test line 2 and the control line; and test lines 1, 2, and the control line appeared when both ibv and ndv were present; only the control line was generated when neither virus was present ( figure 3b ). as figure 4a shows, there were no positive reactions observed when ibv assays were tested with other pathogen templates except for tcov. the results are not unexpected, because tcov and ibv are very closely related in terms of both antigenic and genomic characterizations (guy, 2000) . target sequence (5 -utr) nucleotide identities between tcov and 224 ibv strains are 91.3 to 97.7% (data not shown). moreover, according to the new 2018 taxonomy of viruses published by international committee on taxonomy of viruses, ibv, and tcov are classified as 1 specie (http://www.ictv.global). ndv detection assays were specific to the ndv templates ( figure 4b ). conventional rt-pcr, nrt-pcr, and rt-lamp assays yielded specific products only when tested with target ndv templates. in mrt-lamp-lfd assays, test lines were observed when target templates were contained in the reaction mixture. as for qrt-pcr assays, fluorescent signals were detected only when tested with the ndv templates. the lowest detection limits of ibv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, when detecting ibv alone, were 10 3.8 , 10 0.8 , 10 1.8 , 10 0.8 , and 10 0.8 copies/reaction, respectively ( figure 5a ). the lowest detection limits of ndv conventional rt-pcr, nrt-pcr, qrt-pcr, rt-lamp, and mrt-lamp-lfd assays, detecting ndv alone, were individually 10 3.7 , 10 0.7 , 10 1.7 , 10 0.7 , and 10 0.7 copies/reaction ( figure 5b ). when simultaneously detecting ibv and ndv, mrt-lamp-lfd produced clear visible test lines at concentration of 10 0.8 ibv + 10 0.7 ndv copies/reaction, and the lowest detection limit was the same as that of mrt-lamp-lfd when detecting ibv or ndv alone ( figure 5c ). as figure 6a shows, mrt-lamp-lfd exhibited the highest mean detection rates in the detection of different types of clinical samples when conducting ibv or ndv single detection, 98.65% for ibv and 97.25% for ndv. statistical significance difference studies showed that the mean detection rates of mrt-lamp-lfd were significantly higher than that of conventional rt-pcr assays when detecting ibv or ndv alone (p < 0.05). no positive results were observed when the assays were tested with negative tissues, fadv positive livers, and aiv positive lungs. to further evaluate mrt-lamp-lfd, 10 chickens were experimentally co-infected with ibv and ndv. results showed that mrt-lamp-lfd could not only detect 2 pathogens simultaneously, but also showed higher mean detection rates than the other assays presented here. the mean ibv and ndv detection rates of different samples, detected by mrt-lamp-lfd, were both 95%, and were significantly higher than those detected by conventional rt-pcr and qrt-pcr (p < 0.05, figure 6b) . . specificity study of pcr and lamp assays of (a) ibv and (b) ndv. in specificity study, cdna of 13 ibv, 7 ndv, 1 arv, 1 ibdv, and 1 aiv and dna of 1 gahv-2 and 2 fadv, as well as the synthesized pcr and lamp target sequences including 6 ndv and 1 tcov were used as templates. in the ibv detection assays, positive results were detected with ibv and tcov templates. in ibv qrt-pcr, other viruses include ndv, arv, gahv-2, ibdv, fadv, and aiv. as for ndv detection assays, positive results were observed only when ndv templates existed. in ndv qrt-pcr, other viruses refer to ibv, arv, gahv-2, ibdv, fadv, tcov, and aiv. nc means negative control reactions which were conducted using total rna extracted from allantoic fluid of healthy spf chick embryo as template. markers in the electrophoresis were the same as that in figure 2a . timely and accurate diagnostic methods are very important for the control of infectious diseases, especially for ibv and ndv which are 2 of the most important contagious viruses seriously affecting the poultry industry. furthermore, ibv and ndv produce clinical picture somewhat resembling each other, it is very much crucial not only to detect but also differentiate simultaneously. existing ibv and ndv diagnostic methods, including virus isolation and pcr assays, are specific and sensitive. however, they are not suitable for timely onsite pathogen detection. although portable pcr machines are gradually applied in the field, in most areas, especially in undeveloped and developing countries, these sophisticated equipments are too expensive to be popularized, and the sensitivity of pcr is not satisfactory. multiple rt-lamp-lfd developed in this study could detect and differentiate ibv and ndv, both simultaneously and accurately. when ibv and ndv cdna co-exist in the same reaction system, an ibv and ndv double-positive result was observed. to evaluate the sensitivity of mrt-lamp-lfd, conventional rt-pcr, nrt-pcr, qrt-pcr, and rt-lamp assays detecting ibv or ndv alone were also conducted to compare with mrt-lamp-lfd. when detecting ibv or ndv alone, mrt-lamp-lfd performed as sensitive as nrt-pcr and rt-lamp did, in a directly visual way. it is always thought that qrt-pcr methods provided high sensitivity during pathogen diagnoses. according to the result of detection limit study, nrt-pcr, and lamp assays, established in this study, possessed 10 times higher sensitivity than qrt-pcr. in the first round of amplification of nrt-pcr, the original template was amplified with the outer primers. thus, the number of the fragments containing the inner primer target sequence is greatly improved compared with the original template. as a result, the number of templates in the second round of amplification of nrt-pcr is much higher than that in qrt-pcr. this is the reason why nrt-pcr could detect lower concentration of original templates than qrt-pcr do. similarly to our results, previous study conducted by weng and chen indicated that npcr showed higher sensitivity than real-time pcr when detecting phytophthora infestans (khan et al., 2017) . lamp is one of the most widely used isothermal nucleic acid amplification techniques (inats) . several studies on the diagnostic methods of other pathogens had showed that these inats possessed equal or even higher sensitivities compared with qpcr assays (gao et al., 2018; khan et al., 2017; yang et al., 2015) . our results indicated that lamp assays possessed higher sensitivities than qpcr when detecting ibv and ndv ( figure 5 ). several multiple rt-pcr assays detecting avian respiratory pathogens have been developed in previous studies, while the sensitivity of these multiple assays was lower than single pathogen detection assays (nguyen et al., 2013; pang et al., 2002) . this may be due to the competition among different sets of primers. however, when detecting with ibv and ndv co-existing samples, the lowest detection limit of mrt-lamp-lfd was the same as that of mrt-lamp-lfd when detecting a single pathogen (i.e., 10 0.8 copies/reaction for ibv and 10 0.7 for copies/reaction for ndv), indicating that the sensitivity of mrt-lamp-lfd was not affected when the components of the reaction system became more complex. the purpose of this study was to develop a novel detection tool which could simultaneously accurately detect ibv and ndv on site. previous studies on pcr methods detecting ibv and ndv showed that these pcr methods are specific and sensitive, but the need for expensive thermal cycling equipments makes them not suitable for on-site ibv and ndv detection. portable pcr machines are gradually applied in the field. while, in most areas, especially in undeveloped and developing countries, these sophisticated to further evaluate mrt-lamp-lfd, 10 chickens were experimentally co-infected with ibv and ndv. the mean detection rates of the assays, when detecting different kinds of samples, are labeled above the histograms. the fraction numbers under the x-axis represent (positive sample numbers detected by the assays)/(total sample numbers). * indicate p < 0.05. equipments are too expensive to be popularized. thus, simpler and visible ibv and ndv detecting methods are urgently needed. high specific and sensitive single ibv and ndv rt-lamp assays have been developed in previous studies (chen et al., 2010; pham et al., 2005) . in these assays, products were visualized by electrophoresis or by adding color indicators. when detecting multiple pathogens, the products must be easily differentiated. when visualized by electrophoresis, lamp products could be distinguished by observing bands with different molecular weights, however, this method is not suitable for on-site pathogen detection. by adding color indicators, the change in color could be easily observed, but this change is non-specific. obviously, these two lamp monitoring methods are not applicable for on-site multiple pathogens detection. multiple lamp-lfd has been applied in the detection of some pathogens, and it showed great advances compared with common pcr and lamp assays, such as the requirement for little equipment, short reaction time, and the ability to detect multiple genes or pathogens (chen et al., 2016; lalle et al., 2018) . to our knowledge, rt-lamp-lfd has not been applied to ibv or ndv detection and no studies on an mrt-lamp-lfd technique that detects multiple avian respiratory viruses have been reported. in this study, the products generated in ibv and ndv rt-lamp were differentiated by biotin/fitc and digoxigenin/fitc labeling, respectively. the products could bind with biotin-or digoxigenin-antibodies fixed on different test lines on the lfd strip, and then products were visualized by combining with gold particles modified with fitc-antibodies (figure 1 ). when tested with clinical samples, the mean detection rate of mrt-lamp-lfd was higher than that of the other assays. these results indicate that mrt-lamp-lfd is not only able to detect ibv and ndv simultaneously, but is also suitable for field testing in both technical (more sensitive than pcr and lamp assays) and practical aspects (more simple to put out and no specialized equipment needed). this study combined rt-lamp and lfd conducting a novel ibv and ndv mrt-lamp-lfd detection assay which is specific and sensitive in detecting ibv and ndv simultaneously. furthermore, mrt-lamp-lfd does not require specialized instrumentations, making it suitable for on-site detection. in conclusion, mrt-lamp-lfd is a promising qualitative detection tool, and is even applicable in some low-resource locations. supplementary data are available at poultry science online. table s1 . the tcov strain and 6 other avian virus strains used in the specificity study figure s1 . phylogenetic analyses based on pcr and lamp target sequences of (a) ibv and (b) ndv. strains used in specificity study are in bigger font. wild strains detected during our avian virus monitoring, of which genome sequences have not been submitted to genbank, were labeled by solid circles; artificially synthesized sequences were labeled by solid triangles. according to the trees, 224 ibv strains were grouped into 4 clades, and the ibv strains used in this study distribute in all 4 clades; 331 ndv strains were grouped into 6 clades, and the ndv strains used in this study distribute in all 6 clades. the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity miniaturized isothermal nucleic acid amplification, a review pathogenesis and diagnostic approaches of avian infectious bronchitis global distributions and strain diversity of avian infectious bronchitis virus: a review a review of virulent newcastle disease viruses in the united states and the role of wild birds in viral persistence and spread development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronavirus avian infectious bronchitis virus reverse transcription loop-mediated isothermal amplification for the rapid detection of infectious bronchitis virus in infected chicken tissues point-of-care and visual detection of p. aeruginosa and its toxin genes by multiple lamp and lateral flow nucleic acid biosensor complete nucleotide sequence of newcastle disease virus: evidence for the existence of a new genus within the subfamily paramyxovirinae real-time pcrbased pathotyping of newcastle disease virus by use of taq-man minor groove binder probes a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of salmonella in shellfish turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review evaluation of different pcr-based assays and lamp method for rapid detection of phytophthora infestans by targeting the ypt1 gene loopmediated isothermal amplification-lateral-flow dipstick (lamp-lfd) to detect toxoplasma gondii oocyst in ready-to-eat salad newcastle disease: evolution of genotypes and the related diagnostic challenges multiplex nested rt-pcr for detecting avian influenza virus, infectious bronchitis virus and newcastle disease virus shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick loop-mediated isothermal amplification of dna development and application of a multiplex polymerase chain reaction for avian respiratory agents loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases loopmediated isothermal amplification for rapid detection of newcastle disease virus multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of salmonella in produce brief review of monitoring methods for loop-mediated isothermal amplification (lamp) this research is funded by the national key r&d program of china (2017yfd0500703), the china agriculture research system (cars-40), the national system for layer production technology (cars-40-k14), and the project for science and technology support program of sichuan province (2018nz0009). we declare that we have no conflict of interest. key: cord-006991-2q5ore6g authors: chi, x.; bi, s.; xu, w.; zhang, y.; liang, s.; hu, s. title: oral administration of tea saponins to relive oxidative stress and immune suppression in chickens date: 2017-06-15 journal: poult sci doi: 10.3382/ps/pex127 sha: doc_id: 6991 cord_uid: 2q5ore6g the present study was designed to evaluate the effects of tea saponins on oxidative stress induced by cyclophosphamide in chickens. one hundred twenty chickens were randomly divided into 5 groups. groups 3 to 4 received intramuscular injection of cyclophosphamide to induce oxidative stress and immunosuppression. after that, groups 2 and 4 were orally administered tea saponins in drinking water for 7 d. then, groups 1 to 4 were immunized with a live, bivalent vaccine of newcastle disease virus and infectious bronchitis virus. blood samples were collected for analysis of oxidative parameters and specific antibody titers, and splenocytes were prepared for lymphocyte proliferative assay. the results showed that administration of tea saponins significantly increased total antioxidant capacity, total superoxide dismutase, catalase, glutathione peroxidase, glutathione, ascorbic acid, and α-tocopherol, and decreased malondialdehyde and protein carbonyl. enhanced immune responses, such as lymphocyte proliferation induced by concanavalin a and lipopolysaccharides, and serum newcastle disease virusand infectious bronchitis virus-specific antibodies were also observed in chickens injected with or without cyclophosphamide. in addition, no side effects were found in chickens throughout the study. therefore, tea saponins may be a potential agent to improve imunosuppression induced by oxidative stress in chickens. oxidative stress is caused by the imbalance between pro-oxidants and antioxidants at either cellular or individual level (voljč et al., 2011) . during the normal respiration, oxygen is progressively reduced to yield water (panda and cherian 2013) . however, the incomplete reduction of oxygen during this process leads to formation of chemical entities that have powerful oxidizing properties and is known as reactive oxygen species (ros) . ros are constantly produced in vivo during the course of physiological metabolism in living tissues. over production or insufficient elimination of ros will lead to oxidative stress (yu et al., 2015b) . oxidative stress constitutes an important factor of biological damage and is regarded as the cause of several pathological conditions that affect growth and development (avanzo et al., 2001; iqbal et al., 2001) , and it may predispose to the development of cancer (kim et al., 2014) , diabetes (gomes 2014) , and cardiovascular diseases (chan and chan 2013 various stresses are responsible for decreasing productive and reproductive performance of growing chickens in the commercial poultry industry. when oxidative stress is more severe, the pro-oxidant systems outbalance the antioxidant systems, potentially producing oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately leading to cell death (surai 2000) . such uncontrolled oxidative reactions also can result in severe oxidative stress and metabolic diseases (tavárez et al., 2011) . therefore, decreasing oxidative stress is very important to maintain feed and food quality and enhance bird health and welfare. less effective immune responses were found after vaccination in some cases, such as individuals influenced by oxidative stress and numerous other factors (peyre et al., 2009 ). therefore, it is necessary to improve the immunization with currently available vaccines so as to effectively protect the host from infections. the medicinal use of tea has a long history in asian countries such as china, japan, india and thailand. tea is the most widely consumed beverage, second only to water, with a per capita worldwide consumption of approximately 0.12 liter per d (pastore and fratellone 2006) . tea seeds and leaves contain variety of biological active compounds like polyphenols, saponins, vitamins, oil minerals, and trace elements (cabrera et al., 2003) . the main antioxidants in tea are saponins, 3058 catechins, flavonols, tannins, and phenolic compounds. the antioxidant properties of green tea polyphenols and the pro-oxidant effects of these compounds have been recommended as potential candidates for cancer prevention (yashin et al., 2011) . the beneficial effects of tea have been attributed to its strong antioxidant activity (jang et al., 2007) . recently, saponins isolated from ginseng stem-leaf (gsls) have been found to be an immune-stimulating agent in chickens (zhai et al., 2011a,b; zhai et al., 2014) . gsls not only affected oxidative stress but also improved immune responses in chickens (yu et al., 2015a,b) . therefore, we hypothesized that tea saponins (ts) have both antioxidative and immunomodulatory properties. at the present study, we evaluated the effect of ts on the antioxidative activities as well as the immune responses to a live bivalent vaccine of newcastle disease virus (ndv) and infectious bronchitis virus (ibv) vaccine in chickens in oxidative stress induced by cyclophosphamide (cy). standardized ts were purchased from zhejiang qingtian zhongye natural plant co. ltd. (hangzhou, china) . ts was water-soluble powder with light-yellow color and 82.8% of the saponins are triterpenoid, having a foamility score of 188 mm and ph of 6.0. day-old specific-pathogen-free (spf) chickens (male) were purchased from zhejiang shennong stock breeding inc. (ningbo, china) , and housed in separated units. the house was set at 35 • c for the first 3 d and then adjusted to 26 • c. feed and water were supplied ad libitum. all the birds were treated according to the zhejiang university committee on animal care and use. live bivalent vaccine of ndv (strain la sota) and infectious bronchitis virus (ibv, strain h120) were purchased from zhejiang ebvac bioengineering co. ltd. the antigen and positive control serum used for ndv-specific hemagglutination inhibition (hi) test were purchased from qingdao yebio bioengineering co. ltd. (qingdao, china). the infectious bronchitis virus antibody test kit was purchased from idexx laboratories inc. (westbrook, maine). cyclophosphamide (cy) was purchased from pude pharmaceutical co. ltd. (shanxi, china). concanavalin a (cona), lipopolysaccharides (lps) and thiazolyl blue tetrazolium bromide one hundred twenty chickens were randomly divided into 5 groups, each consisting of 24 birds. chickens in groups 3 and 4 received intramuscular injection of cy at 100 mg/kg body weight (bw) for 3 d to induce immunosuppression. groups 1, 2, and 5 were injected with saline solution in the same way as groups 3 and 4. then, groups 2 and 4 were orally administered ts of 5 mg/kg bw in drinking water for 7 d. similar way to yu et al (2015b) was used to administration of ts. to make the chickens drink the ts water sufficiently, the birds were kept thirsty for 2 h before ts medication. groups 5 were not medicated and served as a control (table 1 ). after that, group 1 to 4 were intranasally immunized twice with a live bivalent vaccine of ndv and ibv at 2-wk intervals. blood samples were collected before cy treatment and 7, 14, and 21 d after immunization for analysis of oxidative parameters, as well as measurement of ndv-specific hi titers and ibv-specific antibody titers. splenocytes were prepared from 6 birds of each group before cy treatment and 7, 14, and 21 d after immunization for lymphocyte proliferative assay. the dose of ts used in this study was based on our previous investigation where 5 mg/kg was found optimal to enhance the serum specific hi response to ndv vaccine when chickens were orally administered ts at the doses of 3, 5, 7, and 9 mg/kg. t-aoc activity was measured by using the ferricreducing antioxidant ability assay (benzie and szeto 1999) . the assay for t-sod activity involved inhibition of nitroblue tetrazolium reduction with xanthine oxidase used as a superoxide generator (sun et al., 1988) . cat was measured by evaluation of hydrogen peroxide based on the formation of stable complex with ammonium molybdate (aebi 1984) . gsh-px was measured by monitoring the reduction of t-butyl hydroperoxide (wheeler et al., 1990) . gsh was assayed by 5,5 -dithiobis-(2-nitrobenzoic acid) reagent (abegg et al., 2012) . vc was measured by turning ferric iron to ferrous iron (zannoni et al., 1974) . ve was estimated by the reducing properties of tocopherol (emmerie and engel 1938) . mda was performed by the spectrophotometric method based on the reaction 2-thiobarbituric acid (mihara and uchiyama 1978) . protein carbonyl was measured by oxidative damage to proteins (pirinccioglu et al., 2010) . serum ndv-specific hi titers were determined as described by zhai et al. (2011a) . briefly, 2-fold serial dilution of serum samples (1:2 to 1:2048) was made in a v-shaped bottom 96-well microtiter plate. afterwards, 4 hemagglutination units of ndv antigen was added to each well. after incubation at 37 • c for 30 min, 25 μl of 1% rooster erythrocyte suspension was added and incubated at 37 • c for 20 min. all samples were tested in duplicate, and the positive and negative serum controls were included on each plate. the hi titers were defined as the reciprocal of the last dilution of serum causing complete inhibition of hemagglutination. the data were expressed as log 2 of the highest dilution that exhibited hi. serum ibv-specific antibody titers were determined using detection kits according to the manufacturers' protocols. test samples were diluted 500-fold (1:500) with sample diluent prior to being assayed and the samples were thoroughly mixed prior to dispensing into the coated plate. all reagents were allowed to come to 18 to 26 • c before use. next, 100 μl of diluted sample was added into appropriate wells and 100 μl of undiluted negative control and positive control were added into duplicate wells. after incubation at 26 • c for 30 min, the solution was removed and washed each well 5 times with approximately 350 μl of distilled water. after that, 100 μl of conjugate was dispensed into each well, then incubated at 26 • c for 30 min. each well was washed as described above and 100 μl of tmb substrate was dispensed. after incubation at 26 • c for 15 min, 100 μl of stop solution was added. the mean optical density (od) was read at 650 nm. the relative level of antibody titer in the unknown was determined by calculating the sample to positive (s/p) ratio as [(mean of sample optimal density) -(mean of negative control optimal density)] / [(mean of positive control optimal density) -(mean of negative control optimal density). endpoint titers were calculated with the equation: log 10 titer = 1.09 (log 10 s/p) + 3.36 (flock check program, idexx). the test was performed as previously (li et al., 2009) . briefly, spleens collected into hank's balance salt solution (hbss) were minced and passed through a fine steel mesh to obtain the cell suspension under aseptic conditions. the cells were washed in hbss 3 times by centrifugation (500 × g at 4 • c for 10 min), and suspended in cold rpmi 1640 supplemented with 0.05 mm 2-mercaptoethanol, 100 iu/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (fcs). cell viability was assessed with trypan blue exclusion stain, and the cell suspension was adjusted to 5.0 × 10 6 /ml. mitogen-induced proliferation was performed by incubating 100 μl of lymphocyte suspension with 100 μl of either cona (40 μg/ml) or lps (35 μg/ml) in each well for the mtt assay. rpmi 1640 medium (100 μl) was added to the cell suspension in control wells. cells were added in triplicate, and incubated in a humidified atmosphere at 5% co 2 at 40 • c for 44 h. after that, 50 μl mtt (2 mg/ml) was added to each well and incubated for an additional 4 h. the plates were centrifuged (1,000 × g for 10 min), and the supernatant was carefully removed. added 150 μl of acidic dimethyl sulfoxide (dmso, 0.04 n hcl) to each well and mixed thoroughly by slightly shaking to solubilize the mtt formazan. the mean optical density (od) was read at 570 nm. the stimulation index (si) was calculated based on the formula: +si = od value of mitogen-stimulated cells divided by od value of non-stimulated cells. analysis of data was performed using spss software (version 20.0, spss inc., chicago, il). one-way analysis of variance (anova) with duncan post hoc test was used for multiple comparisons between groups. values were expressed as the mean ± standard deviation (sd). p-values of less than 0.05 were considered statistically significant. to investigate the effect of ts on oxidative stress, blood samples were collected to measure the serum t-aoc, t-sod, cat, and gsh-px. figure 1 showed that there were no significant difference for serum t-aoc, t-sod, cat, and gsh-px between groups before medication. it was found that activities of t-aoc, t-sod, cat, and gsh-px in birds treated with ts (ts + vaccine) were significantly higher than those without ts treatment (vaccine), and the activities in birds treated with cy (cy + vaccine) were significantly lower than those without cy injection (vaccine). however, the antioxidant enzymes in birds treated with cy serum t-aoc, t-sod, cat and gsh-px. chickens were i.m. injected with cy at 100 mg/kg bw for 3 days to induce immunosuppression and oxidative stress. after that, the birds were orally administered ts (5 mg/kg bw) in drinking water for 7 days. blood samples were collected from 6 birds of each group before cy treatment and 7, 14, 21 days after booster for analysis of t-aoc, t-sod, cat and gsh-px.data were expressed as mean ± s.d. (n = 6). bars with different letters are statistically different (p < 0.05). significantly increased after administered ts (cy + ts + vaccine) when compared to the birds treated without ts (cy + vaccine). to investigate the effect of ts on serum gsh, vc, ve, mda, and carbonyl, blood samples were collected after booster immunization. figure 2 showed that there were no significant difference for serum gsh, vc, ve, mda, and carbonyl between groups before medication; serum gsh, vc, and ve were significantly increased, and mda and carbonyl were significantly decreased after birds were treated with ts (ts + vaccine); serum gsh, vc, and ve were significantly decreased, and mda and carbonyl were significantly increased after birds were treated with cy (cy + vaccine). however, gsh, vc, and ve were significantly increased and chickens were i.m. injected with cy at 100 mg/kg bw for 3 days to induce immunosuppression and oxidative stress. after that, the birds were orally administered ts (5 mg/kg bw) in drinking water for 7 days. blood samples were collected from 6 birds of each group before cy treatment and 7, 14, 21 days after booster for analysis of serum gsh, vc, ve, mda and carbonyl. data were expressed as mean ± s.d. (n = 6). bars with different letters are statistically different (p < 0.05). mda and carbonyl were significantly decreased in birds treated with cy (cy + vaccine) after they had been orally administered ts (cy + ts + vaccine). to investigate the effect of ts on the lymphocyte proliferative responses to mitogens in vitro, chickens were injected with cy to induce immunosuppression and oxidative stress. after that, the birds were orally administered ts and then immunized with a bivalent vaccine of ndv and ibv. splenocytes were prepared from the birds before and after immunization for lymphocyte proliferative assay. figure 3 showed that no significant difference was found for stimulation indexes (si) between groups before cy treatment ( figure 3a) . si was significantly higher in birds treated with ts figure 3 . lymphocyte proliferative responses to cona and lps. chickens were i.m. injected with cy at 100 mg/kg bw for 3 days to induce immunosuppression and oxidative stress. after that, the birds were orally administered ts (5 mg/kg bw) in drinking water for 7 days, and then, intranasally immunized twice with a commercial live bivalent vaccine of ndv and ibv at 2 weeks intervals. splenocytes were prepared from 6 birds of each group before cy treatment and 7, 14, 21 days after booster for lymphocyte proliferative assay measured by the mtt method as described in the text. data were expressed as mean ± s.d. (n = 6). bars with different letters are statistically different (p < 0.05). (ts + vaccine) ( figure 3b and c), and significantly lower in birds treated with cy (cy + vaccine) than the birds without ts treatment (vaccine) ( figure 3b , c and d). however, si in birds treated with cy significantly increased after administered ts (cy + ts + vaccine) when compared to the birds treated with cy (cy + vaccine) ( figure 3b, c and d) . to investigate the effect of ts on the serum antibody responses to vaccination, blood samples were collected to measure the serum antibodies to ndv and ibv after immunization. figure 4 showed that almost figure 4 . antibody responses to a bivalent live vaccine of ndv and ibv. chickens were i.m. injected with cy at 100 mg/kg bw for 3 days to induce immunosuppression and oxidative stress. after that, the birds were orally administered ts (5 mg/kg bw) in drinking water for 7 days, and then, intranasally immunized twice with a commercial live vaccine of ndv and ibv at 2 weeks intervals. blood samples were collected from 6 birds of each group before cy treatment and 7, 14, 21 days after booster for analysis of ndv and ibv specific titers. data were expressed as mean ± s.d. (n = 6). bars with different letters are statistically different (p < 0.05). no specific ndv and ibv titers were detected in birds ( figure 4a ) before immunization. however, antibody titers progressively increased after immunization. significantly increased antibody titers to ndv ( figure 4b , c and d) and ibv ( figure 4d ) in birds treated with ts (ts + vaccine) and decreased titers in birds treated with cy (cy + vaccine) ( figure 4b , c and d) were detected when compared to the birds without ts treatment (vaccine). however, the antibody titers in birds treated with cy significantly increased after administered ts (cy + ts + vaccine) when compared to the birds treated with cy (cy + vaccine). no significant difference for body weight was found at each time point between the birds orally administered ts at 5 mg/kg bw and those without ts administration. and no abnormal behaviors and side effects were observed in chickens throughout the experiment. the present study demonstrated that ts has antioxidant activities in chickens. oral administration of ts enhanced enzymatic and non-enzymatic antioxidant defense mechanisms such as t-aoc, t-sod, cat, gsh-px (figure 1) , gsh, vc, and ve (figure 2a, b and c) , and decreased the protein carbonyl content and mda in oxidative stress induced by cy ( figure 2d and e) . no abnormal behaviors and side effects caused by ts were observed in chickens throughout the study. in chickens, a balance between pro-oxidative and antioxidative activities is important. overproduction or insufficient elimination of ros will lead to oxidative stress (yu et al., 2015b) . there are enzymatic and nonenzymatic antioxidant defense mechanisms in the body. enzymatic antioxidant defenses include total antioxidative capacity (t-aoc), total superoxide dismutase (t-sod), glutathione peroxidase (gsh-px), and catalase (cat). both t-sod and cat can degrade o 2 and decompose h 2 o 2 , and result in a decrease in oxidative stress, which is an effective way of cell protection from damage (nagi and almakki 2009) . glutathione peroxidase (gsh-px) is an enzyme responsible for scavenging ros and its oxidation products to protect the body from lipid peroxidation (lpo) (cui et al., 2010) . the major function of gsh-px, which uses glutathione as a substrate, is to reduce soluble hydrogen peroxide and alkyl peroxides (bebe and panemangalore 2003) . all these enzymes work together to eliminate active oxygen species, and small deviations in physiological concentrations may have a dramatic effect on the resistance of cellular lipids, proteins, and dna to oxidative damage (ince et al., 2012) . non-enzymatic antioxidants are represented by gsh, vc, and ve. gsh may protect cells against oxidative injury. the depletion of gsh due to cy treatment will reduce its cellular level and lead to induction of oxidative stress (yuan et al., 1991; yousefipour et al., 2005) . vc is a water-soluble antioxidant that helps to reduce the effect of oxidative stress (eroglu et al., 2013) . vc can easily react with free radicals in extracellular body fluids, as it is water soluble (bendich 1990) . vc exerts its antioxidant effects in both direct and indirect ways. vc directly scavenges the free radicals formed as a byproduct of metabolic reactions (dawson et al., 1990) . in an indirect way, vc helps recycling of oxidized ve, thus supplying active ve to fight against lpo (netke et al., 1997) . in addition, there are many studies demonstrating that ve protects cell membranes by preventing lpo. α-tocopherol reacts with peroxyl radicals depending on its methylation state of the chromanol ring and the saturation grade of the side chain, forming tocopheroxyl radicals (brigeliusflohé 2009 ). tocopheroxyl radicals are converted to tocopherols by re-acting with ascorbate (may et al., 1998) . ve has been shown to play a role in various enzymes activities by enabling their translocation to the membrane (kempná et al., 2004) or affecting their transcriptional activation process (khor et al., 2000) . in many studies, ve neutralizes lpo and unsaturated membrane lipids because of its oxygen scavenging effect (aldana et al., 2001; john et al., 2001) . cyclophosphamide (cy) is a cytotoxic alkylating agent with a wide of clinical uses and has been proved to be effective originally used in the treatment of some types of cancers and nonmalignant diseases. cy is transformed in the liver into active metabolites (4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, and others), which interfere with cellular dna synthesis in rapidly dividing cells, thus leading to cell death (perini et al., 2007) . in the early study, cy has been found to increase superoxide anion and h 2 o 2 formation in cultured rat hepatocytes (fuhrman et al., 1997) and different kidney cells (hussein et al., 2005) and induce oxidative stress and lipoperoxidation (reiter et al., 1999) . the deleterious effects of cy was, at least in part, due to the increased production of free radicals and ros, which is associated with enhanced ros generation and oxidative stress (maestroni and conti 1989) . thus cy is frequently used to induce oxidative stress models (tripathi and jena 2009; oboh et al., 2011) . in addition, cy has been reported to suppress both cellular and humoral immune responses (bear 1986; hoover et al., 1990) , such as inhibiting lymphocyte activity and specific antibodies to ndv and ibv (reynolds and maraqa 1999; loa et al., 2002; wang et al., 2011) . in this study, injection of cy generated oxidative stress and lowered immune responses by reducing antioxidant enzymes such as t-aoc, t-sod, gsh, and cat, as well as inhibiting lymphocyte proliferation and antibody responses to vaccination. decreased antioxidant activities in animals due to cy were reported in other studies (popovic et al., 2007; singh et al., 2010; ognjanović et al., 2012; yu et al., 2015b) . oral administration of ginseng stem-leaf saponins has recently been reported to reduce oxidative stress induced by cy in chickens (yu et al., 2015b) . similarly, reduced oxidative stress in chickens by oral administration of ts was found in this study. a stressful condition is closely related to the immune system. during oxidative stress, the excessive production of free radicals results in an imbalance in the oxidant system, and suppresses the immune function (kucinski 1990 ). the immune system is particularly sensitive to stress and specific effects of stress have been demonstrated by a number of studies (mastorakos and ilias 2000; wu et al., 2000; mcewen 2001 ). the immune responses can be improved by removing immunosuppression (srikumar 2006 ). in the present study, the increased antioxidant activities by oral administration of ts paralleled the enhanced immune responses to vaccination. ts-treatment significantly increased serum antibodies to ndv and ibv as well as the lymphocyte proliferation induced by cona and lps. newcastle disease and infectious bronchitis virus infection are fatal and could result in large amount of economic losses in poultry industry (callison et al., 2007; ge et al., 2007) . immunization using live, attenuated vaccines is a common practice for the control of ndv and ibv in birds (seto et al., 1974; gelb et al., 1998) . figure 4 showed that oral administration of ts improved antibody responses to ndv and ibv in the birds in healthy and immune-suppressed by cy. the enhanced immune responses could be due to reduction in oxidative stress resulting from oral administration of ts. in conclusion, antioxidative and immunopotentiating effects of tea saponins (ts) on oxidative stress induced by cyclophosphamide in chickens were demonstrated. oral administration of ts at a dose of 5 mg/kg bw significantly improved the antioxidant activity, as indicated by increasing t-aoc, t-sod, cat, gsh-px, gsh, vc, ve, as well as by decreasing mda and the protein carbonyl. the increased antioxidant activity paralleled increased specific antibody responses to ndv and ibd vaccine, lymphocyte proliferative responses induced by cona and lps. considering ts has its antioxidant activity and plentiful resources, ts may be a potential agent to relieve oxidative stress in immunosuppressed chickens in poultry industry. this study was supported by the special fund for agro-scientific research in the public interest (2013203040). the authors wish to thank the students in the laboratory of traditional chinese veterinary medicine (tcvm lab) for their assistance in sample collection. glutathione levels in and total antioxidant capacity of candida sp. cells exposed to oxidative stress caused by hydrogen peroxide catalase in vitro alpha-tocopherol modulates liver toxicity of the pyrethroid cypermethrin effect of vitamin e and selenium on resistance to oxidative stress in chicken superficial pectoralis muscle tumor-specific suppressor t-cells which inhibit the in vitro generation of cytolytic t-cells from immune and early tumor-bearing host spleens exposure to low doses of endosulfan and chlorpyrifos modifies endogenous antioxidants in tissues of rats antioxidant micronutrients and immune responses total antioxidant capacity of teas by the ferric reducing/antioxidant power assay vitamin e: the shrew waiting to be tamed determination of tea components with antioxidant activity development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens angiotensin-generated reactive oxygen species in brain and pathogenesis of cardiovascular diseases anti-oxidation activity of the crude polysaccharides isolated from polygonum cillinerve (nakai) ohwi in immunosuppressed mice relationship between ascorbic acid and male fertility colorimetric determination of dlalpha-tocopherol (vitamin e) hypocholesterolemic effect of lycopene and beta-carotene is related to suppression of cholesterol synthesis and augmentation of ldl receptor activity in macrophages newcastle disease 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quality on broiler performance, meat quality, and lipid oxidation intervention of astaxanthin against cyclophosphamide-induced oxidative stress and dna damage: a study in mice evaluation of different vitamin e recommendations and bioactivity of α-tocopherol isomers in broiler nutrition by measuring oxidative stress in vivo and the oxidative stability of meat a polysaccharide from strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity social isolation stress enhanced liver metastasis of murine colon 26-l5 carcinoma cells by suppressing immune responses in mice determination of antioxidant activity in tea extracts, and their total antioxidant content mechanism of acrolein-induced vascular toxicity improved immune responses to a bivalent vaccine of newcastle disease and avian influenza in chickens by ginseng stem-leaf saponins antioxidative effect of ginseng stem-leaf saponins on oxidative stress induced by cyclophosphamide in chickens glutathione conjugation with phosphoramide mustard and cyclophosphamide. a mechanistic study using tandem mass spectrometry a rapid micromethod for the determination of ascorbic acid in plasma and tissues effect of oral administration of ginseng stem-and-leaf saponins (gsls) on the immune responses to newcastle disease vaccine in chickens enhancement of humoral immune responses to inactivated newcastle disease and avian influenza vaccines by oral administration of ginseng stemand-leaf saponins in chickens enhanced immune responses of chickens to oral vaccination against infectious bursal disease by ginseng stem-leaf saponins key: cord-259959-qzd3hf8y authors: alhatami, abdullah o.; alaraji, furkan; abdulwahab, husam muhsen; khudhair, yahia ismail title: sequencing and phylogenetic analysis of infectious bronchitis virus variant strain from an outbreak in egg-layer flocks in baghdad, iraq date: 2020-07-16 journal: vet world doi: 10.14202/vetworld.2020.1358-1362 sha: doc_id: 259959 cord_uid: qzd3hf8y background and aim: infectious bronchitis (ib) has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens. despite the use of many kinds of vaccines in iraq, it is common to find ib problems in vaccinated chickens. information about the strains that affect iraqi chickens is very limited. therefore, we aimed to detect the currently circulating strains of ib virus that cause frequent outbreaks in egg layers despite the use of vaccination against the virus. materials and methods: isolate detection, sequencing, and phylogenetic analysis were performed using a rapid ib virus antigen kit (32 tracheal swabs), flinders technology associates (fta) card (32 tracheal swabs), and partial gene sequencing (16 positive fta samples). results: the isolated strain was different from other strains, especially the strain isolated in the north of iraq (sulemania strain) and shares 98% homology with an israeli strain (israel variant 2, is 1494). conclusion: although more studies are needed to detect ib virus strains circulating in iraq, this work lays the foundation for making a good strategy to control the disease and selecting vaccines that should be used in farms. an important viral disease affecting the poultry industry globally and locally in iraq is the infectious bronchitis virus (ibv), which causes an acute, highly contagious respiratory disease. the identification of endemic ibv strains promotes better control of the disease and prevents production losses [1] . ibv belongs to the family coronaviridae and the genus gamma-coronavirus. the single-stranded rna (positive-sense, ~30 kb) of this enveloped virus has several open reading frames [2] . although the nucleocapsid (n) gene is highly conserved among coronaviruses, the spike (s) glycoprotein (particularly s1) gene contains highly variable components in these viruses [3] . ibv usually is subjected to changes due to mutations and genetic recombination. therefore, a mutation in the s1 gene may affect virus tissue tropism and virulence [4] . consequently, ibv can spread widely to affect different regions of the world, creating huge difficulties in controlling the disease due to these newly modified strains [3, 4] . even with high numbers of the globally identified genotypes or serotypes of ibv, cross-protection generated by those strains is barely present [5] . the highly variable s1 gene represents the ideal genetic target for monitoring viral evolution occurring in ibv, especially in strains with high rates of serotype correlations [6] . therefore, sequencing the s1 gene of ibv strains detected in the field is critical for control programs and epidemiological purposes. the spike protein is a major structural protein of 1162 amino acids that are cleaved into 535-aa-sp1 (s1, n-terminus) and 627-aa-sp2 (s2, c-terminus). the s1 gene induces serotype-specific based antibody neutralization through its two hypervariable regions [7] . many polymerase chain reaction (pcr)based techniques have been developed to monitor the prevalence of ibv by targeting and analyzing the s1 gene. reverse-transcriptase pcr (rt-pcr) and nucleotide sequencing of the s1 gene are routinely used to detect ibv genotype; in addition to real-time quantitative pcr (rt-qpcr), which also is used to identify ibv from field samples. these molecular methods are highly sensitive and specific compared with other diagnostic methods [8] [9] [10] . since its discovery in 1931, and despite massive routine vaccination, ibv continues to be one of the major concerns in the poultry industry worldwide. this may be due to many reasons, one of which is the continuous emergence of new ibv strains [11] with unique genetic determinants identified in each geographical area. to the best of our knowledge, only one study to date reports the molecular characterization of ibv that resulted in a broiler strain (sul/01/09), identified in the kurdistan region of iraq. therefore, the current report describes the role of ibv during an outbreak of the respiratory disease in an egg-layer farm in the baghdad region of iraq, investigates the genetic characteristics of this field strain by analyzing the s1 gene and compares it with other isolates registered globally for developing significant vaccines to control this disease. this study does not require ethical approval. however, all birds in the current work were treated humanely following international and national criteria of animal care and use. an outbreak occurred during january 2018 in al-janoob company, located in the al-wahda district of baghdad, iraq. this was composed of eight caged houses of egg layers with a total number of 640,000 hens and 170,000 rearing pullets. the outbreak was not reported to the national authority. all flocks were vaccinated with commercial live-attenuated nobilis ib 4-91. respiratory symptoms began in 28-day-old birds of an isa brown flock composed of 88,000 birds per house. clinically, the symptoms were suspected to be ibv infections. necropsies were performed, and gross lesions were evaluated. sixty-four tracheal swabs (two swabs per bird) were collected during january 2018, from birds that showed such respiratory manifestations. thirty-two tracheal swabs were used for serological diagnosis of ibv antigen using a rapid ibv ag test kit (rg1513dd, bionote, korea), which is a chromatographic immunoassay for qualitative ibv antigenic detection in avian swab samples. tests were performed according to the manufacturer's instructions. samples were pooled on flinders technology associates (fta) cards (whatman ® fta ® card technology) with four sample areas per card (total homogenate volume up to 100 μl) containing chemicals for lysing cells, denaturing proteins, and protecting nucleic acids against damage from nucleases, ultraviolet radiation, and oxidation [12, 13] . fta cards containing viral rna were outsourced to anicon labor gmbh (muehlenstraße 13a 49685 hoeltinghausen, germany), where the extraction of the ibv rna was performed using kylt ® rna/dna purification kit through the manufacturer's protocol. rt-pcr runs were generated by anicon labor gmbh. briefly, species-specific and variant-specific rt-pcr methods were performed to detect avian coronavirus (acov, including ibv) and ibv variants. hybridization probe-based chemistry was used with the following primers: and kylt ® ibv-ib80 (anicon labor gmbh). rt-pcr, cfx96, and cfx384 (bio-rad, hercules, ca, usa) systems were used according to the following conditions: 50°c for 10 min and 95°c for 1 min (initial denaturation), then 42 cycles of 95°c for 10 s, 60°c for 1 min, and read. the amplified rt-pcr products were sequenced by anicon labor gmbh. sequences of the iraqi-ibv-strain s1 gene were deposited in the genbank database available from the national center for biotechnology information (ncbi) website to collect accession numbers. ibv s1 gene sequences obtained herein were compared with sequences of ibv deposited globally in the genbank database using the ncbi-based blast search. the identities of the sequences were analyzed by dnastar software (dnastar, madison, wi, usa; https://www.dnastar.com/), and molecular evolutionary genetics analysis (mega) x software (https://www.megasoftware.net/) were used to construct the phylogenetic tree. the flock suffered from signs of infectious bronchitis (ib) disease, and the mortality rates reached 8% for approximately 10 days. birds suffered the following symptoms: coughing, sneezing, and rales with depression. postmortem findings included tracheal congestion, and caseous materials that partially obstructed the trachea and the tracheal bifurcations, and pneumonia and fibrinous airsacculitis ( figure-1 ). kidneys were enlarged and congested and contained an accumulation of urate in the nephrons and ureters. thirty-two (100%) of the examined samples were positive for the presence of the ibv antigen. viral rna from the fta cards (four spots pooled) with the sample number (a1800428.001) showed positive results with species-specific and ibv-variant-specific rt-qpcr (table-1) . available at www.veterinaryworld.org/vol.13/july-2020/15.pdf a 630-bp and 730-bp fragments of the s1 protein-coding gene were sequenced by anicon labor gmbh. the sequences were deposited in the ncbi genbank under accession numbers mh747093 and mh747094 with the name acov strain iq1 and iq2 spike glycoprotein (s1) gene, respectively. the genetic relationship between the s1 gene sequence of the mh747094.1 ibv-iq2 strain and sequences of vaccine and other virulent strains are presented in figure-1 and table2. the mh747094.1 strain was closely related (100% similarity) to eu780077.2_ibv_isolate_is/1494/06 (israeli variant 2), whereas the percentage of sequence identity was 55% compared with gq281656.1ibv_isolate_ sul/01/09_s1 (sulaimania isolate). moreover, the nucleotide similarity was 40% and 28% compared with fj888351.1ibv strain h120 and af 093794.1ibv strain 4/91, respectively. ib has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens [14] . since ibv was identified during the early 1990s, ibv (4/91 type) has frequently been reported in europe and many countries around the world [15] . ibv vaccine strains can perform recombination with field strains, reversing virulence [16] . such characteristics have encouraged verification of the relationship between vaccine and field strains [17] . although a 793/b-serotypebased attenuated vaccine is available, some 793/b-serotype viruses remain active in europe and several countries [16] , and the live-attenuated or killed massachusetts (mass) strain-dependent vaccines are most widely used for vaccination programs throughout the world. nevertheless, there is an increased failure of ibv vaccination programs, particularly against the 4/91 ibv strain, in addition to the circulation of many ibv-vaccine-related strains [18, 19] . most of the ibvvariant strains have distinctive characteristics due to their global distribution; however, some strains are unique to certain regions, and these properties occur for unknown reasons. the current work represents the first identifying and genotyping report of ibv that resembles the israeli isolate in iraq. the results of the sequencing and phylogenetic tree analysis indicate that our isolate shares 98% homology with the israel variant 2 (is 1494). in 2011, zana et al. [20] reported a newly isolated strain along with other regionally identified isolates from other israel strains (is/720/99, is/885), and waleed et al. [21] also registered strains 793/b and mass from infected broilers in the south of iraq. thus, isolates from different parts of iraq demonstrate large differences in homology. in addition, in the sulaimaniah isolate (sul/01/09), the birds displayed nephron pathological lesions, and the virus was detected from kidney samples but not from tracheae. therefore, the sulmania (iraqi strain) that affected broilers differs genetically from our strain that infected layers. furthermore, our isolate shared <82% and 79% in nucleotide sequence with vaccine strains fj888351.1 and kx258195.1, respectively, which may explain the occurrence of infection despite vaccination. therefore, our work lays the main foundation including rna-based s1 protein transcript sequencing and the related phylogenetic analysis, to initiate launching strategies for control of ibv in the field. despite the intensive vaccination program in iraq, ib outbreaks have occurred for many decades. in this study, we isolated a strain of ibv very different from that of local north iraq (sulemania strain) but similar to an israeli strain (israel variant 2, is 1494). genetic characterization of the iraqi circulating ibv strains is critical because of inadequate data from this location. more studies revealing such strains will pave the way for a good strategy of controlling the infections and understanding or developing types of vaccines that should be used in the future. aoa and hma visited the infected farm, collected the samples, and performed the rapid ibv antigen detection. fa and yik visited the infected farm, collected the samples, run the rapid ibv antigen detection, confirmed the results using fta card that was sent to germany, deposited the genetic information into the ncbi database, analyzed the differences between the strains of the virus, and drafted the manuscript. all authors revised the manuscript. all authors have read and approved the final manuscript. pathogenesis and diagnostic approaches of avian 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molecular epidemiology in poland isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east prevalence of avian infectious bronchitis virus in broiler chicken farms in south of iraq the authors thank anicon laboratory in germany for their assistance in the diagnosis of the virus. the work was performed in the college of veterinary medicine, university of al-qadisiyah, available at www.veterinaryworld.org/vol.13/july-2020/15.pdf diwaniyah city, iraq, and the anicon labor gmbh (muehlenstraße 13a 49685 hoeltinghausen, germany). the authors did not receive any funds for this study. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord-269024-re0hyolh authors: bande, faruku; arshad, siti suri; hair bejo, mohd; kadkhodaei, saeid; omar, abdul rahman title: prediction and in silico identification of novel b-cells and t-cells epitopes in the s1-spike glycoprotein of m41 and cr88 (793/b) infectious bronchitis virus serotypes for application in peptide vaccines date: 2016-09-07 journal: adv bioinformatics doi: 10.1155/2016/5484972 sha: doc_id: 269024 cord_uid: re0hyolh bioinformatic analysis was used to predict antigenic b-cell and t-cell epitopes within the s1 glycoprotein of m41 and cr88 ibv strains. a conserved linear b-cell epitope peptide, ytsnettdvts(175–185), was identified in m41 ibv strains while three such epitopes types namely, vsnaspnsggvd(279–290), hpkcnfrpeni(328–338), and netnnagsvsdctagt(54–69), were predicted in cr88 ibv strains. analysis of mhci binding peptides in m41 ibv strains revealed the presence of 15 antigenic peptides out of which 12 were highly conserved in 96–100% of the total m41 strains analysed. interestingly three of these peptides, ggpitykvm(208), wfnslsvsi(356), and yladaglai(472), relatively had high antigenicity index (>1.0). on the other hand, 11 mhci binding epitope peptides were identified in cr88 ibv strains. of these, five peptides were found to be highly conserved with a range between 90% and 97%. however, wfnslsvsl(358), synisaasv(88), and ynisaasva(89) peptides comparably showed high antigenicity scores (>1.0). combination of antigenic b-cells and t-cells peptides that are conserved across many strains as approach to evoke humoral and ctl immune response will potentially lead to a broad-based vaccine that could reduce the challenges in using live attenuated vaccine technology in the control of ibv infection in poultry. infectious bronchitis virus (ibv) is a single stranded, enveloped rna virus belonging to the family coronaviridae, order nidovirales [1] . the virus causes infectious bronchitis (ib), a contagious disease associated with huge economic loses in poultry industry worldwide [2] . of major concern in the control of ib is the continued emergence of variant ibv strains that differ in terms of their tissue tropism, pathogenicity, and cross protection. over the years, serological and molecular studies have been carried out extensively to determine the epidemiology of local ibv strains. remarkably, both classical and variants ibv strains have been reported in different countries [3] . among the widely identified ibv strains are ibv m41 (classical strain), originally recognized in usa [4, 5] , and cr88 (variant strain otherwise known as 793/b or 4/91) which was first reported in europe [3, 6, 7] . over the years, control of ibv infection largely depends on vaccination using live attenuated and killed vaccines. however, one of the challenges with live attenuated ib vaccines is that such vaccines are reported to encourage mutation and recombination, thus leading to the emergence of new variant strains. live attenuated vaccines have also been linked with reversion to virulence, severe disease, and increased mortality rate [8] [9] [10] . on the other hand, killed vaccines induce humoral but not cell mediated immune (cmi) response and in most cases require adjuvants as well as repeated boosting especially in laying chickens and breeder flocks. these challenges therefore necessitate the needs for novel broad vaccines for the control of ib in poultry [11] . to achieve this, stimulation of both humoral (b-cell) and cell mediated immune (cmi) responses is considered very essential for any candidate vaccine [12] . neutralizing antibodies are important in removing freely circulating ib virus, whereas cytotoxic t-lymphocytes (ctl) response is crucial for the control and clearance of virally infected cells. the latter is achieved through mhci immune surveillance as well as antigen presentations which is the function of mhci molecule and both have been associated with the epitope within the s1 glycoprotein [13, 14] . while much has been documented on mhc restricted allele in human and mouse models, little information is available on the biological functions of these molecules in poultry [15] . a large binding groove of bf2 * 2101 mhci molecule identified in b21 chicken line is thought to confer conformational flexibility to the crucial arg9 residue which allows remodeling of key peptide-binding sites and play a role in the resistance against poultry viral infections [16] . chicken mhc b-f molecules have been structurally and functionally linked to mammalian mhc class i molecules and involved in antigen presentation to the cd8 + t lymphocytes, which is crucial in antiviral immune response [17] . interestingly, the s1 glycoprotein of ibv (520 aa) contains different immune epitopes responsible for both antibodies and ctl-based immune responses, thus playing major protective role as viral antigenic determinant [18] . currently, the use of peptide based dna vaccines represents a novel strategy for addressing challenges associated with the control of viral infections [19, 20] . this technology may employ the use of in silico analysis to predict novel b-cells and t-cells immune epitopes for further use in their in vivo applications. one of the innovations in using this technology is the ability to incorporate several epitope peptides directed against different viruses and/or multiple virus strains into one single delivery system with the view to induce broad and specific immune response in single administration [11] . to date, only few epitope based peptide vaccines have been developed and evaluated against ib [20, 21] . the objective of this study therefore is to identify novel b-cells and t-cell epitopes within the s1 glycoprotein of m41 and cr88 ibv strains. the antigenicity of the predicted peptides was also evaluated. nucleotide sequences containing complete s1-gene of homologous to m41 and cr88 ibv strains were retrieved from ncbi database and translated into deduced amino acid sequences for further analysis. consensus amino acid sequences from the two datasets (m41 and cr88) were used to predict b-and tcells epitope peptides within s1 glycoprotein. the criteria used for the selection of m41 and cr88 were simply based on epidemiological relevance of the two strains in terms of their wide geographic distribution as classical and variant ibv strains, respectively. all sequences were assembled with geneious5 software r7 version [22] . the names and accession numbers of ibv sequences used in this study are presented in supplementary file 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/5484972. epitopes associated with bcells were predicted using the bepipred epitope prediction server version 1.0 [23] . similarly, antigenicity index of the predicted epitopes was analysed using vaxijen v2.0 online antigen prediction tool http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html [22] . prediction criteria were set at 75% classifier specificity and 20 epitope lengths with an overlap. the location of the predicted epitope within transmembrane regions was evaluated using tmhmm [24] . only epitope present at the surface of the membrane was selected and further analysed for antigenicity. for this search, target organism was narrowed to viruses and only scores above 0.4 thresholds were considered as a good antigenic epitope. further, conservancy analysis was carried out using iedb tools available http://tools.immuneepitope.org/tools/ conservancy/iedb input [25] . the t-cell epitope prediction was carried out using propred-1 software which covers 47 mhc class-i alleles. both proteasome and immuneproteasome were set at 5% threshold by mhc class i and only peptides with proteosomal cleavage site at the c terminus were considered [26] . immune epitopes were analysed for conservancy and variability using iedb conservation analysis tool: http://tools.immuneepitope.org/tools/ conservancy/iedb input [25] . a total of 86 m41 and 72 cr88 ibv isolates were considered in this study. however, to build the consensus sequence, one isolate from each data set was removed due to some unidentified amino acid at one or two positions. the m41 ibv isolates were found to have an average nucleotides sequence length of 1614 (537 aa) while cr88 isolates had an average length of 1653 (553 aa). analysis of m41 associated linear b-cell epitopes revealed the presence of six unique b-cells peptides within the s1 glycoprotein. of these, only ytsnettdvts 175-185 peptide was found to be antigenic (table 1) . analysis of transmembrane potentials within the m41 ibv s1 gene showed that the predicted linear b-cell epitopes interact with the outside surface membrane especially with maximum threshold occurring at amino acid positions 280 to 290 (figure 1) . interestingly, conservancy analysis showed that ytsnettdvts 175-185 peptide is common in 82.35% of the m41 strains. on the other hand, a total of six antigenic epitope peptides were predicted in cr88 ibv strains; however, three of these peptides, netnnagsvsdctagt 54-69 , vsnaspnsggvd 279-290 , and hpkcnfrpeni 328-338 were demonstrated to be antigenic (table 2) . further, conservancy analysis showed that the three cr88 associated antigenic b-cell peptides had 39.44%, 84.5%, and 43.66% conservancy rates, respectively. as with the m41 ibv strains, most of these epitopes are shown to interact with the surface at varying threshold (yellow) as depicted in figure 2 . in m41 ibv strains, a total of 21 mhci peptides were predicted at different amino acid positions; however, 15 of these peptides were found to be antigenic at various conservancy rates (98.82-100%). three epitope peptides, ggpitykvm 208 , wfnslsvsi 356 , and yladaglai 472 relatively have high antigenicity index (>1.0) as compared to other peptides (table 3 ). in the case of cr88 ibv strains, 18 mhci epitope binding peptides were predicted. of these, 11 peptides were found to be antigenic with most of the epitopes having conservancy rate ranging from 52.11% to 94.37% except peptide occurring at position s69 which is conserved in only 8.45%. interestingly, wfnslsvsl 358 , synisaasv 88 , and ynisaasva 89 demonstrated high antigenicity index compared to other predicted peptides (table 4 ). the use of bioinformatic and/or in silico analyses to understand infectious diseases as well as to predict novel vaccine candidate has been recently extended to poultry [27, 28] . most of the epitopes responsible for virus neutralization have been mapped to be located within the first and third quarters of the linear s1 glycoprotein [13, 16] . the present study identified novel b-cells and t-cells epitopes presence in the s1 glycoprotein of m41 and cr88 ibv strains. predicted antigenic b-cell epitopes were found to be highly conserved and demonstrated strong transmembrane potentials which probably predicts regions of virus-cell interaction. linear bcells epitopes located within the s1 region have been reported to play a role in virus neutralization. using a phage display library, zou et al. [29] identified two neutralizing linear bcells epitopes within the s1 glycoprotein. recent studies have shown that chicken mhci genes are categorized into mhci associated genes (b-f) and mhcii (b-l) associated b-g genes, probably found only in poultry. remarkably, this study has identified 21 and 18 mhci binding peptides located within the s1 glycoproteins of m41 and cr88 ibv strains, respectively. chickens with mhc homozygous b12 and b19 are reported to be more susceptible to infection with ibv-grey strains as compared with b2/b2 and b5/b5 haplotype which often resist infection [30] . recently, tan et al. [21] used bf2 * 4, bf2 * 12, bf2 * 15, and bf2 * 19 chicken mhci haplotypes to predict 21 ctl-peptide candidates in massachusetts and australian t strains. the study revealed that the constructed poly-ctl-epitope dna vaccine was capable of inducing protection in 90% of the vaccinated chicken following challenge with sh1208 ibv strain. in a different study, tian et al., 2008 [31] have identified seven t-and b-cells epitopes within the ibv s1 and s2 and np proteins chimeric dna vaccine derived from these epitope was found to be associated with 80% protection rate. interestingly, the regions through which b-cells and t-cells epitope were predicted in this study correspond to the ibv receptor binding domain (rbd) which play a role in sufficient binding as well as entry of ibv viruses [32] . similarly, epitopes peptides found within rbd have been reported in different study to induce neutralizing antibody response, for example, against human coronavirus and mouse hepatitis virus [33, 34] . this study predicted novel antigenic b-cells and t-cell epitopes within the s1 glycoprotein of m41 and cr88 ibv strains. the characteristic antigenicity index as well as epitope conservancy rates demonstrates potentials of the identified epitope peptides as polyvalent synthetic or dna-based peptide vaccine for application in the control of ibv infection. the use of such vaccines will likely reduce the challenges associated with live attenuated vaccines and allow broad coverage of the target ibv strains. nidovirales: a new order comprising coronaviridae and arteriviridae coronavirus avian infectious bronchitis virus infectious bronchitis virus variants: a review of the history, current situation and control measures genetic and antigenic diversity in avian infectious bronchitis virus isolates of the 1940s detection of massachusetts and arkansas serotypes of infectious bronchitis virus in broilers characterisation of an infectious bronchitis virus isolated from vaccinated broiler breeder flocks proceedings of the angers 1eres journee de la recherche avicole, centre de congres d' angers reversion to virulence of chicken-passaged infectious bronchitis vaccine virus attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus pathogenesis and diagnostic approaches of avian infectious bronchitis progress and challenges toward the development of vaccines against avian infectious bronchitis priming with a dna vaccine and boosting with an inactivated vaccine enhance the immune response against infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry alleledependent association of chicken mhc class i molecules with the invariant chain structures of an mhc class i molecule from b21 chickens illustrate promiscuous peptide binding peptide motifs of the single dominantly expressed class i molecule explain the striking mhc-determined response to rous sarcoma virus in chickens the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens prediction and identification of t cell epitopes in the h5n1 influenza virus nucleoprotein in chicken the protective immune response against infectious bronchitis virus induced by multiepitope based peptide vaccines prediction and identification of novel ibv s1 protein derived ctl epitopes in chicken geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data predicting linear b-cell epitopes using string kernels predicting transmembrane protein topology with a hidden markov model: application to complete genomes development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines in silico prediction of epitopes in virulence proteins of mycobacterium tuberculosis h37rv for diagnostic and subunit vaccine design bioinformatics and evolutionary insight on the spike glycoprotein gene of qxlike and massachusetts strains of infectious bronchitis virus prediction of ctl epitope, in silico modeling and functional analysis of cytolethal distending toxin (cdt) protein of campylobacter jejuni two novel neutralizing antigenic epitopes of the s1 subunit protein of a qx-like avian infectious bronchitis virus strain sczy3 as revealed using a phage display peptide library association of the chicken mhc b haplotypes with resistance to avian coronavirus the immunoreactivity of a chimeric multi-epitope dna vaccine against ibv in chickens mapping of the receptorbinding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e the authors would like to thank the universiti putra malaysia and ministry of science, technology and innovation (mosti), project no. 02-01-04-sf1070 for funding the research. the authors declared no conflict of interests regarding the present publication. key: cord-255619-5h3l6nh6 authors: kuo, shu-ming; kao, hsiao-wei; hou, ming-hon; wang, ching-ho; lin, siou-hong; su, hong-lin title: evolution of infectious bronchitis virus in taiwan: positively selected sites in the nucleocapsid protein and their effects on rna-binding activity date: 2013-03-23 journal: vet microbiol doi: 10.1016/j.vetmic.2012.10.020 sha: doc_id: 255619 cord_uid: 5h3l6nh6 rna recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (ibv), a highly contagious avian pathogen. we have demonstrated that rna recombination can give rise to a new viral population, supported by the finding that most isolated taiwanese (tw) ibvs, similar to chinese (ch) ibvs, exhibit a genetic rearrangement with the american (us) ibv at the 5’ end of the nucleocapsid (n) gene. here, we further show that positive selection has occurred at two sites within the putative crossover region of the n-terminal domain (ntd) of the tw ibv n protein. based on the crystal structure of the ntd, the stereographic positions of both predicted selected sites do not fall close to the rna-binding groove. surprisingly, converting either of the two residues to the amino acid present in most ch ibvs resulted in significantly reduced affinity of the n protein for the synthetic rna repeats of the viral transcriptional regulatory sequence. these results suggest that modulating the amino acid residue at either selected site may alter the conformation of the n protein and affect the viral rna–n interaction. this study illustrates that the n protein of the current tw ibv variant has been shaped by both rna recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the rna-binding capacity of the n protein. among rna viruses, the coronavirus has the largest genome, consisting of a 27.7 kb, single-stranded, positivesense rna. the structural genes of the coronavirus genome encode spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. frequent point mutations in the hypervariable regions of the spike 1 (s1) gene contribute to most of the antigenic determinants of ibv. the n protein participates in binding to the viral rna genome and forms a ribonucleoprotein (rnp) complex. the n-and c-terminal domains (ntd and ctd) of the n protein are mainly involved in rna binding and oligomerization, respectively (jayaram et al., 2006) . recent evidence indicates that the ntd shows strong affinity for the transcriptional regulatory sequence (trs), which lies within the viral leader sequence and also precedes each viral open reading frame (spencer and hiscox, 2006) . this ntd-trs binding is critical for the control of viral life cycle, including viral packaging, genomic replication and viral transcription (grossoehme et al., 2009; hurst et al., 2010) . rna recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (ibv), a highly contagious avian pathogen. we have demonstrated that rna recombination can give rise to a new viral population, supported by the finding that most isolated taiwanese (tw) ibvs, similar to chinese (ch) ibvs, exhibit a genetic rearrangement with the american (us) ibv at the 5 0 end of the nucleocapsid (n) gene. here, we further show that positive selection has occurred at two sites within the putative crossover region of the n-terminal domain (ntd) of the tw ibv n protein. based on the crystal structure of the ntd, the stereographic positions of both predicted selected sites do not fall close to the rna-binding groove. surprisingly, converting either of the two residues to the amino acid present in most ch ibvs resulted in significantly reduced affinity of the n protein for the synthetic rna repeats of the viral transcriptional regulatory sequence. these results suggest that modulating the amino acid residue at either selected site may alter the conformation of the n protein and affect the viral rna-n interaction. this study illustrates that the n protein of the current tw ibv variant has been shaped by both rna recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the rnabinding capacity of the n protein. ß 2012 elsevier b.v. all rights reserved. in addition to maintaining the viral rna in an ordered conformation for replication and transcription, the n protein is also involved in the regulation of cellular transcription, actin reorganization and apoptosis (kopecky-bromberg et al., 2007; surjit et al., 2006) . recently, an antibody against the n protein was used as a diagnostic marker of coronaviral infection (mourez et al., 2007) . moreover, immunization of the n protein alone can elicit sufficiently protective cellular immunity against lethal ibv challenges, indicating that the n protein is an immune-dominant antigen (cowley et al., 2010) . both genetic mutation and recombination contribute to the natural evolution of rna viruses. genetic variants attributed to rna recombination are often found in coronaviruses such as mouse hepatitis virus (mhv) (keck et al., 1987) and infectious bronchitis virus (ibv) (cavanagh, 2007; dolz et al., 2008) . ibv is an important chicken pathogen that causes severe economic losses for global poultry industry (cavanagh, 2007) . genetic recombination in the ibv s1 and n genes has been documented in different field isolates, based on sequence comparisons and phylogenetic incongruence (cavanagh, 2007) . the high frequency of rna recombination in coronaviruses is likely caused by their unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (makino et al., 1986) . in addition to the sporadic generation of new variants, rna recombination may also give rise to new viral populations, as suggested by our study of taiwanese (tw) ibvs (kuo et al., 2010) . in general, tw ibvs are genetically closer to chinese (ch) isolates than to american (us) strains. the only exception is the n gene of the tw ibv, which shows higher similarity to that of us strains. in particular, recombination in the ntd of the n gene has been detected in most analyzed tw ibvs. this phylogenetic incongruence suggests that rna recombination can drive viral evolution at a population level. however, tw ibvs that carry the recombinant ntd may present the same antigenic epitopes, at least in part, as the us massachusetts-or connecticut-serotype vaccine strains, which are routinely administrated in chicken to control ibv in taiwan. this sequence rearrangement may therefore pose a survival disadvantage for the tw recombinants by increasing their susceptibility to vaccine-induced immune surveillance (chua et al., 2004) . whether this recombination event in the tw ibv enables the offspring of the recombinant founder to become dominant and evolutionally preserved is rather unclear. in this study, we show that tw ibv recombinants have undergone positive selection at two amino acids within the putative crossover region of the ntd. replacing either of these two residues with the amino acid present in most ch ibvs significantly attenuated the binding capacity of the n protein to synthetic rna repeats of the viral trs. these findings suggest that the fitness of recombinant tw ibvs has been increased by positive selection conferring a replication/ transcription advantage, even though these variants have been exposed to immune pressure in fowl vaccinated against us-like strains. detail information of the recruited ibv strains is provided in supplementary table 1 (table s1) , including the genbank accession number and the place where the strain was isolated. we analyzed 72 ibv strains, including 26 tw strains, 15 us strains and 31 ch strains. the criteria of recruiting the ibv strains based on the decoded both full-length s1 and n genes. china sd0611 strain is the only exception, whose s1 gene is still not sequenced. among the ibv strains, 8 ibvs in the same clade of me tree in fig. 1b were chosen from each tw, ch and us groups for the bi analysis and positive selection. tw2992/ 02 and 3374/05 were excluded because of their phylogenetic discordance in s1 and 3382/06 was excluded due to the discordance in n. the length of the tested genes is all the same. notably, tw2575/98 is completely sequenced and is the most studied strain in taiwan (kuo et al., 2010) . phylogenetic trees based on me algorithms on the fulllength s1 and full-length n genes were analysis by mega 4.0. bootstrap values, estimated from 1000 replicates of the me analysis, are given. for bi analysis, we compiled the n genes of various ibv strains, including a taiwanese group (tw2575/98, tw1171/92, tw2296/95, tw3374/05, tw2992/02, tw3071/03, tw97-4 and tp/64), an american group (mass 41, h120, cal99, cu-t2, beaudette, gray, connecticut and arkdpi) and a chinese group (ldt3, s14, bj, lx4, ck/ch/ltj/95i, ck/ch/lhb/96i, sh and sd0611). multiple sequence alignment was performed using the clustalw program. the dna sequences were translated into amino acid sequences using the software dambe 4.5.20. phylogenetic trees were constructed with the 24 amino acid sequences of the n genes by bi analysis. the best-fit models and parameters for initial settings of the phylogenetic programs were selected by prottest 1.2.6 for bi algorithms on the basis of the bayesian information criterion. for the ibv n 1-124 data set, the best-fit model of substitution was the jtt model with a gamma substitution parameter of 0.76. for the ibv n 125-409 data set, the best-fit model was the jtt model with a gamma substitution parameter of 0.96. mrbayes 3.1.2 was used for bi analysis. random starting trees were used. a total of 10 million generations of markov chains were run. trees were saved every 100 generations, resulting in 100,000 trees in the initial samples. the burn-in (number of initial trees that were discarded) was set to 25,000. a majority-rule consensus tree was generated from the remaining samples (75,000 trees), and the percentage of samples recovering any particular clade represented the clade's posterior probability. the best-fit model of gtr + i + g was selected for bayesian analysis by the program of modeltest 3.7 using akaike information criterion. in the analyses, 2,000,000 generations of markov chains were run. trees were saved every 100 generations, yielding initial samples with a total size of 20,000. the stationary phase of log-likelihood was reached within 500,000 generations. thus, burin-in (the numbers of the initial trees were discarded) was set to 5000. majority rule consensus trees, constructed from the 15,000 remaining trees, were used to determine the posterior probabilities of each node. genetic recombination was evaluated by the recombination detection program (rdp) 3.0 and the genetic algorithms for recombination detection (gard) program to predict the putative cross-over region and single breakpoint position. detailed information was provided in our previous study (kuo et al., 2010) . based on bi analysis of the 24 ibvs, variable selective pressures were evaluated at individual codon positions of the tw n protein. we applied paired models of variable d n / d s distribution among amino acid sites, including m3 (discrete) versus m0 (one ratio), m2a (positive selection) versus m1a (nearly neutral) and m8 (beta and d n /d s ) versus m7 (beta), in the codon-based phylogenetic models (codeml) program within phylogenetic analysis by maximum likelihood (paml) 4. the likelihood ratio test statistic was calculated as twice the log likelihood (l) difference between the two models and labeled as 2 (l 1 à l 0 ) in table 1 . the value was compared with a chisquare test with one degree of freedom (d.f.), which is equal to the difference in the number of free parameters between the compared models. a homology model of the ntd of the ibv n protein (tp/ 64) was built using the automated mode of the swiss-model program (arnold et al., 2006) . we used the crystal structure of the ntd of the ibv beaudette strain (pdb no. 2btl) as a template to construct a plausible conformation. the deduced models and template were superimposed, showing the overall structure with root mean square deviations of $1.35 å . model fidelity was confirmed using the procheck and adaptive poisson-boltzmann solver modules to examine the main-chain torsion angles and electrostatic distribution, respectively (laskowski et al., 1993) . the ibv n gene was cloned into pet-28a(+) (merck-novagen, usa) via bamh i and xho i sites to synthesize a recombinant ibv n protein with a histidine tag. mutagenesis of n gene was performed according to the instructions provided with the quikchange site-directed mutagenesis kit (stratagene, usa). the sense oligonucleotides used for mutagenesis are as follows: 5 0 -gat aat gaa aat ctt aaa cca agc cag cag cat gg-3 0 , thr to pro at aa 64; 5 0 -cct gat aat gaa aat ctt aaa aat agc cag cag cat gga tac tgg-3 0 , thr to asn at aa 64; 5 0 -gct gca aag ggt gct gat gtt aaa tct aga tct aat c-3 0 , thr to val at aa 123. these mutants were confirmed by nucleotide sequencing. the pet-28 plasmid containing the full-length n protein was transferred into e. coli bl21(de3) competent cells (merck-novagen), and the transformed bacteria were selected and cultured at 37 8c in lb broth containing 25 mg/ml kanamycin. n protein expression was induced by adding 1 mm iptg for 4 h when the od 600 value reached 0.4-0.6. to prevent protein degradation, the induction condition was carried out at 25 8c and supplemented with cocktailed protease inhibitors (sigma-aldrich). the cells were collected, suspended in lysis buffer (50 mm sodium monobasic phosphate, 300 mm sodium chloride, 1 mm imidazole, 1 mg/ml lysozyme) with protease inhibitor and then sonicated. the crude cell lysates were applied to resin affinity columns through a fast protein liquid chromatography (fplc) system (akta prime plus, amersham pharmacia) with buffer containing 8 m urea. resinprepacked columns (amersham pharmacia) were equilibrated with a buffer consisting of 0.1 m nah 2 po 4 , 0.01 m tris-hcl, 8 m urea, and 1 mm imidazole (ph 8.0) and then washed with a buffer consisting of 0.1 m nah 2 po 4 , 0.01 m tris-hcl, 8 m urea, and 10 mm imidazole (ph 6.3). n protein fractions were eluted with a buffer containing 0.1 m nah 2 po 4 , 0.01 m tris-hcl, 8 m urea, and 250 mm imidazole (ph 4.5) at a flow rate of 2.0 ml/ min. purified proteins were further dialyzed and refolded with refolding buffer (50 mm tris-hcl, 50 mm nacl, 0.4 m l-arginine, 1 mm edta, 0.2 mm phenylmethanesulfonyl fluoride, 0.5 mm oxidized glutathione, 5 mm reduced glutathione, ph 8.0). samples were slowly refolded at 4 8c for four days as the urea concentration was lowered from 4 m to 0 m using a dialysis membrane with a molecular cutoff 30-60 kda (millipore). concentrations of the purified n proteins were determined by the bradford assay (bio-rad, usa). in addition, the n proteins were examined by 10% sds-page and stained with coomassie brilliant blue r250 (usb-affymetrix). the binding capacity of n proteins for viral rna was performed on a biacore 3000a spr instrument (amersham pharmacia) equipped with a research-grade sensorchip sa5. this apparatus measures binding capacity by monitoring changes in the refractive index of the sensor chip surface. these changes, recorded in resonance unites (ru), are assumed to be proportional to the mass of the molecules bound to the chip. oligomers of the repeated trs sequence, 5 0 -(cuuaacaa) 4 -3 0 , were synthesized using an automated rna synthesizer, labeled with biotin and purified by gel electrophoresis. the oligomer probes were manually immobilized to the streptavidin-coated biosensor chip. the purified n proteins were dissolved in a solution consisting of 50 mm tris-hcl, 50 mm nacl, 1 mm edta, 0.5 mm oxidized glutathione, and 5 mm reduced glutathione at ph 7.3. the protein was applied to the chip surface at a flow rate of 30 ml/min for 140 s to reach equilibrium. before fitting to the 1:1 langmuir model, binding data were corrected by subtracting the control to account for simple refractive index differences. sensorgrams depicting interactions between rna and n proteins were obtained using bia evaluation 3 software (version 3). one-way analysis of variance (anova) was performed to determine significant differences in n protein rnabinding capacity in three independent experiments. the tukey's post hoc test was further used for multiple comparisons among the data collected for the wild type and variant n proteins. statistical significance was set at p < 0.05. we analyzed 72 ibv strains from taiwan, china and us in this study. the available full-length s1 and n gene sequences were the criteria for recruiting these strains (genbank, accessed on april 13th, 2011). based on a minimum evolution (me) analysis using mega 4.0, the phylogenetic topology of the full-length s1 gene revealed that the most tested tw ibvs are similar to the ch ibvs (fig. 1a) . the tw3374/05 and tw2992/02 strains were the only exceptions, showing greater similarity to the us group. in contrast, analysis of the full-length n genes of the ibv strains by me ( fig. 1a and b) and baysian analyses (supplementary fig. s1 ) demonstrated that the tw ibvs are phylogenetically closer to the us ibvs than to the ch ibvs (fig. 1b) , indicating phylogenetic incongruence between the results for the s1 and n genes. twenty-four strains with complete sequences for the n gene were randomly chosen from the three tw, us and ch viral pools and subjected to phylogenetic analysis on the n protein. putative sporadic recombinant strains, such as tw3374/05 and tw2992/02, are excluded to avoid misinterpretation of further analytic results. one candidate crossover region between the tw and us ibv strains is located between amino acid (aa) 1 and 124 of the n protein (kuo et al., 2010) , as predicted by the recombination detection program (rdp) (martin and rybicki, 2000) used in our previous study (fig. 1c) (kuo et al., 2010) . the p values of the rdp and bootscan algorithms for this recombination were 5.1 â 10 à4 and 1.0 â 10 à5 , respectively (kuo et al., 2010) . because aligning the n genes of severe acute respiratory syndrome (sars) virus, human coronavirus-oc43 (hcov-oc43) and mhv with ibv n sequences gives many gaps across these sequences, an ancestor strain as an outlier sequence was absent in fig. 1a and b (data not shown) . to further confirm our hypothesis of rna recombination, unrooted bayesian inference (bi) algorithm was applied in this study (fig. 1d and e) . bi analysis revealed that the putative crossover region (fig. 1d) in the tw n protein, but not the non-recombinant region (fig. 1e) , belongs to the monophyletic group of us n proteins. moreover, to demonstrate that the trees of fig. 1d and e are representative, we included all the strains in fig. 1b and constructed their bayesian trees (fig. s2) according to the two segments of n-terminal and c-terminal amino acid sequences of ibv n protein (fig. 1c) . the results in fig. s2 illustrated similar topologies as those in fig. 1d and e, strongly supporting the presence of rna recombination between the tw and us ibvs in the n gene sequence (fig. 1d and e and fig. s2 ). to further clarify whether the phylogenetic incongruence is caused by different evolutionary rates in different parts of the n gene, a ml test that is not biased by evolutionary rate variation was applied to recheck the phylogenetic relationships (holmes and rambaut, 2004) . no significant difference was observed between the results from the bi and those from the ml test (supplementary fig. s3 ), confirming that rna recombination probably occurred between tw and us ibvs in the 5 0 -terminal region of the n gene and that the phylogenetic incongruence was not caused by point mutation or variation in local evolutionary rates. although the n protein is evolutionally conserved among ibvs, positive selection may occur at individual amino acid (aa) residues. to investigate this possibility, full-length n genes of the 24 ibvs (shown in fig. 1d and e) were subjected to codon-based phylogenetic models (codeml) within the phylogenetic analysis by maximum likelihood (paml) programs (yang, 2007) . based on bayesian analyses, neutral and positive selection models were compared using likelihood ratio tests. the neutral models (m0, m1a and m7), selection models with a proportion of selected codons (m2a and m8) and a model for d n /d s heterogeneity among aa residues (m3) were applied for tests of selective pressure on n protein residues. the log likelihood values (l n in table 1 ) indicated that positive selection models (m2a, m3, m8) fitted the tested region better than neutral models (m0, m1a, m7). the nested comparisons between neutral and positive models, including m0 versus m3 (m0/m3), m1a/ m2a and m7/m8, confirm the better fitness of positive models, suggesting that positive selection occurs at certain sites during the evolution of the tw n protein (p < 0.001 in these three comparisons, chi-square test) (table 1 ). both aa 64 and aa 123 of the tw n protein were consistently highlighted by positive selection models (m2a, m3, m8) as putative selection sites with high posterior probability values (p a and p b values of both sites >0.95 in m3 and m8 models, respectively) ( table 1) . the detail profile of these two positively selected sites of all tested ibvs in this study was summarized in table 2 . using the available database of fully sequenced n proteins (72 ibv strains), we determined that thr residues are present at the aa 64 and 123 positions in 80.8% (21/26) and 65.4% (17/26) of the tw ibv strains, respectively (table 2) . however, thr residues are present at the aa 64 and 123 positions in only 46.7% (7/15) and 33.3% (5/15) of the us ibv strains, respectively. in the ch ibvs, the prevalence of thr residues at these two positions is further reduced to 29.0% (9/31) and 22.6% (7/31), respectively. this information supports the codeml prediction that both positions have undergone positive selection in tw ibvs. to further demonstrate the result of positively selected residues in table 1 (24 ibv strains), 58 ibv strains in table 2 were recruited and few strains with partial sequences were excluded for codeml analysis. as shown in table s2 , both aa sites at 64 and 123 are predicted to be positively selected using m8 model (probability > 0.95), strengthening the finding of the occurrence of positive selection at aa 64 and aa 123 of n protein among tw ibvs. notably, the selected sites are located within the putative crossover region of the n protein in the tw ibvs (fig. 1c) , linking the rna recombination with the positive selection events. this observation also reflects the notion that particular residues of tw ibv recombinants have evolved under positive selection pressure in vaccinated flocks. in addition, the putative positively selected residues are located in the ntd, suggesting that viral progeny with strong rna-binding affinities may have been selected during the adaptive evolution of ibv in taiwan. the ntd of the ibv n protein participates in the binding to viral rna and the formation of the rnp complex. the crystal structure of the ntd, based on the beaudette strain, shows a u-shaped conformation composed of a fivestranded antiparallel b sheet with positively charged amino acids clustered throughout the groove (fan et al., 2005) . flexible loops and turns are around the inner core of the b sheet of the ntd. the positively selected sites, aa 64 and 123, are located in the external a turn and loop, respectively (arrow heads, fig. 2a) . notably, neither site is close to the rna-binding groove (arrow, fig. 2a ). the locations of these two residues indicate that they do not directly participate in viral rna binding. software predictions by rnabindr (terribilini et al., 2007) , including ensemble, pssmseq and pssmstruct algorithms, also support this observation (data not shown). regarding the aa 64 position, pro is present in the beaudette model strain, and thr is present in the tw tp/64 strain. interestingly, replacing pro with thr at residue 64 transforms the a-turn conformation into a looped structure after protein modeling (fig. 2b) , indicating the structural flexibility of this ntd residue. this result also suggests that the epitope character of this region of the tw t t bj aap92682 p v arkdpi aax39764 t v tw2296/95 aat39490 t t ck/ch/lhb/961 abc02826 s v beaudette aaa70242 p t tw2575/98 abg36794 strains in gray are recruited for bi and codeml analyses. ibv n protein may be vulnerable to alteration under immunological pressure. for the other selected site, aa 123, where a thr is present in the tw tp/64 strain and a val is present in the beaudette strain, no obvious conformational change was noted after homology modeling. nevertheless, a single mutational change from a hydrophilic amino acid (thr) to a hydrophobic one (val) could modulate the surface charge of the protein and its efficiency at forming high-order oligomers (fan et al., 2005) . to investigate the importance of the selected amino acid residues, the tw tp/64 strain, which was the first ibv strain to be identified in taiwan in 1964, was chosen to represent wild type tw ibv and subjected to an analysis of rnabinding activity. the tp/64 strain shares high genetic similarity to most tw ibv isolates (fig. 1a and b and table 2 ). three n protein mutants, including t64p (aca to cca, thr to pro), t64n (aca to aat, thr to asp), and t123v (act to gtt, thr to val), were generated by site-directed mutagenesis and confirmed by sequencing (fig. 3a) . the production of full-length n protein of tp/64 (arrow head in fig. 3b ) was successfully induced by adding isopropyl b-d-1-thiogalactopyranoside (iptg) in e. coli (fig. 3b) . the n protein of tp/64 (wild type, wt) and the mutant n proteins (t64p, t64n, t123v) were purified by ni-column and further examined by the staining with coomassie blue in sds-page (fig. 3c) . the detected size of full-length n protein was around 57 kda ( fig. 3b and c) , higher than the theoretical prediction (45 kda). the upper shift of band location of expressed n protein might be caused by the intrinsic charged aa components of the n protein but not post-translational modification in e. coli due to the same detected molecular weight (mw) of n protein produced by an in vitro transcription and translation system (fig. s4 ). in addition, this upper shift of the n protein in sds-page were reported in a previous ibv n protein study (yu et al., 2010) and other coronaviral n proteins (hurst et al., 2009 (hurst et al., , 2010 . the rna-binding capacity of the n protein was determined by surface plasmon resonance (spr) analysis. previous studies showed that the coronaviral n protein has a high affinity for the trs sequence (grossoehme et al., 2009) . a repeated trs sequence has been used as a probe in the spr experiments to measure the interaction of the n protein with viral rna (huang et al., 2009; nelson et al., 2000) . the applied viral rna probe consisted of repeated ibv trs sequences, 5 0 -(cuuaa-caa) 4 -3 0 and was biotin-labeled. one hundred resonance units (ru) were immobilized onto a streptavidin-coated biosensor chip for detecting the binding capacity of purified ibv n proteins. compared to the wild type n protein, all three mutants showed significantly reduced trs-binding capacity at 0.1 and 0.5 mm protein concentrations ( fig. 4a -c,) (p < 0.01, one-way anova, tukey's post hoc analysis). when the protein concentrations were elevated from 0.05 mm to 0.5 mm, proportional increases in their rna-binding capacities were detected (fig. 4c) . the t64p variant, which mimicked the 64 aa position of us beaudette strain, showed about half the rna-binding capacity of the wild type tw n protein (fig. 4c ) (p < 0.01, one-way anova, tukey's post hoc analysis). likewise, the t64n and t123v variants, which represented the most common configurations of aa 64 and aa 123 in the ch n protein (52.2% and 91.3%, respectively) ( table 2) , showed only 30-40% of the rna-binding capacity of the tw tp/64 strain (fig. 4c ) (p < 0.01, one-way anova, tukey's post hoc analysis). the anova analyses also revealed that the rna-binding activity between the pairs of mutants (t64p, t64n and t123v) has no significant difference. taken together, these results indicate that both aa 64 and 123 are critical for 5 0 -(cuuaacaa) 4 -3 0 binding, and the modulation of these two residues may affect the binding affinity of the n protein for viral genomic or subgenomic rna. while the ibv n protein has generally been conserved and negatively selected during viral evolution, we have identified two positively selected sites at aa 64 and 123 in the n protein of ibvs. these two residues are located in the putative recombinant region of the ntd domain and are critical for binding to trs repeats. to our knowledge, this is the first report on viral evolution linking an rna recombination event with positive selection. this study also provides the first functional assay to illustrate the importance of positively selected sites in the n protein for coronaviral rna binding. sporadic genetic recombination in the sequences of interests will change branch lengths and the topology of phylogenetic tree (yang et al., 2000) . these two parameters are assumed to be constant across the tested sequences for the analyses of codeml, especially when the positive selection is evaluated by branch-site method based on a likelihood ratio test (scheffler et al., 2006) . to avoid false result of the positive selection in this study, the possible recombinant ibv strains, such as tw3374/05, tw2992/02, tw3381/06 and tw3382/06, are excluded ( fig. 1a and b ) and the selected strains are chosen from the population in a same clade. in addition, the candidate of positive selected sites (table 1) is evaluated by a codon-substitution model but not a branch-site likelihood model. finally, the fidelity of the mathematic prediction by codeml is further validated by the experimental function assay, demonstrating that the positively selected sites are critical for the viral rna-binding activity. in addition to forming part of the rnp, the coronaviral n protein participates in the formation of the replicationtranscriptional complex. specifically, the n protein's ntd binds to the trs of the leading sequence and regulates trs-ctrs (complementary trs) helical unwinding (grossoehme et al., 2009) , suggesting its critical role in genomic duplication and subgenomic expression. in this study, we showed that substituting either of the positively selected residues with the amino acid present in most ch ibvs dramatically reduced the binding capacity of the n protein for synthetic trs repeats (fig. 4) . although neither selected site is located in the rna-binding groove of the ntd, the modified residues may alter the secondary structure or surface charge distribution of the n protein and consequently affect rna-ntd interactions. here, phylogenetic evaluation of viral proteins not only helps us to reconstruct the evolutionary paths of viral species but also provides new insights into functional residues in viral proteins. during viral propagation, variants with enhanced cellular tropism, viral transmission or replicative advantage show enhanced fitness in infected hosts (domingo and holland, 1997) . amino acid mutations in human immunodeficiency virus (hiv) gag (banke et al., 2009 ) and pol genes (huang et al., 2002) , which are mainly involved in genomic duplication, were positively selected in patients and conferred fitness under the pressure of anti-hiv drug treatment. in addition, positively selected residues in capsid proteins have been reported in rabbit hemorrhagic disease virus (esteves et al., 2008) , hepatitis c virus (kurbanov et al., 2010) and foot-and mouth disease virus (haydon et al., 2001) . however, the importance of these selected sites has not yet been functionally evaluated. in this study, spr binding assays of point-mutant ibv n proteins demonstrated that the positively selected residues in the tw ibv n protein may improve binding efficiency for viral trs repeats. sophisticated approaches using viral replicons or recombinant infectious clone should further illuminate the detailed roles of these selected sites in coronaviral propagation. given that vaccine-based immunization imposes strong selective pressure on viral evolution, we did not rule out the possibility that the avian immune system has reshaped the recombinant n protein of tw ibv through positive selection. both s and n proteins are known to be major antigenic determinants of ibv (cavanagh, 2003) . administration of n proteins via intraperitoneal injection can elicit protective adaptive immunity against an ibv challenge (cavanagh, 2003) . we speculate that the tw ibv, sharing epitopes in the ntd of the n protein with the us-serotype ibv because of an rna recombination event, may have become more vulnerable to attack by the adaptive immune system in fowl vaccinated against the connecticut or other us-like ibv strains. to counteract this adverse effect, it is possible that residues in the a-turn (aa 63-67) and surrounding peptides located in an externally exposed loop (or a turn) have been positively selected to attenuate antigenic recognition by host b lymphocytes. this speculation is supported by the fact that almost all the test tw ibvs were isolated from vaccinated flocks and were under immune selection pressure. in addition, a recent study on mhv infection, which emphasized that epitope-escape coronaviral strains can be quickly selected by genetic deletion or mutation under strong immunological pressure (chua et al., 2004) . in addition, it has been suggested that the a-turn region (aa 63-67) may form an antigenic epitope in the ibv n protein (ignjatovic and sapats, 2005) . future works will aim to determine whether the mutations at aa 64 and 123 of the n protein in adapted strains result in altered epitope determinants and consequently provide competitive benefits for quasispecies in hosts by allowing the selected tw strains to escape immune surveillance. taken together, our data support the conclusion that positive selection has occurred in specific residues of the recombinant ibv n protein. this selection may promote the viral fitness in infected hosts, at least in part, by modulating the rna-binding capacity of the n protein's ntd. diagram illustrating the proposed evolution of ibv in taiwan is provided in fig. 5 . further investigation is required to illustrate the details that how the recombinant configuration of the n gene in the original founder was maintained and consequently fixed in the current tw ibv population. the swiss-model workspace: a web-based environment for protein structure homology modelling positive selection pressure introduces secondary mutations at gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus effects of an epitope-specific cd8+ t-cell response on murine coronavirus 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analyzing and predicting rna-binding sites in proteins paml 4: phylogenetic analysis by maximum likelihood codon-substitution models for heterogeneous selection pressure at amino acid sites a novel b-cell epitope of avian infectious bronchitis virus n protein we are grateful to dr. drena dobbs and michael terribilini at iowa state university for supplying rnabindr rna-binding predictions for the ntd of the tw ibv n protein. this work was supported by national science council in taiwan (nsc 99-2321-b-005-012-my3). this work was also supported in part by the ministry of education, taiwan, r.o.c. under the atu plan. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2012.10.020. key: cord-285942-mb1xwdqw authors: feng, k. y.; chen, t; zhang, x; shao, g. m.; cao, y; chen, d. k.; lin, w. c.; chen, f; xie, q. m. title: molecular characteristic and pathogenicity analysis of a virulent recombinant avain infectious bronchitis virus isolated in china date: 2018-10-01 journal: poultry science doi: 10.3382/ps/pey237 sha: doc_id: 285942 cord_uid: mb1xwdqw abstract a virulent infectious bronchitis virus (ibv), designated as ck/ch/gd/qy16 (referred as qy16), was isolated from a diseased chicken farm in guangdong province, china, in 2016. the complete genome of the strain was sequenced and analyzed. the results show that the genome of qy16 consists of 27,670 nucleotides, excluding poly (a) tail, and that its genome organization is 5’ utr-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3’ utr-poly (a) tail. sequence comparison among qy16 and other ibv strains was conducted and its results demonstrate that the s1 gene of qy16 has the highest nucleotide sequence identity with that of 4/91, and the other part of its genome is highly similar to that of yx10. the results of the phylogenic analysis show that the entire genome of qy16 and most of the qy16 genes are located in the same cluster as those of yx10, except for the s1 gene which is located in the same cluster with that of 4/91. it has been further confirmed by the rdp and simplot analysis that qy16 is a recombinant strain deriving from yx10 (as the major parental sequence) and 4/91 (as the minor parental sequence), and that the recombination occurs in a region which includes the 3’-terminal 1b sequence (85 nt) and the 5’-terminal s1 protein gene sequence (1,466 nt). the results of the vaccination-challenge test suggest that qy16 is a nephropathogenic strain of ibv and that the vaccine strains–h120 and 4/91—cannot provide effective protection against it. these results indicate that the continuing evolution of ibv strains by genetic drift and genetic recombination may lead to ibv outbreaks even among the vaccinated chickens in china. avian infectious bronchitis (ib) is an acute and highly contagious disease caused by the avian infectious bronchitis virus (ibv). it has a major impact on the poultry industry worldwide and causes severe economic losses. ibv affects chickens of all ages and varieties, and causes upper respiratory diseases, nephritic syndromes, egg production decrease, and secondary bacterial infections (yu et al., 2001) . ibv belongs to the genus coronavirus of the family coronaviridae in the order nidovirales. its viral genome consists of a linear, non-segmented, positive-sense, and single-stranded rna of approximately 27.6 kilobases (kb), the genome of ibv contains 6 genes (gene 1 to 6) and can be transcribed into a nested set of mrnas (mrna 1 to 6) by a unique discontinuous transcription mechanism of coronavirus (sawicki and sawicki, 1998) . the gene 1 encodes 2 polyproteins-polyprotein 1a (pp 1a) and polyprotein 1ab (pp 1ab), which is an extension of pp 1a by a minus 1 frameshift translation mechanism (brierley et al., 1987) . the 2 polyproteins can be cleaved by viral proteases into 15 non-structural proteins (nsp) that participate in the genome replication, transcription, and viral infection (hodgson et al., 2004; armesto et al., 2009 ). the gene 2 encodes a structural protein spike (s) glycoprotein that can be translated into a precursor spike glycoprotein (s0) and later be cleaved into 2 subunits s1 and s2 by cellular proteases (cavanagh et al., 1992) . the s1 subunit forms the tip of a spike, whereas the s2 subunit anchors the s1 to the viral membrane. the s1 subunit, with virus neutralizing epitopes and serotype-specific sequences (cavanagh, 1983; cavanagh et al., 1986) , plays an important role in viral attachment to host cells and the 3519 induction of neutralizing antibodies (koch et al., 1990) . the gene 3 encodes nsp 3a and 3b, and a structural protein envelope protein (e), whereas the gene 4 encodes a structural protein membrane glycoprotein (m) and nsp 4b and 4c. the gene 5 encodes nsp 5a and 5b. the m and e are 2 membrane-binding proteins, whereas the 4 nsps (3a, 3b, 5a, and 5b) have been proved to be dispensable for virus replication but affect the efficiency of the replication and immunogenicity of the virus (hodgson et al., 2006) . the gene 6 encodes a structural protein nucleocapsid protein (n) and a nsp 6b. the n protein has the ability to bind the ribonucleoprotein generated by the viral rna of ibv and plays a key role in viral replication and assembly as well as in cellular immunity (ignjatovic and galli, 1994; ignjatovic and galli, 1995) . the nsp 4b, 4c, and 6b have rarely reported in literature although they were present in most ibvs and turkey coronavirus (tcov) (cao et al., 2008; gomaa et al., 2008; thor et al., 2011; xue et al., 2012; reddy et al., 2015) . although ibv vaccines have been used worldwide in most of the commercial chickens, ib still breaks out frequently and causes severe production problems. there are several reasons for the difficulties in preventing and controlling ibv. firstly, the viral rna-dependent rna polymerase (rdrp) of ibv is not able to proofread its rna, leading to rna insertion, deletion, and substitution in the process of rna replication. secondly, due to the unique template switch during discontinuous rna synthesis in ibv transcription, the nature recombination of the viral rna frequently occurs among different strains (zuniga et al., 2010) . in addition, there were at least 30 serotypes of ibv identified worldwide, yet most of the available ibv vaccines can hardly provide effective cross-protection against strains of different serotype (cook et al., 2012) . as such, variants of the virus that varies in pathogenicity or serotype incessantly emerge, leading to different outbreaks of the disease. in china, ibvs have been identified since 1982 and have struck all provinces of the country (han et al., 2011) . in previous studies, the phylogenetic analysis of the s1 gene of ibv indicates that the strains of ibv isolated in china could be divided into at least 7 genotypes including qx-type, 4/91-type, tw-type, masstype, hn08-type, ldt3-type, and tc07-2-type (feng et al., 2014) . furthermore, there are ibv variants isolated as a result of gene recombination whose serotypes or pathogenicity may be different from any one of the parental strains (feng et al., 2017) . despite the extensive use of attenuated or inactivated ibv vaccines (cook et al., 2012; jackwood, 2012) , ib still breaks out frequently even among the vaccinated flocks. it can be attributed to the low efficacy of the current vaccines against the emergence of new serotypes or antigenic variants of ibv in china. in this study, we sequenced the complete genome of a novel virulent ibv strain ck/ch/gd/qy16 (hereafter referred to as qy16). sequence comparison, phyloge-netic analysis, and recombination analysis were carried out to compare qy16 with other reference strains including. an animal experiment was conducted to explore the efficacy of 2 vaccines in protecting the chickens against the new strain. the results of this study present a valuable insight into the evolution of ibv in china, give a better understanding of the virus at the molecular level, and demonstrate the needs to improve the efficacy of the vaccines against the ibv infection in the poultry industry. the ibv ck/ch/gd/qy16 strain (referred as qy16) was isolated from the kidneys of ibv-infected yellow feather broilers in a farm in qingyuan city guangdong province, china, in november 2016. the infected chickens were mainly less than 30-day-old, and typical clinical sign was nephritis that caused 30% mortality rate were identified. the specimens were frozen and thawed 3 times before treated with phosphatebuffered saline containing 200 u/ml penicillin and 200 μg/ml streptomycin and then centrifuged at 10,000 × g for 5 min. after keeping in 4 • c for 3 h, the viruses were propagated through blind passage 5 times. during each blind passage process, 9-day-old embryos of specific pathogen-free (spf) chickens were inoculated with 0.2 ml of the supernatant (first blind passage) or allantoic fluid (second to fifth blind passage) via the allantoic cavity. after incubation at 37 • c for 48 h post-inoculation, the allantoic fluids were harvested and stored at -70 • c. the virus in the eggs after the fifth passage was selected for genome sequencing, pathogenicity, and vaccination-challenge test. the virus isolation and follow-up experiments were carried out in biosafety level 2 biocontainment laboratory. the genomic rna of the strain was extracted through using the axyprep body fluid viral dna/rna miniprep kit (axygen, china) according to the manufacturer's instructions. reverse transcription (rt) was performed using primescript ii 1st strand cdna synthesis kit (takara, japan) following the manufacturer's protocol. polymerase chain reaction (pcr) was conducted using a set of primers. the s1 gene of the stain was amplified using a sense primer 5 -aag act gaa caa aag acc gac t-3 and an antisense primer 5 -caa aac ctg cca taa cta acat a-3 (supplementary table s1 ). the size of the amplified segment was about 1,760 bp, including the entire s1 gene and the protease cleavage motif. the complete genome sequence of the strain, excluding the 5 and the 3 terminal, was amplified as 22 overlapping genome fragments using ibv consensus primer sets (supplementary table s1) designed based on the alignment of the genome nucleotide sequences of ibv strains. pcr was conducted in a 50 μl reaction volume containing 2 μl of cdna, 2 μl of the specific primer (10 pmol of each), 21 μl of dh 2 o, and 25 μl of primestar max premix (2×) (takara, japan). the thermocycler was set at 35 cycles. during the process, the cdna was desaturated at 98 • c for 10 s, annealed at 55 • c for 5 s, and extended at 72 • c for 20 s. after the 35 cycles, a final 10 min dna extension step was conducted at 72 • c. the 5 and 3 rapid amplification of cdna ends (race) was performed using designed race primer (supplementary table s1 ) and the smarter race 5 /3 kit (clontech) following the manufacturer's instructions. the pcr products were tested by electrophoresis in 1% agarose gels and purified by using the axyprep dna gel extraction kit (axygen, china). the gelpurified pcr products were ligated to pcloneez topoisomerase cloning vector (clonesmarter) and transformed into dh5a escherichia coli competent cells (takara, japan). cells carrying the recombinant plasmid were selected on luria-bertani agar plates containing ampicillin (100 μg/ml). positive clones were screened by pcr following the same steps as the abovementioned pcr amplification. three positive clones were sequenced by sang-gong technology & services co., ltd (shanghai, china). the obtained 24 overlapped nucleotide sequences of the strain (including 5 and 3 untranslated region [utr]) were assembled into a complete genome sequence using seqman program in the lasergene package (dnastar, madison, wi). comparison and analysis of the genome sequences, open reading frames (orfs), and the deduced proteins of the strain and the reference ibv strains were conducted using editseq and megalign programs also in this package. after aligning the genome sequence of the strain with the reference ibv sequences using the clustalw multiple alignment algorithm, phylogenetic analysis of the nucleotide sequences of the strain's complete genome and specific genes was run with the neighbor-joining method using mega version 7.0 (www. megasoftware.net). the bootstrap values were determined from 1,000 replicates of the original data. the putative recombinant sequence of the strain and its parental strains were detected by using recombination detection program (rdp) software version 4.80 with the recombination detection methods (rdp, geneconv, bootscan, maxchi, chimaera, and siscan). the detection of recombination breakpoints through at least 5 of these methods was taken as confirmatory for any putative recombination event. the potential recombination events and breakpoints were further verified by simplot version 3.5.1. the nucleotide identity comparison was carried out using the kimura (2-parameter) method with a transition-transversion ratio of 2, and the window width and step size were 200 and 20 bp, respectively. forty 3-day-old spf chickens were divided into 4 groups of 10 and kept in 4 isolators with negative pressure. food and water were provided ad libitum. chickens in group 2 and 3 were vaccinated oculonasally with commercially available h120 and 4/91 vaccines, respectively, with a dose of 10 3.5 eid 50 per chick. chickens in group 1 and the control group were mock inoculated with sterile allantoic fluid. at 14 d postinoculation (dpi), chickens in groups 1, 2, and 3 were challenged with strain of qy16 by the oculonasal administration with a dose of 10 5.0 eid 50 per chicken, whereas those in the control group were mock inoculated again with sterile allantoic fluid. all chickens were observed daily to track the morbidity and mortality among them for 20 d after challenge. dead chickens were dissected for the observation of their organs. blood samples from all the chickens in each group were collected at 7 and 14 d post-vaccination and at 7, 14, and 20 d post-challenge, and the serum was stored at -80 • c before the test. the serum samples were assayed at a single dilution using a commercial enzyme-linked immunosorbent assay designed for the detection of total antibodies (idexx corporation) according to the manufacturer's instructions and the seropositivity rates were calculated. cloacal swabs collected from all the chickens in each group at 7, 14, and 20 d post-challenge were used for virus recovery. each swab sample was prepared as 10% w/v tissue suspension in phosphate-buffered saline, clarified by centrifugation at 12,000 g at 4 • c for 10 min, and filtered through 0.22 mm membrane filters (millipore). three 9-day-old spf chicken embryos were inoculated with 0.2 ml filtered supernatant of each sample. the inoculated eggs were incubated at 37 • c and were candled daily. the allantoic fluid was collected from 2 of the inoculated embryos 72 h postinoculation. detection of the virus was performed by extracting rna from the allantoic fluid and amplifying s1 gene by rt-pcr. the rt-pcr procedure uses the primescript one step rt-pcr kit (takara, japan) according to the manufacturer's instructions. the rt-pcr products were analyzed on 1.0% agarose gels. forty-four representative ibv strains published in genbank were selected for the sequence and phylogenetic analysis of the s1 gene of ibvs, and 16 ibv strains were used for the complete genome and specific genes (besides s1 gene) alignment, sequence comparison, phylogenetic, and recombination analysis. the accession numbers of these reference strains are listed in supplementary table s2 . the s1 gene of qy16 is composed of 1,617 nt with cleavage motifs his-arg-arg-arg-arg (hrrrr). basic local alignment search tool was used to compare the nucleotide and protein sequences of qy16 with the sequence databases. the s1 gene of qy16 has the highest nucleotide identity and amino acid (aa) identity with that of 4/91 (97.5% and 96%, respectively). the s1 gene of qy16 was compared to that of 44 available representative ibv strains and the nucleotide identity range from 60 to 97.5%. the qy16 showed high-sequence identity of s1 gene with 4/91-type strains (4/91, ta03, 7/93, ck/ch/lhlj/140,906, and ck/ch/lhb/130,630), and the nucleotide identity ranges from 95 to 97.5%, whereas the amino acid (aa) identity ranges from 94 to 96%. the nucleotide identities between the s1 gene of qy16 and those of the vaccine strains used in china including h120, h52, and ldt3-a are respectively 78.7%, 77.6%, and 85%, whereas the aa identities between them are respectively 76%, 76%, and 84.3%. the qy16 showed lowest sequence identity of s1 gene with strain de072 and the nucleotide identity is only 60%. phylogenetic analysis was conducted using the nucleotide sequences of the full s1 gene of qy16 and those of 44 reference strains ( figure 1 ). it is shown that the chinese strains can be divided into 6 genotypes according to the previous studies (feng et al., 2017) . the strain qy16 clusters to the 4/91-type that includes reference strains such as 4/91, ta03, and 7/93, and distinct from other genotypes. the complete genome sequence (including the 5 -and 3 -terminal segments) of qy16 was obtained through assembling 24 overlapping sequences via the dnastar software. the complete genome sequence has been submitted to genbank under the accession no.mg197727. the complete genomic sequences of qy16 are comprised of 27,670 nt, excluding poly (a) tail, and there are also 6 different genes and 13 orfs flanked by 5 and 3 utrs. the genome organization and location is as follows. gene 1 has 19,892 nt, consisting of 2 overlapping orfs, orf1a (11,859 nt) and orf1b (7,959 nt), and it encodes 2 polymerase proteins including1a and 1ab, which can be incised into 15 nsps and are related to rna replication and transcription. the predicted papain-like proteinase (plpro) and 3c-like proteinase (3clpro) cleavage sites that define the nsp2 to 16 boarders are shown in table 1 . there are 50 nt overlaps between gene 1 and gene 2. with a length of 3,498 nt, gene 2 has a single orf, and it encodes a spike (s) glycoprotein that is cleaved into 2 subunits s1 and s2, which are with 1,617 and 1,878 nt, respectively (encoding 539 and 625aa). between gene 2 and gene 3, there is only 1 nt overlap. there are 675 nt in gene 3 that encodes 2 nsps including 3a and 3b as well as a structural protein e. 3a, 3b, and protein e are with 174, 192, and 327 nt, respectively (encoding 57, 63, and 108aa). the number of nt overlaps between 3a and 3b, and 3b and e are respectively 1 and 20. there are 29 nt overlaps between gene 3 and gene 4. with a length of 1,045 nt, gene 4 contains 3 orfs, and it encodes a structural protein m protein, and 2 nsps 4b and 4c which are with 678, 285, and 162, nt respectively (encoding 225aa, 94aa, and 53aa). overlaps (80 nt) are identified between 4b and 4c. as for gene 4 and gene 5, it has been found that there are 4 nt overlaps. containing 443 nt, gene 5 consists of 2 overlapping orfs, orf5a and orf5b, which are 198 and 249 nt in length, respectively (encoding 65 and 82aa). there are 58 nt overlaps between gene 5 and gene 6. gene 6 encodes n protein and nsp6b which are respectively with 1,230 and 222 nt (encoding 409 and 73aa). the 5 and 3 utr of the genome are 525 and 274 nt in length, respectively. the complete genome sequence of qy16 was compared to the complete genomic sequences of 16 ibv reference strains available in genbank. as shown in table 2, the sequence comparison analysis reveals that the nucleotide identity of complete genome between qy16 and these strains ranges from 85.9 to 96.4%. qy16 has the highest resemblance to yx10 with 96.4% identity and is least similar to m41 with 85.9% identity. the sequence identity between qy16 and reference strains in terms of their different genes or regions was also calculated in table 2 ; the nucleotide identity of the 5 -utr, 1a, 1b, s1, s2, 3ab, e, m, 4bc, 5ab, n, 6b, and the figure 1. consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the s1 gene of qy16 (red dots) and 44 reference strains. the trees were computed using the neighbor-joining method with 1,000 bootstrap replicates by the mega7 program. the 16 strains labeled with blue triangles were used for the complete genome sequence comparison, the phylogenetic analysis, and the recombination analysis. 99 .6%, respectively. qy16 has the highest nucleotide identity with strain yx10 in most of their genes or regions (5 -utr, 1a, 1b, s2, 3ab, e, m, 4bc, 5ab, and the 3 -utr). in terms of the 6b gene, qy16 is most similar to the strain dy07. in terms of the n protein, it has the highest identity with saibk. in terms of full s gene, qy16 has the highest identity (90.5%) with that of the 4/91. with regard to the s1 gene, qy16 has the highest identity (97.5%) with the 4/91 and 80.7% identity with the yx10, whereas regarding the s2 gene, it has the highest identity (97.6%) with yx10 and 85% identity with the 4/91. the putative cleavage site of the qy16 s protein is his-arg-arg-arg-arg (hrrrr) which is similar to that of the qx-type strains of ibv (dy07, gx-ly5, lx4, sczy3, and yx10). in addition, the analysis of the nucleotide identity between nsps encoded by the gene 1 of qy16 and other ibv strains was conducted (table 3 ). the nsp3, nsp5, and nsp12 were predicted as plpro, 3clpro, and rdrp, respectively. as shown in the table 3 , the nsp2, nsp3, nsp4, nsp5, and nsp14 of qy16 have the highest identity with the those proteins encoded by yx10, whereas its nsp6, nsp7, nsp9, and nsp11 have the highest identity with the those proteins encoded by gx-yl5. the nsp12, nsp15, and nsp16 encoded by qy16 have the highest identity with those of strain ck/ch/gd/gz14, whereas its nsp10 and nsp13 have the highest identity with those of sczy3. the nsp8 and nsp11 of qy16 have the highest identity with those of bj and yn, respectively. the transcription-regulating sequence (trs) comparison shows that the core sequence (cs) ctt/gaacaa is conserved for most ibvs. the cs of the gene 1, gene 4, and gene 6 of qy16 is cttaacaa, whereas the cs of its gene 2 and gene 3 is ctgaacaa. however, it has been detected that the css of the gene 5 of qy16, lx4, a2, bj, and sczy3 have an additional 6 nucleotide (g/aagaaa) insertion and 2 nucleotide (cg) deletion (figure 2) , whereas the css of the gene 5 of ck/ch/gd/gz14, saibk, dy07, cq04-1, gx-yl5, and yn have an additional 3 nucleotide (aaa) insertion compared to the homologous sequences of other ibv strains (figure 2 ). in addition, the overseas strains 4/91, beaudette, and m41 were found to have no insertion. the cs is located in the initiation codons of the genes 1 to 6 of qy16 upstream 461, 52, 23, 77, 7, and 93 nt, respectively. in order to study whether the insertions of cs in trs of gene 5 exist extensively in chinese strains, the comparison of trs of gene 5 among qy16, 42 chinese isolates, and 36 oversea isolates was conducted (supplementary figure s1) . the result shows that 44% (19/43) of chinese strain have 5 to 6 nt insertion (g/aa/cgaaa or cttaa) and 2 nt (cg) deletion, 37% (16/43) of chinese strain have 3 to 4 nt insertion (aaaa or aaa) without deletion, and only 8 strains without insertion. by contrast, only a british and a korean strains have 6 nt insertion (actaaa or gagaaa) and 2 nt (cg) deletion, 3 strains isolated from thailand have 3 nt insertion (aaa), most overseas strains are without insertion. in addition, the overseas strains with insertion in trs of gene 5 mainly isolated from the countries surrounding china (korea and thailand), and the strains may have close relationship with chinese ibv strains. it suggests that an insertion in the trs of the gene 5 is common among the chinese ibv but uncommon among oversea ibv strains. in order to assess the genetic relatedness between qy16 and the reference ibv strains, phylogenetic trees were constructed for the nucleotide sequences of the complete genome and specific genes of qy16 and the 16 reference strains. as shown in figure 3 and supplementary figure s2 , the complete genome, 1a, 1b, s2, e, m, n, 3ab, 4bc, 5ab, and 6c, of qy16 is in the same cluster as those of yx10, whereas the full s gene, similar to the s1 gene, is located in the same cluster as those of 4/91 and evolutionarily distant from those of yx10. with the combination of the results of the sequence comparison and phylogenetic analysis, it is reasonable to assume that qy16 has a close evolutionary relationship with ibv yx10 and 4/91. in order to detect possible recombination events within the qy16 genomic sequence, rdp analysis was conducted and the complete genomes of 16 reference strains were used as putative parent strains. the results indicate that qy16 is possibly a recombinant strain formed by a major parent strain yx10 and a minor parent strain 4/91. it has been proved that the recombination breakpoints of qy16 are located at the 20, 311 to 20, 343, and 21,810 to 21, 844 nt. the region 1 to 20,310 and 21,845 to 27,678 nt of qy16 is 97.5% similar to that of yx10, and the region 20,334 and 21,811 nt of qy16 showed 98.9% similarity with that of 4/91. the p-value of rdp method is 3.97 × 10 −217 . to confirm the results of the rdp analysis, genomic sequences analysis of qy16, yx10, and 4/91 was carried out using the simplot software. the result indicates that the recombination signal and the breakpoint of qy16 are located at 20,336 and 21,836 nt. furthermore, the recombination region consists of a 3 -terminal orf 1b with 85 nt and a 5 -terminal s1 protein gene with 1466 nt (there are 50 nt overlaps between the2 genes), and the region between these 2 breakpoints is a putative recombinant region, which is also consistent with the rdp results ( figure 4a ). the phylogenetic tree of the complete genomic sequences of the strains and that of their detected recombinant regions were also constructed and analyzed, demonstrating that there is a topological alternation in the recombinant region of qy16, which further confirms the recombination event ( figure 4b ). all the chickens were observed clinical signs after the vaccination or the challenge, and none of the chickens showed any clinical signs before the challenge. as shown in table 4 , at 3 to 14 d post-challenge, chickens in group 1 showed severe clinical signs such as depression, ruffled feathers, sagging head and wings, diarrhea, lassitude, and huddling. however, no obvious respiratory sign or muscle lesions appeared in them. four chickens died during 5 to 12 d post-challenge with gross lesions present as typical nephritis symptoms such as swollen specked kidney, distended ureter filled with uric acid, severe dehydration, and weight loss. it suggests that qy16 is a nephropathogenic ibv strain. at 15 d postchallenge, there was neither clinical symptom nor death in the remaining chickens. however, half (3/6) of cloacal swabs collected from the survival chickens in group 1 were positive at 20 d post-challenge, and the virus recovery result indicates that the survival chickens had been releasing the virus incessantly through the cloaca. as shown in table 4 , groups 2 and 3 are with different levels of morbidity rate, mortality rate, and virus recovery rate. the morbidity rate, mortality rate, and virus-positive rate (at 20 d post-challenge) are respectively 90, 40, and 33% in group 2 where chickens were inoculated with the h120 vaccine and are respectively 40, 20, and 13% in group 3 where chickens were inoculated with the 4/91 vaccine. the results suggest that both the h120 vaccine and the 4/91 vaccine do not provide effective and full protection against qy16. as expected, none of the chickens was either ailed or present as virus positive in the control group. the results of the serological responses induced by qy16 and the other 2 ibv vaccines are presented in table 4 . only 30% of chickens in group 2 and 40% in group 3 had seroconverted at 7 d post-vaccination, but at 14 d post-vaccination more than 90% of the vaccinated chickens in group 2 and group 3 had seroconverted. after 14 d post-challenge, all the survival chickens in group 1 had seroconverted. the frequent outbreaks of ibv cause severe problems to the poultry industry in china, despite the intensive use of anti-ibv vaccines. due to its vast territory, china is caught in complicated situations where multiple ibv genotypes and serotypes coexist (liu et al., 2006) . the analysis of sequence of ibv is main focus on s1 gene in previous studies. although it plays an important role in the viral attachment to host cells, and the induction of neutralizing antibodies (koch et al., 1990) , the s1 gene is only a small part of the ibv genome and thus cannot represent all the biological characteristics of the virus. it is, therefore, necessary to sequence and analyze the complete genome of some isolated strains, especially the recombinant ones. in this study, the ibv strain qy16 was isolated from yellow feather broilers vaccinated with h120 and 4/91 vaccines. it has been found that the s1 gene of qy16 is highest identical to 4/91 and is located in the same cluster with it in the phylogenic tree. whether qy16 is re-isolated from the 4/91 vaccine strain or is the recombinant result originating from 4/91, it needs to be further confirmed by sequencing and analyzing its complete genome. the genome organization of qy16 is 5 utr-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3 utr, which is different from that of most ibv genomes (5 utr -1a-1b-s-3a-3b-e-m-5a-5b-n-3 utr) as previously reported. although 4b, 4c, and 6b are present in the most of the ibv genomes, they have rarely been studied, and their functions were thus unknown (reddy et al., 2015) . the gene 1 accounts for two-thirds of the genome, and encodes 2 overlapping proteins pp1a and pp1ab. pp1ab is an extension of pp1a by a minus 1 frameshifting translation mechanism (brierley et al., 1987) . the pp1a/1ab is cleaved into 15 nsps (nsp2 to nsp16) by plpro and 3clpro encoded by the virus. these nsps, forming a transcription/replication complex that involves in the viral genome's transcription, replication, and the viral infection, are important functional proteins for the virus. the cleavage products and cleavage sites of the pp1a/1ab of qy16 are highly identical to those of reported reference ibv strain a2 (liu et al., 2009) , sczy3 (zhao et al., 2013) , and tcov (gomaa et al., 2008) . it suggests that pp1a/1ab is highly conserved in ibv. the cs (ctt/gaacaa) of the trs is another conserved sequence of ibv. the cs can be the binding center of the leader sequence and the subgenomic mrna, and can promote the transcription of mrna2 to 6 (sola et al., 2005) . in this study, the cs sequence 20,335, 21,837 to 27,670, and 20, 336 to 21,836 nt among qy16, yx10, 4/91, and other 14 reference strains. phylogenetic trees were constructed using the neighbor-joining method (bootstrapping for 1,000 replicates). the recombinant strain qy16 was labeled with green squares, the major parental strain yx10 with blue squares, and the minor parental strain 4/91 with red squares. of qy16 is found to be conserved as ctt/gaacaa except for the gene 5 which is with a 6 nt insertion (gagaaa) and 2 nt deletion (cg). it has been found in qy16, lx4, and yx10, whereas another 6 nt (aa-gaaa) insertion was reported in the ibv strain (liu et al., 2009 )-a2 and bj, and sczy3. furthermore, a 3 nt (aaa) insertion was found in the strains including saibk, ck/ch/gd/gz14, dy07, cq04-1, gx-yl5, and yn which are all chinese isolates. in addition, the overseas strains 4/91, beaudette, and m41 were found to have no insertion. the further comparison among qy16, 42 chinese isolates, and 36 overseas isolates suggests that an insertion in the cs of trs of the gene 5 is common among the chinese ibv but uncommon among oversea ibv strains. whether these insertions could be a genetic marker between chinese and overseas strains needs to be further verified. the results of the sequence comparison and phylogenetic analysis show that qy16 has the highest identity with 4/91 in terms of the s1 gene and is located in the 1 10/10 4/10 0/10 0/10 4/7 6/6 6/6 7/7 5/6 3/6 2 9/10 4/10 3/10 9/10 8/8 6/6 6/6 8/8 4/6 2/6 3 4/10 2/10 4/10 10/10 9/9 8/8 8/8 5/9 2/8 1/8 control 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1 chickens in groups 2 and 3 were vaccinated with h120 and 4/91 vaccines, respectively, and challenged with qy16. chickens in group 1 were only challenged with qy16. chickens in the control group were not exposed to any viruses. 2 same cluster with it as well. nevertheless, its complete genome and most specific genes (except for s1) are of high similarity to yx10 rather than 4/91. these results suggest that qy16 may be a recombinant strain of 4/91 and yx10. the recombinant analysis including rdp, simplot, and phylogenetic analysis further confirms that qy16 originates from the recombination of a major parent strain yx10 and a minor parent strain 4/91. the qx-type strain yx10 was isolated in zhejiang province, china, in 2010, and it is reported to be involved in recombinant variant in recent years (wu et al., 2016; feng et al., 2017) . the 4/91 pathogenic strain was isolated in the uk and causes tremendous economic losses to the poultry industry in european (gough et al., 1992; cook et al., 1996) . the 4/91 vaccine obtained through attenuating the pathogenic strain has been widely used in the global poultry industry. despite the fact that the 4/91 strain has become an important gene donor in genetic evolution of ibv in china (han et al., 2011; zhang et al., 2015) , it is difficult to identify the 4/91 pathogenic or vaccine strain involve in the formation the recombinant ibv strains. in this study, the 4/91 vaccine is proved to be a minor parent strain of qy16 and the recombination region is predicted to include the 3 1b terminal (85 nt) and most part of the s1 gene (1,466 nt). it is believed that the recombination "hot spots," including c(t/g)taacaa (jia et al., 1995; lee and jackwood, 2000) , cttttg (wang et al., 1993) , ctttt(c/t) (mondal and cardona, 2007) , and other a-t-rich regions (zhao et al., 2013) , are located adjacent to the putative crossover sites. in our study, these putative crossover sites are found to be nowhere near the breakpoint, and it is difficult to determine whether these motifs are associated with the recombination. as the recombination region of qy16 includes most of the s1 gene, the pathogenicity study and vaccination-challenge tests were conducted to examine the efficacy of the h120 and 4/19 vaccines against qy16. the result shows qy16 causes 40% death among 17-day-old spf chickens compared with the reported mortality rate of 25% among 15-day-old spf chickens caused by its major parent strain yx10 (xue et al., 2012; feng et al., 2015) . the pathogenicity of qy16 has apparently enhanced in this regard as this result shows. the clinical symptoms of the chickens infected with qy16 are typical nephritis symptoms which are similar to the situations caused by yx10 but different from the muscle lesions caused by the 4/91 pathogenic strain. meanwhile, it has been proved by our study that the h120 vaccine cannot provide effective protection against the qy16 challenge, and it is probably because of the difference between their serotypes. the diversity of serotypes among the mass-type vaccines (h120, h52, w93, etc.) and chinese isolates has been confirmed by previous studies (li et al., 2012; feng et al., 2017) , which can provide a possible explanation for h120's failure in serving as an effective vaccine against qy16. our results also prove that the 4/91 vaccine cannot provide complete protection against qy16 challenge either. on one hand, recombinant qy16 and 4/91 may have difference in serotypes. although the s1 gene of qy16 is highly similar to that of 4/91, the difference in the s1 gene and other genes may still lead to the great change in the serotype and pathogenicity. on the other hand, a broader degree of cross-protection provided by a vaccine against different viral strains is related to the cell-mediated immune responses to the shared t-cell epitopes among some heterologous strains seo et al., 2000) . as most of the t-cell epitopes of ibv remain in the m and n protein (ignjatovic and galli, 1994) , the diversity in m and n genes may contribute to low cross-protection efficacy, in this case 4/91 vaccine's low protection efficacy against qy16. in summary, the complete genome and specific gene sequences of qy16 have different levels of sequence identity with other ibv strains. the recombinant strain qy16 is proved by our study to derive from yx10 (as the major parental sequence) and 4/91 (as the minor parental sequence). it can be inferred from the results of the pathogenicity study and vaccination-challenge test that ck/ch/gd/qy16 is a type of nephropathogenic strain and that the vaccine strains h120 and 4/91 cannot provide complete protection against it. these results indicate that the continuing evolution of ibv strains by genetic drift and genetic recombination may lead to ibv outbreaks even among the vaccinated chickens in china, which calls for the further development of new vaccines to help prevent and control the disease. supplementary data are available at poultry science online. supplementary table s1. primers used for qy16 genome amplification. supplementary table s2 . the accession numbers of ibvs used for s1 gene, complete genome and specific genes alignment, sequence comparison, phylogenetic and recombination analysis, supplementary figure s1 . results of the sequence comparison of qy16, 42 chinese isolates, and 36 overseas isolates in terms of the trs of their gene 5. supplementary figure s2 . phylogenetic trees constructed by using the neighbor-joining method based on the complete genome and different regions of the genome of the strains (bootstrapping for 1,000 replicates with its value >60%). (a) 3ab, (b) 4bc, (c) 5ab, and (d) 6b. the qy16 sequence is labeled with red dots. the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity an efficient ribosomal frameshifting signal in the polymerase-encoding region of the coronavirus ibv complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus coronavirus ibv: structural characterization of the spike protein location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry the long view: 40 years of infectious bronchitis research a survey of the presence of a new infectious bronchitis virus designated 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nucleocapsid protein gene sequences coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription the authors declare that there are no conflicts of interest. key: cord-265681-ab8j4o1u authors: boroomand, zahra; asasi, keramat; mohammadi, ali title: pathogenesis and tissue distribution of avian infectious bronchitis virus isolate irfibv32 (793/b serotype) in experimentally infected broiler chickens date: 2012-04-01 journal: scientificworldjournal doi: 10.1100/2012/402537 sha: doc_id: 265681 cord_uid: ab8j4o1u infectious bronchitis (ib) is one of the most important viral diseases of poultry. the aim of this study was to investigate the distribution of avian infectious bronchitis virus isolate irfibv32 (793/b serotype) in experimentally infected chicken. ninety-one-day-old commercial broilers were divided randomly into two groups (seventy in the experimental and twenty in the control group). chicks in the experimental group were inoculated intranasally with 10(5) eld50/0.1 ml of the virus at three weeks of age. the samples from various tissues were collected at1, 2, 3, 5, 7, 11, 13, 15, and 20 days postinoculation. chickens exhibited mild respiratory signs and depression. viral rna was detected in the kidney, lung and tracheas on days 1 to 13 pi, in the oviduct between, days 3 and 13, in testes between days 1 and 11 pi, and in the caecal tonsil consistently up to day 20 pi. the most remarkable clinical signs and virus detection appeared on day 1 pi. data indicated that the number of infected chickens and viral rna detection from tissues was reduced with increasing antibody titer on day 20 pi. the results demonstrated that the irfibv32 virus has wide tissue distribution for respiratory, urogenital, and digestive systems. infectious bronchitis virus (ibv) is, by definition, the coronavirus of the domestic fowl. although it does indeed cause respiratory disease, it also replicates at many nonrespiratory epithelial surfaces, where it may cause pathology, for example, kidney and gonads [1, 2] . strains of the virus vary in the extent to which they cause pathology in nonrespiratory organs. replication at enteric surfaces is considered to not normally result in clinical disease, although it does result in faecal excretion of the virus [3] . infectious bronchitis (ib) is one of the most important diseases of chickens and continues to cause substantial economic losses to the industry. infectious bronchitis is caused by ib virus (ibv), which is one of the primary agents of respiratory disease in chickens worldwide. all chickens are susceptible to ibv infection, and the respiratory signs include gasping, coughing, rales, and nasal discharge. sick chicks usually huddle together and appear depressed. the severity of the symptoms in chickens is related to their age and immune status. other signs of ib, such as wet droppings, are due to increased water consumption. the type of virus strain infecting a flock determines the pathogenesis of the disease, in other words, the degree and duration of lesions in different organs. the upper respiratory tract is the primary site of infection, but the virus can also replicate in the reproductive, renal, and digestive systems [4] . the conventional diagnosis of the ibv is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. since two or three blind passages are often required for successful primary isolation of ibv, this procedure could be tedious and time consuming [5] . alternatively, ibv may be isolated by inoculation in chicken tracheal organ cultures. furthermore, ibv may be detected directly in tissues of infected birds by means of immunohistochemistry [6, 7] or in situ hybridization [8] . the reverse transcription-polymerase chain reaction (rt-pcr) has proved useful in the detection of several rna viruses [9, 10] . outbreaks of the disease can 2 the scientific world journal occur even in vaccinated flocks because there is little or no cross-protection between serotypes [2, 11] . the necessity of ib prevention in chicken regarding the nature of the virus with a high mutation rate in the s1 gene dictates the necessity to develop effective vaccines. the first step is to study the virus strains distributed in the geographical region and determine their antigenicity and pathogenicity in order to choose a suitable virus strain for vaccination. this virus was isolated from a flock suspected of ib suffering from severe respiratory distress and experiencing high mortality [12] . the objective of the present study was to clarify some aspects of pathogenesis of the disease caused by irfibv32 (793/b serotype) in experimentally infected broilers. rt-pcr test was performed to detect the presence of the virus in body tissues and samples. the clinical signs, gross lesions, and antibody response of the affected chicks were also monitored. the virus isolate used in this study was irfibv32 (genbank: hq123359.1) [12] . it was obtained from shiraz veterinary university and was propagated two times in 9to 11-day-old embryonated chicken eggs. the embryo lethal dose (eld50) was calculated according to the reed and muench [13] formula. design. ninety-one-day-old commercial broiler chicks were divided randomly into two groups (seventy chicks in the experimental and twenty chicks in the control group). they were reared separately in the animal research unit of the veterinary school of shiraz university and received feed and water ad libitum during the experiment. all experiments were conducted after institutional approval of the animal use committee of shiraz university. prior to challenge, all birds were serologically tested using enzyme-linked immunosorbent assay (elisa) and they were negative for antibodies to infectious bronchitis virus antigens. furthermore, five birds from the experimental group were killed and their organs were investigated for virus detection. at the age of 20 days, all birds in the experimental group were challenged intranasally and with allantoic fluid containing 10 5 eld50/0.1 ml of the virus. the remaining 20 birds were left as unchallenged control. all the chickens were monitored daily for 20 days for clinical signs, antibody responses to ibv, and mortality. on days 1, 2, 3, 5, 7, 11, 13, 15, and 20 postinoculation (pi), four chickens from the experimental group and two chickens from the control group were randomly selected and used for sample collection. all were bled before humanly euthanasia. gross lesions were recorded, and their trachea, lungs, kidneys, caecal tonsil, testes, and oviduct were aseptically collected for virus detection using rt-pcr assay ( table 1) . sera of the birds were collected on 0, 5, 11, 15, and 20 days pi for elisa test. all tissue samples were immediately stored at −70 • c until used. rna of the samples was extracted using the accuzol userś manual (bioneer corporation, republic of korea) according to the manufacturer's protocol. briefly, appropriate tissue (50-100 mg of tissue) was homogenized with 1 ml of accuzol, and then 200 µl chloroform was added into the mixture and the mixture was centrifuged at 12000 rpm at 4 • c for 15 min. the upper phase was added to an equal volume of isopropyl alcohol and stored at −20 • c for 10 min then centrifuged at 12000 rpm at 4 • c for 10 min. after the washing step, by using 80% ethanol and centrifuging at 12000 rpm at 4 • c for 5 min, the pellet was dissolved in a final volume of 50 µl distilled water (dw) and stored at −70 • c until used. the cdna was synthesized using accupowder rt premix kit (bioneer corporation, republic of korea) according to the manufacturer's instruction. the primers were specific for 3 utr gene [14] . five µl of total rna and one µl of each 10 pmol primer were used for cdna preparation. pcr was performed to amplify a 276-bp fragment of the 3 utr gene of avian infectious bronchitis virus. the same primers were used in the pcr master mix containing: 2.5 µl pcr buffer 10x, 0.75 µl mgcl 2 (50 mm), 0.5 µl dntps (10 mm), 1 µl of each 10 pmol primer (utr1 and utr2), 3 µl cdna, 15.75 µl water, and, at the end, 0.5 µl taq dna polymerase (5 iu/µl) was added. the program in ependorf thermal cycler was 95 • c for 3 min and 35 cycles including 95 • c for 45 sec., 55.6 • c for 45 sec, 72 • c for 50 sec, and a postpolymerization step at 72 • c for 7 min. the products were analyzed in 1% agarose gel containing ethidium bromide, using an ultraviolet transilluminator. assay. the enzyme-linked immunosorbent assay (elisa) technique was used to measure antibody levels to avian infectious bronchitis virus (ibv). ten chickens in the inoculated and control groups were bled at days 0, 5, 11, 15, and 20 pi. serum samples were collected and tested by biochek poultry immunoassays infectious bronchitis virus antibody test kit (catalogue code ck119). microtitre plates had been precoated with inactivated ibv antigen. chicken serum samples were diluted and added to the microtitre wells where any anti-ibv antibodies and other serum proteins were then washed away. anti-chicken igg labeled with the enzyme alkaline phosphatase was then added to wells and bound to any chicken anti-ibv antibodies that had bound to the antigen. after another wash to remove unreacted conjugate, substrate was added in the form of pnpp chromogen. a yellow color was developed if anti-ibv antibody was present, and the intensity was directly related to the amount of anti-ibv antibody present in the sample. the absorbance at 405 nm measured after 1 h in a ultra-microplate reader (biotek, el80). descriptive statistics was used to summarize the data generated from the study. data were presented as mean ± se to compare antibody titers between the infected and the control group on different days following inoculation. lesions, and mortality. some chickens in the inoculated group showed mild tracheal rales, coughing, and gasping at 24 hours pi. the signs were less severe after 4 days pi. in addition, the birds appeared lethargic, reluctant to move from the 1st to the 4th day pi. no mortality was observed in any of the groups during the experiment. these symptoms disappeared at 4 days pi. on days 1, 2, 3, 5, 7, and 10 pi, two chickens from control group and four from test group were randomly selected; necropsied and gross lesions were recorded. slight hyperaemia and oedema in tracheal mucosa were observed in the euthanased birds from 1 to 3 days pi (figure 1 ). pale and swollen kidneys were observed from 5 to 10 days pi. no gross lesions were observed in brain, gizzard, proventriculus, intestine, and heart muscle as well as liver and spleen. no clinical signs and gross lesions were observed in the uninfected control chickens. the presence of the virus was checked in all samples obtained from the inoculated and control groups at different dpi. the rt-pcr test was performed for virus detection (figure 2 ). no virus was detected from the birds killed before infection. table 2 shows the virus distribution in tissues following virus inoculation. the virus was found in the trachea, lung, kidney, caecal tonsil consistently for 13 days pi, and also in the kidney, caecal tonsil at day 15, as well as being detected in the caecal tonsil at day 20 when the virus was not detected in any other tissue. the virus was also detected in the testes consistently for 11 days. the virus was detected on days between 3 and 13 pi from the ovary and oviduct. virus was not found in any tissues of the control. result. the sera from chickens of different groups were screened for antibodies levels against ibv using elisa. for the result to be valid, the mean negative control absorbance should be read below 0.3 and the difference between the mean negative control and the mean positive control should be greater than 0.15 (titre range: 833 or less, negative antibody status, and 834 or greater, positive antibody status). table 3 and figure 3 show the mean antibody (ab) titer in the challenged and control group. increase of ab titers occurred 11 days following inoculation. in the challenged group, sera were negative at 0, 5, and 11 days pi but they were positive (≥834) at 15 and 20 days. the ab titer was 827.66 at 11 days, but it developed to 1657.87 at 20 days. sera of the control was negative (≤833) on all of the days pi. in this study, the pathogenesis of the infectious bronchitis virus isolate irfibv32 which was recently isolated in iran [12] , tissue tropism, and dissemination of the virus throughout the body were evaluated following intranasal (in) inoculation of commercial broiler chickens by rt-pcr. after 24 hours postinoculation, the challenge infected chickens exhibited mild tracheal rales, coughing, and gasping. these signs were not visible from day 4 pi. in the present study, clinical signs and gross lesions were in agreement with the findings that have been described previously [15] . twenty four hours after exposure, purcell and mcferran [16] noted cloudy and edematous abdominal and posterior thoracic air sacs, which 3 days later thickened and filled with clear bubbly exudates. we did not observe any gross changes in the air sacs, which were thin, clear, and transparent. the lungs also appeared normal in all birds, similar to the observations of grgić et al. [17] . in this study, irfibv32 induced mild lesions in trachea and kidneys. these findings are in accordance with some reported observations [17] [18] [19] . terrigino et al. [20] reported that inoculated birds showed severe conjunctivitis, associated with abundant lacrimation, oedema, and cellulitis of the periorbital tissues at 48 hours following challenge with ibv. we observed predominant lesion in the trachea and kidneys. these results were similar to the findings that have been described previously [15, [20] [21] [22] . diagnostic laboratories usually first isolate the virus in embryonated eggs and use the allantoic fluid to detect ibv specific rna by rt-pcr. to reduce the time and labor needed for diagnosis, we first assessed the feasibility of the n-gene specific rt-pcr to detect the virus directly in tissues without virus isolation. stachowiak et al. [4] assessed the feasibility of the ngene specific rt-pcr to detect the virus directly in tissues without virus isolation. trachea, lung, kidney, and cecal tonsils from birds were analyzed. representative rt-pcr results are shown, and, as expected, ib virus was detected more frequently in the tracheal tissues than in the lungs, kidney, or cecal tonsils. in the current experiment, the virus was detected from all organs of the infected birds except the oviduct at 24 hours pi. the virus was detected consistently in the trachea, lung, kidney, and caecal tonsil consistently for 13 days and persisted longer in the kidney and caecal tonsil than in the respiratory tissues. the virus disappeared from the kidney after 15 days, although it was found in the caecal tonsil and intestine consistently for 20 days pi. the virus was also detected in the testes consistently for 11 days. previous studies confirmed ibv can also replicate in the testes [23] . the virus disappeared from the ovary and oviduct after 13 days, from testes after 11 days. these findings indicate irfibv32 isolate has a broad tissue distribution that includes respiratory, digestive, congenital, and urinary tract tissues; predominantly in urinary and digestive tracts. this result is in agreement with the previous studies about ibv distribution [6, 22, 24, 25] . chen and itakura [24] reported that the tracheal lesions recovered faster in ibv inoculated groups. the nephropathogenic property of their ibv was longer lasting than the respiratory involvement. in our investigation, virus was detected from kidneys more often than the tracheas. terrigino et al. [20] isolated virus from kidney, trachea, ovary, and oviduct following inoculation of the qx strain of infectious bronchitis virus. mahdavi et al. [22] demonstrated the viral rna antigen and tissue tropism of serotype 793/b (4/91) isolated in iran, using the immunohistochemistry method. they reported that the 793/b serotype of ibv has a greater affinity and pathogenicity for the kidney than to other tissues, but, in our study, the distribution of viral rna in caecal tonsils and guts was more than the other tissues. virus detection in the caecal tonsil and intestine the scientific world journal 5 could indicate this isolate of ibv tropism for the digestive tract. however, in our experiment, no relevant gross lesions were detected in the digestive tract following ibv challenge. cavanagh [3] also reported that infection of enteric tissues usually does not manifest itself clinically. lucio and fabricant [26] showed m41 strain can infect a variety of tissues and some isolates may be recovered frequently from the digestive tract. in another study, ibv was not detected from intestine and cecal tonsils using immunofluorescence technique [27] . abdel-moneim et al. [9] detected ibv antigen in the proventriculus, skin, sclera of the eye, spinal cord, and the central nervous system in infected embryos by immunohistochemistry. lee et al. [28] investigated the tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization. viral rna was detected at 2 days after infection in epithelial cells of the trachea, lung, intestine, and bursa. in chickens, the virus has been routinely isolated from the trachea, lung, and caecal tonsils but the persistence of the virus in the bursa was of interest. their results clearly confirm the strict epitheliotropic nature of ibv. in this research, elisa technique was used to measure antibody titer to ibv. elisa detected moderate levels of the antibody to ibv on 11 days pi and high levels of antibody against ibv on day 20 pi. sera were positive for ibv at 15 days pi. ghadakchi et al. [29] showed that elisa could be reliable, repeatable, and sensitive for monitoring vaccination schedules and the rapid detection of the early rise of antibodies against ib. emikpe et al. [30] evaluated prevalence of antibodies to infectious bronchitis virus in southwestern nigeria using elisa. their finding indicated that antibody titer to ibv increased following ibv infection. chen and itakura [24] reported that the clinical signs and gross and histological lesions in the trachea and kidneys due to ibv infection were more severe and of longer duration in dually infected chicks (with infectious bursal disease virus and ibv) than in ones inoculated with ibv. our field observation indicates that flocks that are infected by ibv have shown an increase in mortality in recent years that could be due to increased pathogenicity of the virus or due to other undetected field infections. this study demonstrated that ibv alone cannot cause severe and devastating disease, but ibv-infected birds can be susceptible to superinfectant bacteria and coinfection with endemic nonhighly h9n2 avian influenza virus. future work should aim to determine if the available and used ib vaccines provide sufficient protection against this ibv isolate. avian infectious bronchitis virus: a possible cause of reduced fertility in the rooster severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus infectious bronchitis virus surveillance in ontario commercial layer flocks the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus histopathology and immunohistochemistry of renal lesions due to infectious bronchitis virus in chicks comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant dna immunohistochemistry for detection of avian infectious bronchitis virus strain m41 in the proventriculus and nervous system of experimentally infected chicken embryos lna probe-based real-time rt-pcr for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions isolation and identification of a new isolate of avian infectious bronchitis virus irfibv32 and study of its pathogenicity a simple method of estimating fifty per cent endpoints universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction serological and gross findings in broilers with aiv and ibv the histopathology of infectious bronchitis in the domestic fowl pathogenicity of infectious bronchitis virus isolates from ontario chickens comparison of the nephropathogenicity of four strains of infectious bronchitis virus pathogenicity of australian strains of avian infectious bronchitis virus pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes isolation, identification and molecular characterization of ibv variant from out break of visceral gout in commercial broilers experimental histopathologic study of the lesions induced by serotype 793/b (4/91) infectious bronchitis virus detection the 4/91 strain of infectious bronchitis virus in testicular tissue from experimentally infected rooster by reverse transcription-polymerase chain reaction histopathology and immunohistochemistry of renal lesions due to avian infectious bronchitis virus in chicks uninoculated and previously inoculated with highly virulent infectious bursal disease virus the histopathology of infectious bronchitis in fowls infected with a nephrotropic "t" strain of virus tissue tropism of three cloacal isolates and massachusetts strain of infectious bronchitis virus the pathogenesis of nephritis in chickens induced by infectious bronchitis virus tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe standardization of an enzyme-linked immunosorbent assay for detection of infectious bronchitis virus antibody prevalence of antibodies to infectious bronchitis virus (ibv) in chickens in southwestern nigeria this research was funded by the research committee, school of veterinary medicine of shiraz university, shiraz, iran. key: cord-000924-wwuqxx1r authors: yan, fang; zhao, yujun; hu, yongting; qiu, jianyang; lei, wenxin; ji, wenhui; li, xuying; wu, qian; shi, xiumin; li, zhong title: protection of chickens against infectious bronchitis virus with a multivalent dna vaccine and boosting with an inactivated vaccine date: 2013-03-24 journal: j vet sci doi: 10.4142/jvs.2013.14.1.53 sha: doc_id: 924 cord_uid: wwuqxx1r the protective efficacy of dna plasmids encoding avian infectious bronchitis virus (ibv) s1, n, or m protein was investigated in chickens. chickens were inoculated monovalently (with plasmid pvax1-16s1, pvax1-16m, or pvax1-16n alone) or multivalently (combination of the three different plasmids, pvax1-16s1/m/n). a prime-boost immunization protocol against ibv was developed. chickens were immunized with the multivalent dna vaccine twice and then boosted with an inactivated vaccine once. antibody titers of the chickens immunized with pvax1-16s1/m/n were much higher than those of the monovalent groups (p < 0.01). a protective rate up to 90% was observed in the pvax1-16s1/m/n group. the serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent dna vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. additionally, the prime-boost group also showed the highest level of ibv-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent dna vaccine group, and the prime-boost group completely protected from followed viral challenge. avian infectious bronchitis (ib) is a major disease in the poultry industry worldwide. this disease frequently occurs in vaccinated and non-vaccinated flocks, and has caused severe economic loss over the last few years [11] [12] [13] [14] 17, 21] . protection failure is mainly due to the numerous ib virus (ibv) serotypes and frequent emergence of new variants [23, 26] . given the economic effects of ibv on the commercial poultry industry, preventing infection with this virus has been continually pursued. traditional ibv vaccines include inactivated or live attenuated vaccines [2] [3] [4] . however, both types of vaccines have unique disadvantages. inactivated vaccines can induce the production of relatively high antibody titers but a very low level of cytotoxic t lymphocyte (ctl) responses [5] . live attenuated vaccines can initiate humoral and cellular immune responses, but lead to the spread the live vaccine viruses [7, 15] . it is therefore necessary to develop novel vaccines and/or optimize current vaccination procedures. ibv belongs to the coronaviridae family and contains a positive single-stranded rna genome encoding four major structural proteins: a small envelope protein (e), integral membrane protein (m), phosphorylated nucleocapsid protein (n), and spike glycoprotein (s) [19] . the s protein is cleaved into two subunits (s1 and s2). the s1 protein is very important for inducing protective immunity and has been successfully used to construct ibv dna vaccines [6, 9] . the n protein is conserved and induces ctl as well as activated b cell responses, which are critical for preventing ibv infection in poultry [8, 20] . the m glycoprotein can induce the production of detectable antibodies and delayed type hypersensitivity responses [8] . hence, all of these proteins are primary targets for developing dna vaccines to elicit immune responses. in the present study, we evaluated the protective effect of three plasmids expressing the s1, n, and m proteins of the virulent ibvsx16 strain that we previously constructed [22] . chickens were immunized monovalently with each individual plasmid (pvax1-16s1, pvax1-16m, and plasmids pvax1-16s1, pvax1-16n, and pvax1-16m encoding the s1, n, and m proteins of the virulent ibvsx16 strain, respectively, were described in our previous publication [22] . the virulent ibvsx16 strains used to challenge immunized chickens in this study were isolated from the kidneys of ib-infected chickens from the shanxi province by department of infectious disease and immunology, college of animal science and technology, shanxi agricultural university [21] . virus stocks containing 1 × 10 3 egg infective dosage (eid 50 ) of ibvsx16 with 100 μl were used to inoculate the allantoic cavities of 10-day-old specific pathogen free (spf) embryonated chicken eggs (shandong specific-pathogen-free chicken research center, china) that were then kept at 37 o c for 48 h. allantoic fluid containing the virus was harvested after 48 h post-inoculation, stored at −80 o c until use. chill eggs at 4 o c for at least 2 h to kill the embryo and to reduce the contamination of the allantoic fluid with blood during harvesting. remove sticky tape and swab each egg with cotton wool soaked with 70% alcohol to disinfect and remove condensation from the shells. dip the forceps or scissors in absolute alcohol and flame to sterilize. remove the eggshell above the air space. discard embryos that are visibly contaminated. remove a sample of allantoic fluid from each egg. the 50% eid 50 /ml of the viral stocks were calculated as previously described by reed and muench [25] . titer of the ibvsx16 strains was 10 9 eid 50 /ml. a total of 140 seven-day-old spf chickens (shandong specific-pathogen-free chicken research center, china) were housed in spf environment at the laboratory animal and resources facility, shanxi agricultural university. the inactivated vaccine was made by adding 37% formaldehyde (final concentration, 0.1%) to allantoic fluid containing ibvsx16 and incubating at 37 o c for 24 h. the inactivated vaccine 200 μl was inoculated into the allantoic cavity of 10-day-old spf embryonated chicken eggs. the embryos were incubated at 37 o c and examined twice daily for their viability. the allantoic fluids were harvested after 72 h and two blind passages were conducted to examine the efficiency of ibvsx16 inactivation. one part of the inactivated allantoic fluid was then emulsified with two parts (v/v) of paraffin oil (hangzhou oil refinery, china). our animal research in our study had been approved by shanxi province animal disease control center (china). the plasmids used were amplified in escherichia (e.) coli dh5α cells (takara, japan), and extracted using a pureyield plasmid maxiprep system (promega, usa). seven-day-old chickens were randomly divided into seven groups of 20 chickens each and immunized intramuscularly on 7, 21, and 35 day-old, using different vaccination strategies (table 1 ). each group of chickens was injected with 100 μg (1 μg/μl) monovalent dna vaccine (pvax1-16s1, pvax1-16m, and pvax1-16n) respectively and 0.5 ml inactivated ibv vaccine. pvax1-16s1/m/n was a multivalent dna vaccine containing 100 μg of each plasmid (equivalent molar ratios for each dna component) and therefore delivered the same dose of each expression plasmid targeting the s1, n, or m, respectively, as each monovalent vaccine. all the chickens were immunized intramuscularly with the vaccines. peripheral blood samples were also collected from five randomly selected chickens from each group from the jugular vein into heparinized capillary tubes (laiwu yaohua pharmaceutical packing, china) when the birds were 7, 21, 35, and 49 days old. titers of antibodies against ibv in the serum samples were measured using an enzyme-linked immunosorbent assay (elisa) kit (idexx laboratories, usa) according to the manufacturer's instructions. the optical density at 650 nm (od 650 ) was measured using a microplate reader (model 680; bio-rad, usa). each serum sample, including the negative and positive controls, was analyzed in triplicate. negative and positive sera for ibv were obtained from china institute of veterinary drugs control. titers of virus neutralizing antibody against ibv were also measured as previously described [25] . briefly, serial 2-fold dilutions of serum samples were mixed with 100 eid 50 of ibv16 strain and kept at room temperature for 1 h. 200 μl the virus-serum mixtures in each dilution were inoculated into the allantoic cavity of 10-day-old spf chicken embryos in ten replicates. the embryos were also inoculated with 100 eid 50 alone in parallel. seven days after inoculation, characteristic ibv lesions such as dwarfing, stunting, or curling of embryos were examined. the virus neutralizing antibody titer of each sample was recorded at the highest serum dilution value which protected 50% of the embryos from death. a t cell proliferation assay was performed as previously described [1] with some modification. peripheral blood mononuclear cells (pbmcs) were isolated by lympholyte-mammal (cedarlane, canada). briefly, 2 ml freshly heparinized whole blood was diluted with hanks' balanced salt solution in a ratio of 1：1 and carefully add on top of 2 ml lympholyte-mammal separation medium, centrifuge at 400 × g/min for 20 min, aspirate coat of leukocyte, add 4 ml kpmi 1640 (invitrogen, usa), centrifuge at 400 × g/min for 20 min, discard supernatant and wash precipitation cell twice by the same method. the cells were resuspended in rpmi 1640 medium (invitrogen, usa) supplemented with 5% fetal bovine serum (invitrogen, usa), 100 μg/ml penicillin (invitrogen, usa), and 100 μg/ml streptomycin (invitrogen, usa) at a concentration of 1 × 10 7 cells/ml. the freshly isolated pbmcs (10 three weeks after the third vaccination, all chickens were challenged with 10 3 eid 50 of ibvsx16 through a nasal-ocular route. the chickens were observed daily for 14 days post-challenge. deceased chickens were necropsied and evaluated for ibv infection. the surviving chickens were euthanized humanely by pectoral muscle injection of ketamine hydrochloride in 22 mg/kg of body weight 14 days post-infection. the trachea and kidneys were collected for virus detection. the number of ibv-positive chickens was confirmed by reverse transcriptasepolymerase chain reaction (rt-pcr). all procedures for rna isolation and rt-pcr were previously described [15] . viral rna was isolated and purified from trachea and kidneys collected from chickens challenged with ibvsx16 using trizpl reagents (invitrogen, usa) according to the manufacturer's instructions. amplification of the specific fragments of n gene by rt-pcr was performed. the rna pellet was dissolved in 10 ml of depc-treated water. the statistical analysis was performed with spss (ver. 17.0; ibm, usa). differences between the groups were analyzed with a one-way repeated measurement anova and least significance difference test. the concentrations of serum anti-ibv antibodies were measured with a serum-neutralizing assay and an elisa. mean serum ibv-neutralizing antibody titers of the different groups immunized with different vaccines as shown in fig. 1 . values are expressed as the log 10 inverse mean titer ± sd for 10 chickens in each group. different letters above the columns indicate significant differences (p ＜ 0.01) while identical letters indicate that there was no significant difference (p ＞ 0.05) within each time point. the titers measured at each time point were significantly different in each group (p ＜ 0.01). as expected, sera from the pre-primed chickens and birds immunized with the pvax1 vector had no detectable levels of antibodies against ibv according to the serum-neutralizing assay (fig. 1) . antibody titers for chickens immunized with pvax1-16s1, pvax1-16m, pvax1-16n, or pvax1-16s1/m/n increased from day 7 to day 49 (fig. 1) . antibody titers of the multivalent pvax1-16s1/m/n-immunized chickens were significantly higher than those of all chickens immunized with the monovalent dna vaccine on days 21, 35, and 49 (p ＜ 0.01). these results indicate that the triple-gene plasmid mix induced a greater antibody response than the single-gene plasmid. antibody titers of the pvax1-16s1/m/n + inactive vaccine group were much higher than those of the pvax1-16s1/m/n group (p ＜ 0.01) on days 21 and 35. however, no significant difference (p > 0.05) in antibody titers between the two groups was observed on day 49. these findings were also confirmed with an elisa assay as shown in fig. 2 . data are expressed as the mean ± sd for 10 chickens in each group. different letters above the columns indicate significant differences (p ＜ 0.01) while dna vaccine and an inactivated vaccine 57 fig. 3 . lymphocyte proliferation rates of chickens from the different groups administered different vaccines. data are expressed as the mean od570 values ± sd (n = 5). columns are labeled with letters. different letters indicate statistically significant differences (p ＜ 0.01) between different treatments within the time point while columns with the same letters indicate that no significantly differences (p ＞ 0.05) were observed. absence of a letter indicates that there were no significant differences (p ＞ 0.05) between any of the time points. although not indicated in the graph, the differences between corresponding treatments from different day groups are significant (p ＜ 0.01). kinetic changes in a 570 values for the animals are shown in fig. 3 . data are expressed as the mean od 570 values ± sd (n = 5). columns are labeled with letters. different letters indicate statistically significance differences (p ＜ 0.01) between different treatments within the time point while columns with the same letters indicate that no significantly differences (p > 0.05) were observed. absence of a letter indicates that there were no significant differences (p > 0.05) between any of the time points. similar to the antibody responses we observed, pbmc proliferation in response to ibv and cona stimulation was also observed in chickens immunized with pvax1-16s1, pvax1-16m, pvax1-16n, and pvax1-16s1/m/n on day 21 and further increased on day 35. a 570 values for the monovalent and multivalent groups were much higher than those for the pvax1 dna vector control and inactivated vaccine groups when the birds were 35 days old. the a 570 values for the chicken immunized with the pvax1-16n construct were higher than those for birds immunized with the pvax1-16s1 or pvax1-16m constructs (p ＜ 0.01). on day 49, proliferative responses of the pvax1-16s1/m/n and pvax1-16s1/m/n + inactivated vaccine groups were significantly higher than those of the other groups (p ＜ 0.01). these results indicate that immunization with a combination of the three-gene dna vaccine and an inactivated vaccine not only elicited the strongest antibody response, but also induced the highest ibv-specific cellular proliferation rates in the chickens. the responses of all experimental groups to stimulation with cona were similar to ones provoked by ibv (data not shown). percentages of cd3+cd4+ and cd3+cd8+ t lymphocytes among the various groups are presented in fig. 4 . columns are labeled with letters. different letters indicate statistically significant differences (p ＜ 0.01) while letters in common indicated that no significant differences (p ＞ 0.01) were observed in each t cell subgroup. no significant difference in the two t lymphocyte subgroups was found between the control pvax1 and inactivated vaccine group. in contrast, a significant difference in the percentage of cd3+cd4+ and cd3+cd8+ t lymphocytes (p ＜ 0.01) was observed between the pvax1-16s1, pvax1-16m, pvax1-16n, and pvax1-16s1/m/n groups compared to the pvax1 group. levels of cd3+cd4+ and cd3+cd8+ t lymphocytes were significantly higher in the pvax1-16n group than in the pvax1-16s1 or pvax1-16m groups (p ＜ 0.01). the highest percentage of cd3+cd4+ and cd3+cd8+ t lymphocytes were detected in the to evaluate the protective efficacy of the dna vaccines and prime-boost vaccination strategy we developed, all chickens were challenged with virulent ibvsx16 virus. mortality and protection rates for the different groups are shown in table 2 . the number of chickens positive for ibv was determined by rt-pcr analysis of birds that died from viral infection and ones that were euthanized. mortality was determined as the number of dead chickens divided by the total number of chickens in each group. protection rate was measured as a percentage of the number of chickens negative for ibv over the total number of chickens. chickens in the pvax1-inocluated group developed clinical signs of infection including coughing, nasal discharge, and dyspnea. mortality rate of the pvax1injected groups was 35% 14 days post-challenge. in contrast, the protection rates were 75% for the pvax1-16s1 group, 60% for the pvax1-16m group, 80% for the pvax1-16n group, 90% for the pvax1-16s1/m/n group, 80% for birds given the inactivated vaccine (80%), and 100% for the pvax1-16s1/m/n + inactivated vaccine animals. the mortality rate was 5% for the pvax1-16s1/ m/n group, 10% for the inactivated vaccine group, and 0% for birds treated with pvax1-16s1/m/n + inactivated vaccine. in the present study, a prime-boost vaccination regimen that completely protected chickens against challenge with a virulent ibvsx16 strain was developed. immune responses were evaluated in chickens immunized with the dna vaccines either alone or with a mixture of the three vaccines. findings from the present study agree with those of a previous investigation showing that all three ibv proteins selected for this study have their own unique and important roles in eliciting ibv immune responses [24] . the dna vaccination with pvax1-16m alone provoked the weakest immune response demonstrated by the lowest antibody titers (p ＜ 0.01) in comparison with pvax1-16s1 and pvax1-16n. in contrast, pvax1-16s1 induced the highest antibody titers among the three constructs. chickens immunized with pvax1-16n had significantly higher levels of ibv-specific cellular proliferation (p ＜ 0.01) and t lymphocytes (p ＜ 0.01) than birds immunized with pvax1-16s1 and pvax1-16m. the challenge assay also proved that the dna vaccine targeting the n protein is more effective. chickens that received a combination of the three dna vaccines also mounted stronger immune responses than the birds immunized with each dna vaccines alone, suggesting that a combined dna vaccination with s1, m, and n genes may provide stronger protection against ibv. cd4+ t cells may directly produce antiviral cytokines, which increasing b cell activity and increasing the proliferation, maturation, and functional activity of cd8+ ctls. the cd8+ ctl plays a critical role in controlling ibv infection [18] . at 49 days of age, chickens immunized with plasmid constructs groups had percentages of cd4+cd3+ and cd8+cd3+ t lymphocytes subgroups higher than those of birds administered the control pvax1 vector or inactivated vaccine (p ＜ 0.01). increased number of t lymphocytes indicates that administration of the dna vaccine resulted in effective cellular immunization and promoted virus clearance [2] . as for the choice of a booster, the inactivated vaccine is prepared from the whole virus particle, so the immune responses induced by inactivated ibv vaccines could respond to the whole virus. the inactivated vaccine has a better capability than choosing dna plasmid as the booster to evoke memory b cells [16] . the inactivated vaccine as a booster is also considered to be able to balance the th1 and th2 immune responses [10] , which could enhance both specific cell-mediated immunity and neutralizing antibody inductions with increased t cell response. in the current study, the dna vaccine stimulated a relatively strong ibv-specific cellular response but low antibody titers. when the dna vaccine and inactivated vaccine were administered in combination, both higher cellular proliferation rates and antibody titers were observed among the dna vaccine-primed chickens as seen in previous studies [10] . chickens that received the dna vaccine with the inactivated vaccine were fully protected from ibvsx16 challenge. none of these animals developed clinical signs or harbored detectable levels of virus in the trachea or kidney after challenge. these results clearly demonstrate that only the combined administration of dna vaccine and inactivated vaccine, but not either alone, offered full protection from challenge with the ibvsx16 strain. our results directly demonstrated that the multivalent dna vaccine targeting the s1, m, and n proteins of ibv developed humoral and cellular immune responses against ibv in chickens. we also showed that our prime-boost immunization strategy induced strong immune responses and provided effective protection from challenge with a high dose of virulent ibv. dna vaccines are easy to prepare and inactivated vaccines are readily available. administration of dna vaccines with an inactivated vaccine is a safe and effective immunization technique. given its superior efficacy, this combination represents a valuable strategy for protecting against ibv infection and can combat poor cross-protection among the numerous ibv serotypes along with the frequent emergence of new variants. optimum conditions for the chicken lymphocyte transformation test development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review avian coronavirus diseases and infectious bronchitis virus vaccine development severe acute respiratory 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infectious bronchitis virus isolates in korea between identification of a newly isolated avian infectious bronchitis coronavirus variant in china exhibiting affinity for the respiratory tract phylogenetic analysis of avian infectious bronchitis virus 60 fang yan et al. strains isolated in japan avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination prime-boost immunization using a dna vaccine delivered by attenuated salmonella enterica serovar typhimurium and a killed vaccine completely protects chickens from h5n1 highly pathogenic avian influenza virus detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand cross-reactive cellular immune responses in chickens vaccinated with live infectious bronchitis virus vaccine coronaviruses: structure and genome expression enhancement of the immunogenicity of an infectious bronchitis virus dna vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2 isolation and biological properties of avian infectious bronchitis virus isolated from shanxi province cloning and expression of the s1, n and m gene of ibv phylogenetic analysis of s1 gene of infectious bronchitis virus isolates from china multivalent dna vaccine enhanced protection efficacy against infectious bronchitis virus in chickens animal virology genetic analysis revealed lx4 genotype strains of avian infectious bronchitis virus became predominant in recent years in sichuan area the study was funded by the scientific and technological research program of shanxi province (grant no. key: cord-260799-kx6hfpu0 authors: mahmood, zana h.; sleman, rizgar r.; uthman, aumaid u. title: isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east date: 2011-05-12 journal: vet microbiol doi: 10.1016/j.vetmic.2010.12.015 sha: doc_id: 260799 cord_uid: kx6hfpu0 infectious bronchitis virus (ibv) was isolated from trachea and kidney tissues of eight broiler farms in kurdistan region of north iraq from 2008 to 2010. the birds were suffering from respiratory and nephropathological symptoms and lesions. a 1116 bp hyper mutable spike glycoprotein (s1) gene was amplified and sequenced using conventional rt-pcr. sequence analysis and blast homology search in genbank data base indicate that two of the farms were infected with the 4/91 strain, one with an unidentified ibv and five were infected with sul/01/09. the birds in the latter five farms were suffering from nephropathogenic lesions, however, the virus was isolated from kidney but not from trachea in these cases. the birds were vaccinated regularly with 4/91 or ma5 vaccine. the deduced amino acid sequence of the isolated and amplified s1 subunit (372 aa) of sul/01/09 was differed in 27–28% from that of all three vaccine strains (4/91, ma5, and h120) used in the region. this dissimilarity is most likely the cause of poor efficacy of vaccines used in the region, at least in five of these farms. amino acid sequence comparison and phylogenetic tree analysis with other published ibv genotypes indicate that this newly isolated virus together with other regionally related and recently published isolates from israel (is/720/99, is/885) and egypt (egypt/benisuef/01) belong to a new genotype. this is the first report of identification and genotyping of ibv isolate in iraq, which indicate the circulation of 4/91 along with a new variant (sul/01/09) of ibv in vaccinated broiler farms. avian infectious bronchitis (aib) is an acute and highly contagious viral disease of the respiratory system in chickens. it is of significant economic importance because it results in high mortality and poor weight gain in broilers as well as decreasing egg production and quality in layers. in addition to the respiratory and reproductive system, the virus has been associated with nephritis (dhinakar raj and jones, 1997; butcher et al., 2002; cavanagh and naqi, 2003) . the disease is caused by infectious bronchitis virus (ibv), a member of the group 3 of coronavirus species. cdna representing the entire ibv genome has been cloned and sequenced (boursnell et al., 1987) , it comprises of approximately 27.6 kb, a single stranded rna virus of positive sense, encoding several proteins which are associated with rna replication and transcription. all coronaviruses have four structural proteins, spike protein (s), small membrane envelope protein (e), membrane protein (m), and nucleoprotein (n). the spike protein of ibv undergoes post translational cleavage to form s1 and s2 subunits (boursnell et al., 1984; cavanagh et al., 1986) . veterinary microbiology 150 (2011) 21-27 infectious bronchitis virus (ibv) was isolated from trachea and kidney tissues of eight broiler farms in kurdistan region of north iraq from 2008 to 2010. the birds were suffering from respiratory and nephropathological symptoms and lesions. a 1116 bp hyper mutable spike glycoprotein (s1) gene was amplified and sequenced using conventional rt-pcr. sequence analysis and blast homology search in genbank data base indicate that two of the farms were infected with the 4/91 strain, one with an unidentified ibv and five were infected with sul/01/09. the birds in the latter five farms were suffering from nephropathogenic lesions, however, the virus was isolated from kidney but not from trachea in these cases. the birds were vaccinated regularly with 4/91 or ma5 vaccine. the deduced amino acid sequence of the isolated and amplified s1 subunit (372 aa) of sul/01/ 09 was differed in 27-28% from that of all three vaccine strains (4/91, ma5, and h120) used in the region. this dissimilarity is most likely the cause of poor efficacy of vaccines used in the region, at least in five of these farms. amino acid sequence comparison and phylogenetic tree analysis with other published ibv genotypes indicate that this newly isolated virus together with other regionally related and recently published isolates from israel (is/720/99, is/885) and egypt (egypt/benisuef/01) belong to a new genotype. this is the first report of identification and genotyping of ibv isolate in iraq, which indicate the circulation of 4/91 along with a new variant (sul/01/09) of ibv in vaccinated broiler farms. ß 2010 elsevier b.v. all rights reserved. serotype-specific determinant of ibv is thought to be located in the hypervariable regions (hvrs) of s1 glycoprotein (cavanagh et al., 1988) , and it is the s1 subunit of the spike protein which induces neutralizing antibody (cavanagh et al., 1988; koch et al., 1990) . in addition to the well-known massachusetts (mass) serotype of ibv, many other serotypes, distinct from mass, have been isolated in africa (el houadfi et al., 1986; abdel-moneim et al., 2006) , asia (rajeswar et al., 1998; bochkov et al., 2006) , australia (ignjatovic et al., 2006) and europe (capua et al., 1994; worthington et al., 2008) . the majority of these strains are endemic for certain geographic regions. three closely related variants of ibv isolates in israel (is/ 720/99 and is/885) and egypt (egypt/beniseuf/01) have been genotyped by sequencing of the s1 subunit (meir et al., 2004; abdel-moneim et al., 2006) . in spite of the use of three different vaccines (h120, ma5, attenuated 4/91) in poultry farms in iraq, outbreaks have been observed with high mortality in broiler farms having nephropathogenic lesions. since outbreaks of ibv still occur in vaccinated flocks and the virus strains isolated are frequently different from serotypes of the vaccine strains used (liu and kong, 2004; cavanagh et al., 2005; li et al., 2010) , continuous identification of the genotype and production of new generations of vaccines are crucial. in order to investigate ibv genotypes in iraq, where the disease is endemic and widely spread in vaccinated and unvaccinated poultry farms mainly associated with kidney damage and urolethiasis, identification and molecular characterization of ibv (sul/01/09) isolated from eight infected broiler farms was conducted and the deduced amino acid sequence of the s1 subunit of the virus was compared with geographically related isolates. samples: tissue samples from eight suspected ibv outbreaks in sulaimani broiler farms were collected. trachea, lung and kidneys were obtained from 3 to 4 birds in each farm and transferred to the laboratory on ice. the birds suffered from respiratory symptoms and lesions as well as kidney damage (enlargement, congestion, and uroletheasis). the flocks were vaccinated with 4/91 or ma5 vaccine. three different strains of ibv vaccines (h120, ceva), (ma5, intervet) and (4/91, intervet) were used as positive control. serological test: blood samples were collected from 10 birds of each of the eight broiler farms and repeated one week later. the serum was separated; enzyme linked immunosorbent assay (elisa) was conducted for the detection of ibv antibody in clinically suspected farms using ibv antibody test kit (synbiotics, usa). this elisa kit is highly specific in which a titer of (>5 â 10 3 ) indicates the present of ibv infection. rna extraction: 50-100 mg tissue from trachea, lung and kidney of the birds were homogenized in liquid nitrogen. trizol 1 reagent was used to extract the rna from tissue samples according to the manufacturer's instructions. the rnas were dissolved in 40 ml rnase-dnase free distilled water and directly used for subsequent rt-rcr or stored at (à20 8c). virus isolation: tissue homogenate (200 ml) from trachea and kidney of each pcr positive sample was inoculated in the allantoic cavities of 3 spf chicken egg embryos (9-11 days) and incubated for 3 further days at 37 8c and candled daily. after 5 passages, allantoic fluids were collected and rna was extracted for rt-pcr (momayez et al., 2002) . oligonucleotides: oligonucleotides used in this project are illustrated in table 1 . cdna synthesis: superscript tm ii rt protocol (invitrogen) was used according to the manufacturer's instruction, briefly, 1 ml total rna (1 mg/ml) was mixed with 1 ml reverse primer (10 pmole/ul), r-n1221 for n gene or r-s1982 for s gene (table 1) , 1 ml dntps (10 mm each) and rnase free distilled water in a total volume of 12.5 ml mixture. the mixture heated to 65 8c for 5 min then directly chilled on ice. 4 ml from 5â first-strand buffer and 2 ml dithiotheritol (0.1 mm), 0.5 ml (40 units/ ml) rnase inhibitor were added to the mixture. after incubating the mixture at 42 8c for 2 min, 1 ml (200 units/ml) superscript tm ii reverse transcriptase was added and further incubated at 42 8c for 50 min, and finally, the enzyme was inactivated at 70 8c for 15 min. the cdna was stored at 20 8c for the following pcr amplifications. detection of the virus by n gene amplification: a primer pair (f-n784 and r-n1221) was used to detect ibv in clinical samples amplifying 437 bp of n gene. this pair of primers was designed specific to a conserved region of nucleocapsid (n) gene to ensure a wide detection range. the pcr amplification reaction was carried out in 25 ml mixture containing 2.5 ml of 10â pcr reaction buffer, 0.7 ml of 10 mm dntps, 0.5 ml of each of 10 pmol forward table 1 sequences, genome location and the references of primers used in this study. sequences 5 (f-n784) and reverse (r-n1221) primers, 0.1 ml of red hot taq dna polymerase (5 unit/ml) (thermoscientific, uk), 1 ml cdna, the mixture is finalized to 25 ml by the addition of 19.7 ml dnase-rnase free distilled water. amplification was performed with a thermo cycler (eppindorf, usa) at 94 8c for 5 min initial denaturation step, 35 cycles (94 8c for 1 min, 52 8c for 30 s and 72 8c for 1 min) and a final extension at 72 8c for 10 min. pcr amplification of s1 gene: rt-pcr was applied for the first 1230 base of s1 gene which contains the 3 hyper variable regions (hvrs) of s gene (dolz et al., 2006; cavanagh et al., 2005) . the amplification reaction was carried out in 25 ml reaction mixture containing 2.5 ml of 10â pcr reaction buffer, 1 ml of 10 mm dntps, 0.5 ml of 10 pmol forward (f-s1 uni2+) and reverse (r-xce2à) primers, 0.3 ml of 5 u/ml thermoprime taq dna polymerase, and 1 ml cdna. amplification was carried out with a thermal profile at 94 8c for 5 min initial denaturation then 35 cycles (94 8c for 1 min, 50 8c for 30 s and 72 8c for 2 min) amplification with a final extension of 72 8c for 10 min. the primer pair (f-col via2 and r-col via2) (table 1 ) was used to amplify housekeeping gene of chicken collagen via2 as control for cdna synthesis. nucleotide sequencing: about 50 ml of 1230 bp pcr product of amplified s1 subunit from 8 broiler farms were purified using pcr purification kit (qiagen, germany), and sequenced with two forward (f-s1uni2+ and f-xce1+) and one reverse (r-xce2à) primers in innovations biochemical laboratory (ibl), vienna-austria. genbank accession numbers: genbank accession number of the nucleotide and amino acid sequence of sul/01/09 isolate s1 gene reported in this study is (gq281656). genbank accession numbers of ibv sequences used in the analysis are: israel/720/99 (ay091552), is/885 (ay279533), egypt/beniseuf/01 (af395531), ir-1061-ph (ay544778), ir-1062ga (ay544777), 4/91 (af093794), spain/00/338 (dq064814), italy-02(aj457137), qxibv (af193423), h120 (m21970), ma5 (ay561713), m41 (ay561711), connecticut (li8990), and australia (ay775779). clinical signs and virus isolation: all eight clinically suspected farms were serologically positive, even vaccinated farms had a very high titer of antibody ab to 1.4 â 10 4 , in most of the farms the elisa titer was increased in the second sampling, which indicate that reaction is not due to the vaccination. the ibv-ab-test kit used allows differentiating between vaccinated and nonvaccinated farms, depending on the antibody titer. the virus has been detected in all 8 poultry farms. the mortality in these farms was approximately 30% in the first 5 days of the onset of the disease and the birds suffered mainly from kidney damage and urolitheasis. in addition to kidney damage, respiratory signs and lesions were found in two broiler farms, where the disease was further complicated with other infections such as airsacculitis due to e. coli infection, in which 4/91 was isolated, with a mortality rate of approximately 50-60%. these two farms lucked the hygienic measures and the birds had been vaccinated with 4/91 vaccine two weeks before the onset of the disease. the sequences of the isolated viruses from these two vaccinated broiler farms were identical with vaccine strain 4/91. a local isolate sul/01/09 has been detected in five broiler farms, where the birds were suffering from nephropathologic lesions. on culturing the virus in egg embryos, one third of the embryos died after each passage of 4/91 strain. dwarfism has been observed in both 4/91 and sul/01/09 infected embryos. s1 sequence analysis: the first 1116 nucleotides encoding s1 spike glycoprotein subunit including the hyper variable regions were sequenced. comparative analysis of the nucleotides and the deduced amino acid sequence of s1 subunit were performed to assess the relation of sulaimani (sul/01/09) isolate with other sequences and to find similarities with vaccine strains h120, ma5 and 4/91. it has been found that sul/01/09 isolate is 27-28% different from all vaccine strains. however, it only deviates in 6% from egypt/benisuef/01 isolate and 5% from is/885 and israel/720/99 isolates from israel (table 2) . nucleotide sequences of these four isolates are identical with about 96-97% similarity (table 2) . only a few restriction enzymes can be used to differentiate between these closely related isolates (fig. 1) , these enzymes can be used for restriction fragment length polymorphism (rflp) to identify and differentiate between them. multiple sequence alignment of 14 different strains and isolates have been performed using clustal w version 1.83 (www.genome.jp) (fig. 2) , which indicate that the regions at the positions 27-36, 95-114, 164-178, 207-247 and 335-363 of s1 subunit based on 4/91 strain (accession no. af093794) are significantly conserved regions in almost all compared sequences. hyper variable regions (hvrs) of 14 selected s1 amino acid sequences from geographically different regions are located in positions 50-78, 115-148, and 284-292 (fig. 2) . the phylogenetic tree of the aligned amino acids was also produced using mega5 beta version 6.1 online software (www.megasoftware.net) which shows a typical relatedness between sul/01/09 isolate and reference ibv strains from israel, but not with isolates from our neighbor country iran (fig. 3) . poultry farms in iraq are suffering from huge economic losses caused by avian infectious bronchitis disease. in spite of the use of three different vaccine strains (h120, ceva), (ma5, intervet) and (4/91, intervet) the nephropathogenic type of the disease and even 4/91 infection is appearing in vaccinated farms. since iraq suffered from three wars and long time embargos in the last three decades, little has been done to investigate the spread of the disease and the genotype of the causative agent of ibv. a nephropathogenic ibv (sul/01/09) isolate was identified from the tissue of kidney of vaccinated broiler flocks with a mortality rate of approximately 30% at the time of sampling. sequence analysis of sulaimani isolate s1 gene revealed its close relatedness only to three field isolates in israel and egypt (table 2 ). since high rate of antiserum immunized with different homologous field genotype could neutralize each other (shimazaki et al., 2009; wang and huang, 2000) , which suggest genotypes based on s1 sequence analysis to some extent correlate with serotypes of ibv strains, therefore isolates from egypt, iraq and israel can be considered as the same novel middle east genotype, and most likely to be the same serotype. in spite of the long border and a large trade relation with iran, especially import of chicken and chicken products since 2003, it is not clear why iraq's isolate (sul/01/09) differs from isolates from iran (ir-1061-ph, and ir-1062-gh) in approximately 27% amino acids. further investigation is necessary to exclude the existence of iranian isolates in other parts of iraq. since diverse coronaviruses have been detected recently in wild birds (hughes et al., 2009) and it is common among mallard ducks (muradrasoli et al., 2009) , it is very likely that such identical isolates transmitted between iraq, israel and egypt through wild migrated birds, but not through poultry product trade, which does not existing between these countries. further investigation of ibv in migrated wild birds is necessary to elucidate this measure. the low nucleotide and amino acid similarities between the (h120, ma5) massachusetts and 4/91 vaccine with sulaimani isolate may account for the occurrence of the ibv outbreak in vaccinated flocks. production of a new vaccine based on the middle east genotype is necessary to fig. 3 . phylogenetic tree of sul/01/09 isolate and other selected strains and isolates from genbank representing different geographic regions show the relationship among the deduced s1 amino acid sequence of isolates from the middle east (is/885, israel/720/99, and sul/01/09), reference strains from usa (h120, ma5, m41, and connecticut), isolates from europe (spain/00/338, and italy-02), isolates from neighbor country iran (ir-1061-ph, ir-1062-ga), uk strain (4/91), australian t strain, and qxibv from china. protect the poultry industry in iraq. isolation of 4/91 from vaccinated broiler farms may be due to the involvement of the vaccine strain, which has been previously reported (farsang et al., 2002) . multiple sequence alignment and phylogenetic tree analysis separate the isolates into different groups which are genotypically related to each other. most of the serotypes are different from each other in the first hvr (positions 50-78) of s1 spike glycoprotein by more than 20%. this region seems to be conserved in each geographically isolated virus, which can be used for genotyping of the virus. it is most likely that this region is a serotypespecific determinants of ibv and contains antigenic epitope, which is serologically important for serotyping of ibv. amino acid sequences of european, north american or middle east groups are highly homologous in the first hyper variable region of s1 subunit of each group, but differ with other groups (fig. 2) . this data indicates that in addition to 4/91 a new nephropathogenic ibv is circulating in iraq, which belongs to the ibv variant previously isolated in middle east. s1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt molecular analysis of the 793/b serotype of 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during alternate passage in chickens and embryonated eggs infectious bronchitis virus: immunopathogenesis of infection in the chicken antigenic and molecular characterization of isolates of the italy 02 infectious bronchitis virus genotype the isolation and characterization of six avian infectious bronchitis virus isolated in morocco molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain genetically diverse coronaviruses in wild bird populations of north england isolation of a variant infectious bronchitis virus in australia that further illustrates diversity among emerging strains antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during identification of a novel nephropathogenic infectious bronchitis virus in israel isolation and identification of infectious bronchitis virus from commercial chickens broadly targeted multiprobe qpcr for detection of coronaviruses: coronavirus is common among mallard ducks (anas platyrhynchos) seroprevalence study of infectious bronchitis among poultry in tamilnadu serological studies of infectious bronchitis vaccines against japanese field isolates of homologous and heterologous genotypes relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus a reverse transcriptasepolymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from this work was carried out with financial support from kurdistan institute for strategic studies and scientific researches, university of sulaimani, and directorate of veterinary in sulaimani, iraq. key: cord-252048-ftbjsoup authors: mckinley, enid t.; jackwood, mark w.; hilt, deborah a.; kissinger, jessica c.; robertson, jon s.; lemke, cornelia; paterson, andrew h. title: attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus date: 2011-04-22 journal: virus res doi: 10.1016/j.virusres.2011.04.006 sha: doc_id: 252048 cord_uid: ftbjsoup the full-length genomes of 11 infectious bronchitis virus (ibv) field isolates from three different types of the virus; massachusetts (mass), connecticut (conn) and california (cal) isolated over a 41, 25 and 8 year period respectively, were sequenced and analyzed to determine the mutation rates and level of polymorphisms across the genome. positive selection was not detected and mutation rates ranged from 10(−4) to 10(−6) substitutions/site/year for mass and conn ibv types where attenuated live vaccines are routinely used to control the disease. in contrast, for cal type viruses, for which no vaccine exists, positive selection was detected and mutation rates were 10 fold higher ranging from 10(−2) to 10(−3) substitutions/site/year. lower levels of genetic diversity among the mass and conn viruses as well as sequence similarities with vaccine virus genomes suggest that the origin of the mass and all but one of the conn viruses was likely vaccine virus that had been circulating in the field for an unknown but apparently short period of time. the genetic data also identified a recombinant ibv isolate with 7 breakpoints distributed across the entire genome suggesting that viruses within the same serotype can have a high degree of genetic variability outside of the spike gene. these data are important because inaccurate measures of genetic diversity and mutation rates could lead to underestimates of the ability of ibv to change and potentially emerge to cause disease. avian infectious bronchitis virus (ibv) a gamma-coronavirus in the coronaviridae family causes a highly contagious upperrespiratory tract disease of domestic chickens characterized by coughing, sneezing and tracheal rales. serotype-specific attenuated live vaccines are routinely used to control the disease, yet ibv remains one of the most widely reported respiratory diseases of chickens worldwide resulting in significant economic loss due to reduction in weight gains and feed efficiency, and condemnations at the processing plant. the virus can also cause permanent oviduct damage in layers and some strains of the virus cause nephritis, which when severe can result in significant mortality. infectious bronchitis virus is an enveloped, positive-sense single-stranded rna virus with a genome size of approximately 27 kb (holmes, 1991) . the 5 two-thirds of the genome, approximately 21 kb, encodes two large overlapping open reading frames (orfs), 1a and 1ab, which encode 15 non-structural proteins (nsps), nsps 2-16 (ibv does not have nsp 1) including a papainlike protease 2 (the pl1 protease is truncated and non-functional in ibv), a main protease, the rna-dependent rna-polymerase (rdrp) and several other non-structural proteins involved in making up the replication transcription complex. the remaining one-third of the genome encodes four structural proteins; spike (s), envelope (e), membrane (m) and nucleocapsid (n), and a number of nonstructural proteins. the spike glycoprotein of ibv forms projections on the surface of the virion. spike is post-translationally cleaved into s1 and s2 subunits with the s1 subunit forming the outermost portion and s2 forming a stalk like structure that is embed in the viral membrane. the s1 subunit contains hypervariable regions that play a role in attachment to host cell receptors, membrane fusion and entry into the host cell, and it contains conformationally-dependent virus-neutralizing and serotype-specific epitopes (cavanagh et al., 1998; niesters, 1987 it is extremely difficult to control ibv because little or no cross protection occurs between numerous different types of the virus. coronaviruses evolve by a process of natural selection working on mutations and recombination events (domingo and holland, 1997; moya et al., 2004; vijaykrishna et al., 2007) . these factors contribute to diverse subpopulations of the virus that continually emerge to form new and variant ibv types. in the absence of a specific vaccine, the mutation rate for the hypervariable region in the s1 gene of the 793/b type of ibv was reported as 3 × 10 −3 substitutions/site/year (cavanagh et al., 1998) while the mutation rate for the ga98 virus, which emerged as a result of selection pressure driven by vaccine usage, was reported to be 1.5 × 10 −2 substitutions/site/year in the hypervariable region of s1 (lee and jackwood, 2001) . sequence alignments and phylogenetic reconstructions indicate that recombination events play a significant role in coronavirus evolution. recombination in ibv was demonstrated with coinfection of different ibv types in embryonated eggs (kottier et al., 1995a) . sequence analysis of the s1 gene from field isolates of ibv also revealed evidence of multiple recombination events (jia et al., 1995; wang et al., 1993) and that recombination occurred between vaccine viruses (mondal and cardona, 2007) . in a recent study, recombination in ibv, which replaced the spike gene with an unknown sequence, likely from another coronavirus, resulted in the emergence of turkey coronavirus (tcov) a new enteric disease of young turkeys (jackwood et al., 2010) . characterization of ibv is based on analysis of the spike glycoprotein by either serotyping or genotyping methods (cavanagh and gelb, 2008) . however, pathogenicity in ibv is associated with spike as well as genes outside of spike (ammayappan et al., 2009; cavanagh, 2007; hodgson et al., 2004) . because typing is focused on the analysis of spike, there is no clear understanding of whether viruses with similar spike proteins also share high sequence similarities in other regions of the genome especially in regions associated with pathogenicity. this gap in knowledge results from the lack of complete genome sequences from viruses of the same serotype. the objective of this study was to determine the levels of polymorphism across the entire genome of ibv isolates with similar spike genes and to examine the mutation rates for viruses with and without vaccine selection pressure. we sequenced the full-length genome from 11 ibv field isolates of three different serotypes, massachusetts (mass), connecticut (conn) and california (cal) isolated over a 41, 25 and 8-year period respectively. the genome sequences were aligned and phylogenetic analysis was conducted to examine genetic relationships between the viruses. estimates of the rates of mutation were calculated and evidence of recombination was investigated for each of the full-length genome sequences. to our knowledge, this is the first study to identify polymorphisms and determine mutation rates using full-length genome sequences of ib viruses isolated over a multi-year time span. in this study, we sequenced mass viruses isolated in 1965 mass viruses isolated in , 1972 mass viruses isolated in , 1979 mass viruses isolated in and 1985 obtained from dr. jack king (southeast poultry research laboratory, athens, ga) and one isolated in 2006 obtained from dr. pedro villegas (poultry diagnostic and research laboratory, athens, ga). in addition, conn viruses isolated in 1966 , 1972 , 1983 and 1991 obtained from dr. king, and cal viruses isolated in 1995 and 2003 obtained from dr. peter woolcock (california animal health and food safety laboratory system, university of california, fresno, ca) were sequenced. the specific viruses were all isolated from broiler chickens with clinical signs of disease. the viruses were propagated in embryonated eggs as described (gelb and jackwood, 2008) . the strains and embryo passage number are; ibv/mass/65 (passage 1), ibv/mass/72 (passage 7), ibv/mass/79 (passage 2), ibv/mass/85 (passage 15), ibv/mass/06 (passage 3), ibv/conn/66 (passage 3), ibv/conn/72 (passage 7), ibv/conn/83 (passage 15), ibv/conn/91 (passage 1), cal99/ne15172/95 (passage 3), cal/ca557/03 (passage 5). 2.2. viral rna extraction, amplification and genome sequence determination viral rna extraction, reverse transcription and polymerase chain reaction (rt-pcr), library construction and sequencing were conducted as previously described (jackwood et al., 2010) . briefly, the viruses were filtered through a 0.8 m filter then through a 0.22 m filter (millipore, billerica, ma) and viral rna was purified using the high pure rna isolation kit according to the manufacturer's recommendation (roche diagnostic corporation, foster city, ca). the rt-pcr amplifications were performed with the takara rna la pcr kit (takara bio inc., otsu, shiga, japan) using a random primer and an amplification primer in a strand displacement amplification reaction following the manufacture's protocol. the sequence of the random reverse transcription primer was 5 -agcgggggttgtcgaatgtttgannnnn-3 , and the amplification primer sequence was 5 -agcgggggttgtcgaatgtttga-3 . both primers were obtained from integrated dna technologies, inc. (idt, coralville, ia). a master mix for the rt reaction was prepared, which included mgcl (5 mm), 10x rna pcr buffer (1×), dntp mixture (1 mm), rnase inhibitor (1 units/l), reverse transcriptase (.25 units/l), 5 degenerate primer (2.5 m), and rna (5.75 l/reaction) then 10 l per sample was aliquoted in a thermocycler tube. the rt reaction conditions were 10 min at 30 • c for the primer annealing then 1 h at 50 • c for extension followed by a five-minute incubation at 99 • c for inactivation of the enzyme and a 5-min period at 5 • c. a pcr master mix, which included at the final concentrations: mgcl (2.5 mm), 10x la pcr buffer (1×), sterilized distilled water (32.25 l), takara la taq (1.25 u/50 l), and 5 primer (0.2 m) was prepared and 10 l of the rt reaction was added to 40 l of the mix. the amplification reaction consisted of a 94 • c step for 2 min followed by 30 cycles of 94 • c for 30 s, 60 • c for 30 s, and 72 • c for 3 min. ten pcr reactions were combined for each virus and purified using the qiaquick pcr purification kit (qiagen, foster city, ca) then run on a 1% agarose gel. the pcr products were size selected by removing amplicons between 500 and 1500 bp from the gel, followed by purification using the qiaquick (qiagen) gel purification kit. the pcr products were cloned into the pcr-xl-topo vector (invitrogen, life technologies, carlsbad, ca) according to the manufacturers recommendations and transformed into one shot topo electrocompetent e. coli cells (invitrogen) using 30 l of competent cells mixed with 2 l of the ligation reaction. electroporation settings were 20 kv and 200 using a biorad gene pulser (bio-rad, hercules, ca), and the electroporated cells were incubated at 37 • c in 480 l of super optimal broth medium for 1 h on a rotary shaker. the cultures were mixed with 70% glycerol and frozen at −80 • c until plated on q-trays (genetix, boston, ma) containing liquid broth agar cat#3002-032 (mp biomedicals, llc, solon, oh) with 50 g/ml of kanamycin. the q-trays were pre-warmed at 37 • c before the entire culture (approximately 500 l) was spread on the plates and incubated overnight at 37 • c, then robotically picked with a q-bot (genetix, boston, ma). plasmid dna from the libraries of cloned cdna fragments for each virus were isolated using an alkaline lysis method modified for the 96-well format, and incorporating both hydra and tomtek robots (http://www.intl-pag.org/11/abstracts/p2c p116 xi.html). cycle sequencing reactions were performed using the bigdye tm terminator ® cycle sequencing kit version 3.1 (applied biosystems, foster city, ca) and mj research (watertown, ma) thermocyclers. finished reactions were filtered through sephadex filter plates into perkin-elmer microamp optical 96-well plates. a 1/12strength sequencing reaction on an abi 3730 was used to sequence each clone from both the 5 and 3 ends. each viral genome was sequenced to approximately 10× coverage. the accuracy of the sequence is insured by generating sequence reads for both strands. gaps and areas with <2× coverage were identified and specific primers were synthesized (idt) for rt-pcr amplification and sequencing of the ambiguous areas. the rt-pcr was conducted as described above, and the reaction conditions were 42 • c for 60 min, 95 • c for 5 min, then ten 10 cycles of 94 • c for 30 s, 50 • c for 30 s, 68 • c for 90 s, followed by 25 cycles of 94 • c for 30 s, 50 • c for 30 s, 68 • c for 90 s + 5 s/cycle added. the final elongation step was 68 • c for 7 min then the reaction was cooled to 4 • c. the pcr products were directly sequenced in both directions using the amplification primers at a concentration of 15 ng/reaction and the abi prism bigdye terminator v3.0 (applied biosystems, foster city, ca). the amount of cdna added to the reaction ranged from 20 to 30 ng and the sequencing reactions were analyzed on an abi 3730 (applied biosystems). the extreme 5 end of each genome was obtained using the 5 race system for rapid amplification of cdna ends, according to manufactures protocol (invitrogen, life technologies, carlsbad, ca). the primer design was based upon an alignment of ibv sequences. the race primers are designated; ibv 5 race sp1 (5 -cgtatagaaaaacaaagcgtcac-3 ), ibv 5 race sp2 (5 -gtcactgtctattgtatgtctgctc-3 ) and ibv 5 race sp3 (5 -tagccgaccttatgcgagaacg-3 ). the extreme 3 end of each genome was obtained using primers designated; m41 3 end reverse (5 -gctctaactctatactagccta-3 ) and genome 3 f 900 bp (5 -tgacaagatgaatgaggaaggtat-3 ). sequences for some viruses were verified and sequence gaps were filled by amplification using overlapping primer pairs. briefly, the primer design tool located at http://www2.eur.nl/fgg/kgen/primer/overlapping primers.html was used with a template genome sequence of a virus with the same serotype as the virus to be sequenced. thirty-two overlapping primer pairs (available upon request) were synthesized (idt) for each virus and used to amplify the genome. the amplicons were sequenced directly using the amplification primers and the abi prism bigdye terminator v3.0 (applied biosystems) as described above. the sequence reads for each virus were assembled using the seqman program (dnastar, inc. madison, wi) and the orf finder at the national center for biotechnology information (ncbi, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict open reading frames. genome annotation was entered using the annotation editor seqbuilder (dnastar, inc.). the blastn program (http://www.ncbi.nlm.nih.gov/blast/) was used to search genbank (national center for biotechnology information, http://www.ncbi.nlm.nih.gov/) for similar ibv sequences. the megalign program implemented in dnastar was used to align sequences and to calculate percent identity and number of substitutions between selected sequences. whole genome alignments were generated using clustalw and phylogenetic trees were constructed with the neighbor-joining method, minimum evolution method, maximum parsimony method, and upgma with 1000 bootstrap replicates (mega4, http://www.megasoftware.net/index.html (tamura et al., 2007) ). to reconstruct evolutionary trees, maximum likelihood (tree-puzzle, http://www.tree-puzzle.de/) and bayesian analysis (beast 1.4, http://beast.bio.ed.ac.uk/) were conducted. a codon-based test of positive selection (z-test, mega4) was used to analyze the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) in the spike glycoprotein. because only 2 cal type viruses were sequenced herein, additional reference sequences for the cal type viruses were included in the codon-based test (z-test) of positive selection; cav/cav56b/91 (af027509), cav/cav9437/95 (af027510), cal99/cal99/99 (ay514485) and ca/ca1737/04 (eu925393). to determine mutation rates, the full length genomes as well as the coding sequences for orf1ab, s1, e, m and n gene from each virus within the same virus type were aligned using clustalx 2.0.8 (larkin et al., 2007) and manually edited in jalview (waterhouse et al., 2009) . the bayesian markov chain monte carlo (mcmc) implemented in beast v1.4.8 (drummond and rambaut, 2007) was used to estimate mutation rates of the aligned sequences and the sampling dates for each isolate were used as calibration points. the mutation rates were analyzed using the general time-reversible (gtr) (rodriguez et al., 1990 ) substitution model with gamma-distributed among site rate variation with four gamma rate categories (yang, 1994 ). the srd06 model was used to partition codon positions and an uncorrelated lognormal relaxed clock model was selected. each mcmc chain was run for 10 million states and sampled at every 1000 states. tracer v1.4.1 (http://tree.bio.ed.ac.uk/software/tracer/) was used to confirm convergence of mcmc chains with 10% of each chain discarded as burn-in. complete genome nucleotide sequences for cal99/ne15172/95, ibv/ck/ca99/99, ca/ca557/03, ibv/conn/66, ibv/conn/72, ibv/conn/83, ibv/conn/91, ibv/mass/65, ibv/mass/72, ibv/mass/79, ibv/mass/85, ibv/mass/06 and the ibv/ck/a2 china strain (used as out-group) were aligned using clustalx 2.0.8 (larkin et al., 2007) . to identify recombinants as well as major and minor parents, the data set was scanned using a recombination detection program (rdp) v2 with implemented algorithms geneconv, bootscan, maxchi, chimera and siscan. similarity plot and bootscan analyses were performed using the simplot program (lole et al., 1999) to further identify recombination events and recombination breakpoints. the window width and step size were set to 1000 bp and 40 bp, respectively. genome sequences generated in this study were submitted to the genbank database and assigned the follow the full-length genome sequence of 11 ibv isolates from the field obtained over a 41 year period (5 mass table 1 ). genomic libraries and rt-pcr fragments from overlapping specific primer pairs were used to assemble genomic sequences with seqman pro and genome annotation was performed using seqbuilder (dnastar, inc., v.8.0.2, madison, wi), to reveal a typical gamma coronavirus gene order for all of the viruses; 5 utr, orf1a/ab, spike glycoprotein, orf3a, orf3b, envelope protein, membrane protein, orf 4b, orf 5a, orf 5b, nucleocapsid protein, orf 6b, 3 utr and a poly(a) tail. the full-length genome sequences were aligned using clustalw and all the phylogenetic trees constructed with the neighbor-joining method, minimum evolution method, maximum parsimony method, and upgma using mega 4.0.2 had similar topography (tamura et al., 2007) . a representative tree with the 11 viral genomes sequenced herein and other ibv full-length genomes available in genbank is presented in fig. 1 . the nucleotide sequence similarities between the genomes of the viruses sequenced herein are from 93.8% to 94.9% for the cal isolates, 99.8% to 99.9% between the conn isolates and 92.4 to 100% between the mass isolates. to determine the relationship between different virus types for sequences in specific areas across the genome, phylogenetic analysis of the nucleotide sequences of the 5 and 3 utrs and the amino acid sequences of individual orfs was conducted (fig. 2) . the spike glycoprotein, which is used to define ibv isolate type, shows individual groupings for conn, mass and cal viruses with the exception of the ibv/mass/06 virus, which is positioned as an offshoot of the conn group. similar groupings were observed for the envelope and nsp 3 (pl2) protein trees, whereas ibv/mass/06 clearly grouped with conn type viruses in the membrane, nucleocapsid, nsp 5 (mpro) and nsp 11/12 (rdrp) protein trees (fig. 2) . a blastp search showed the ibv/mass/06 virus spike glycoprotein to have 98% similarity to h120 (acc. no. acq55230), which is a commonly used mass type vaccine. a blastp search and phylogenetic analysis of the other mass spike glycoprotiens identified mass type attenuated viruses (acc. nos. eu283075, eu283081, eu283082), which grouped together (data not shown). a blastp search and phylogenetic analysis of the conn spike glycoproteins identified conn attenuated viruses (acc. nos. eu283059, eu283061 and eu283062), which also grouped together (data not shown). the phylogenetic analysis of the 5 utr showed individual groupings for conn, mass, and cal viruses, however the ibv/mass/06 virus grouped with the cal viruses (fig. 2) . in the 3 utr, the cal/ca557/03 is an offshoot of the mass group, the ibv/mass/06 virus is an offshoot of the conn group, and cal99/ne15172/95 was clearly distinct from the other viruses. a blast analysis of the cal99/ne15172/95 virus 3 utr showed that it was 99% identical to the de072 strain of ibv (acc. no. af203002), a delaware type virus. although the 3 utrs sequence divergence was as high as 13.5% among the viruses, sequence alignment of the stem-loop (s2m) region in the 3 utr revealed identical sequences among cal99/ne15172/95, ibv/conn/66, ibv/conn/72, ibv/conn/83, and ibv/conn/91, one nucleotide difference (nt. position 9: a to g) in the cal/ca557/03 and ibv/mass/79 viruses, and two nucleotide differences (nt. positions 9: a to g and 26: a to c) in the ibv/mass/65, ibv/mass/72 and ibv/mass/85 viruses while ibv/mass/06 had an major a and a minor g at nt. position 36 (data not shown). the optimal tree with the sum of branch lengths is shown. the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. a bootstrap consensus tree was constructed from 1000 replicates (percentage of replicate trees in which associated strains clustered together are presented at nodes). the p-distance scale is presented at the bottom of the figure. the analyses were constructed using mega4 (tamura et al., 2007) . to further examine the relationship of the ibv/mass/06 virus with the other viruses examined in this study as well as selected ibv genomes available in genbank we conducted a recombination detection program (rdp) analysis, which indicated that the ibv/mass/06 isolate was a recombinant with conn and cal viruses as the potential major parents (data not shown). the recombination events in ibv/mass/06 were identified using simplot, which showed that the ibv/mass/06 genome shared a high sequence similarity, 97-100%, with cal and conn isolates in different regions of orf1ab, with mass in the s1 gene and with conn isolates in the remainder of the 3 end of the genome (fig. 3a) . at the extreme 3 end of orf1ab, between nucleotides 15,500 and 19,800, the ibv/mass/06 virus did not show any significant sequence similarity with any of the genomes in this study. a blast analysis of that region showed 99% similarity with ibv strain h120 (acc. no. fj807652). the simplot bootscan analysis revealed a total of seven breakpoints located at approximate nucleotide positions 1530, 5895, 8188, 11,200, 12,266, 19,918 and 21,975 in the ibv/mass/06 virus (fig. 3b) . the sequences located between the breakpoints were designated regions i-viii, respectively. regions one through five were located in nsp2, nsp3, nsp4, nsp9 and nsp11/12 respectively. region vii contained the entire s1 glycoprotein gene, whereas the s2 glycoprotein gene was in region viii. evidence of recombination in the ibv/mass/06 isolate was further confirmed when phylogenetic trees constructed from the regions delineated by the breakpoints showed incongruence (fig. 4) . the ibv/mass/06 isolate grouped with mass type viruses in region vii, which contained the s1 gene. however, ibv/mass/06 grouped with cal type viruses in regions i, iii and v, and with conn type viruses in regions ii, iv and viii. in region vi, ibv/mass/06 did not group with any of the genomes included in this study. as indicated above, a blast search of the ibv/mass/06 virus sequence in that region showed that it was most closely related to the h120 mass type vaccine virus. fig. 2 . infectious bronchitis virus amino acid sequences a through j as indicated in the figure, were aligned with clustalw and the phylogenetic trees were constructed using the neighbor-joining method. the optimal trees with the sum of branch lengths are shown. the trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances (the p-distance scale is presented at the bottom of each tree). bootstrap consensus trees were constructed from 1000 replicates and the percentage of replicate trees in which associated strains clustered together are presented at the nodes. the analyses were conducted using mega4 (tamura et al., 2007) . a codon-based test of positive selection (z-test, mega4), which measures the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) showed that positive selection was occurring in the cal viruses and between ibv/conn/66 and other conn viruses but not in mass type viruses (table 1 ). the beast v1.4.1 software, used to estimate the mutation rates for individual coding regions within each virus type, indicated that the cal isolates had mutation rates that were 2-4 orders of magnitude higher than the mass and conn isolates (data not shown). data for the fulllength genomes and the s1 glycoprotein are presented in table 2 . the mutation rate for the orf1ab, s1 and n gene was calculated to be 10 −3 substitutions/site/year and 10 −2 for the e and m genes from the cal viruses, whereas the conn and mass viruses had mutation rates that were 10 fold lower and ranged from 10 −4 to 10 −6 in all of the coding regions except for the e gene in the conn viruses, which did not show any polymorphisms. the majority of emerging infectious diseases are caused by rna viruses. presumably, high rates of mutation and rapid replication drive their evolution. infectious bronchitis virus, like most rna viruses, undergoes a high rate of genetic change that contributes to diverse subpopulations of the virus that continually emerge to form new variants of existing strains as well as completely new types of the virus. new variants and types of ibv are defined by the sequence of the spike glycoprotein and/or by specific antibodies against spike, and for many ibv types, only spike sequences are available. to examine sequence changes not only in spike but also over the entire length of the genome, we sequenced the full-length genomes of 5 mass type, 4 conn type and 2 cal type ibv field viruses that were isolated over a 41, 25 and 8 year period respectively. all of the viruses were obtained from chickens with clinical signs of the disease. alignments of the full-length genome sequences showed that the mass viruses had ≥92.4% identity (≥93.7% if the recombined ibv/mass/06 is not included), the cal viruses had ≥93.8% identity and the conn viruses varied the least, at ≥99.8% identity. examination of the mutation rates for the orf1ab, s1, e, m and n genes showed, in general, that the cal viruses had rates between 10 −4 and 10 −2 substitutions/site/year, whereas the conn and mass viruses had lower mutation rates that ranged from 10 −4 to 10 −6 substitutions/site/year. in addition, the e gene in the conn viruses showed no signs of change. although the egg passage level of all the viruses used in this study was relatively low (≤15 passages) it is possible some genetic changes could have been introduced as a result of propagation in embryonated eggs. however phenotypic change is likely to be minimal since it typically takes 75 or more egg passages to attenuate ibv and in a study that examined genetic changes in the highly variable s1 gene following 10 passages in embryonated eggs, five viruses had no changes, two viruses had only 2 nucleotide changes one virus had only 1 change (mckinley et al., 2008) . it has been shown that vaccine usage can result in faster evolutionary rates in ibv field viruses (lee and jackwood, 2001; wang fig. 4 . the nucleotide sequences a through h as indicated in the figure, were aligned with clustalw and the phylogenetic trees were constructed using the neighbor-joining method (saitou and nei, 1987) . the optimal trees with the sum of branch lengths are shown. the trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances (the p-distance scale is presented at the bottom of each tree). bootstrap consensus trees were constructed from 1000 replicates and the percentage of replicate trees in which associated strains clustered together are presented at the nodes. the analyses were conducted using mega4 (tamura et al., 2007) . mutation and evolutionary rates of the genome and s1 genes for california (cal), connecticut (conn) and massachusetts (mass) isolates. mutation rate (nucleotide changes/year) evolutionary rate (amino acid changes/year) genome a s1 gene b genome a s1 gene b cal 1.0 × 10 −2 9.4 × 10 −4 2.0 × 10 −2 2.4 × 10 −3 conn 1.5 × 10 −4 1.7 × 10 −4 2.6 × 10 −4 3.5 × 10 −4 mass 1.9 × 10 −3 2.4 × 10 −4 3.3 × 10 −3 5.8 × 10 −4 a viruses used in the genome analysis were the cal, conn and mass viruses shown in fig. 1 . b viruses used in the s1 gene analysis are the same viruses shown in table 1 . et al., 1993) . thus, it was surprising that mutation rates for mass and conn type viruses were lower than mutation rates for cal type viruses because live attenuated vaccines have been used against the mass and conn type viruses in poultry for many years, whereas no vaccine exists for the cal type viruses. one possible explanation for the differences in mutation rates is that the mass and conn viruses were possibly re-isolated vaccine viruses that have been circulating in the field for an unknown but relatively short period of time, with the exception of the ibv/conn/66 virus, which did show positive selection when compared to the other conn viruses. in contrast, because no vaccine exists for cal viruses, those viruses represent true field isolates that have been circulating for known periods of time. the possibility that the origin of the mass and conn isolated viruses is vaccine, is supported by lower levels of genetic diversity observed for many of the genes within each virus type (fig. 2) as well as the genetic similarity of the viruses, previously characterized as attenuated viruses. it has been shown that ibv can be shed for long periods of time and that vaccine viruses can persist in the field and revert to cause disease (farsang et al., 2002; jackwood et al., 2009; matthijs et al., 2008; naqi et al., 2003) . thus, it is possible that vaccine viruses are more likely to be isolated from the field because they may be more abundant having displaced field viruses of the same type or because they are more easily isolated since they are adapted to grow in embryonated eggs. because the mass and conn type viruses were isolated from clinically diseased chickens suggests that if they were originally vaccine viruses then they have likely acquired the sequence changes necessary for pathogenicity. further studies would need to be conducted on vaccine viruses and pathogenic strains to gain a better understanding of the molecular changes necessary for coronavirus reversion to pathogenicity. another explanation for lower mutation rates in mass and conn viruses could be that some of the mutations observed are incidental, meaning that they are not deleterious and provide no selective advantage to the virus rather than due to selection pressure. this possibility was suggested in another study where no evolution was detected in the 5 hypervariable region of the s1 gene for a number of 793/b type viruses isolated over a 11 year period (cavanagh et al., 1998) . this however does not explain why polymorphisms were observed for the cal type viruses. recombination contributes to the genetic diversity of coronaviruses and can lead to the emergence of new viruses and outbreaks of new diseases (holmes, 2009; jia et al., 1995; woo et al., 2009) . although the ibv/mass/06 virus is identified as a mass type virus based on characterization of the spike glycoprotein, it was shown herein to be a recombinant virus with conn and cal viruses as major parents. eight regions defined by 7 breakpoints distributed across the entire genome were identified. region vii was similar to other mass type viruses, which was expected because that region includes the entire s1 gene. however, cal like sequences were found in regions i, iii and v, conn sequences were found in regions ii, iv and viii, and region vi was found be similar to the h120 mass type vaccine strain. high frequencies of recombination including recombination events between vaccine and field strains have been reported for ibv (brooks et al., 2004; cavanagh et al., 1992; fang et al., 2005; jackwood et al., 2010; kottier et al., 1995b; lee and jackwood, 2000; yu et al., 2001) . in addition, ibv pathogenicity was shown to be polygenic, involving spike as well as replicase proteins (ammayappan et al., 2009; armesto et al., 2009; cavanagh, 2007; hodgson et al., 2004) . although ibv type is defined by the spike glycoprotein, our data shows that viruses within the same type can have vastly divergent sequences in other regions of their genome including regions that contain genes important for pathogenicity. if recombination occurs outside of the spike gene where a pathogenic field virus acquires attenuated vaccine virus sequences, the outcome would likely be a relatively benign virus regardless of the virus types (spike sequences) involved. furthermore, if a vaccine virus circulating in a flock recombines with a pathogenic virus outside of the spike gene, resulting in the vaccine virus becoming pathogenic, the outcome is still likely to be of little or no consequence since the birds would presumably be immune to the unchanging spike. however, in the event that a pathogenic field virus recombines with either a vaccine virus or another pathogenic virus and acquires a new or unique spike glycoprotein gene, the outcome could be the emergence of a new virus capable of causing disease. this was recently shown for tcov (jackwood et al., 2010) . although new coronaviruses can emerge and cause disease as a result of recombination it appears that the emergence of most variant or new ibv types is due to accumulation of mutations in the spike glycoprotein over time (cavanagh et al., 2005; jackwood et al., 2005 jackwood et al., , 2007 lee and jackwood, 2001; nix et al., 2000) . thus, until pathogenicity genes can be specifically identified, monitoring the spike gene in ibv appears to be the most reliable measure of genetic change leading to the emergence of new viruses capable of causing disease. this is the first study to identify genetic changes and mutation rates using full-length genome sequences of ibvs isolated over many years. our data show that mutation rates are lower and that positive selection was not occurring for ibv types where attenuated live vaccines are used compared to an ibv type were no vaccine exists, suggesting that the virus isolates examined were re-isolated vaccine viruses that have been circulating in the field for a relatively short-period of time. this observation is extremely important because it could lead to incorrect estimates of mutation rates for rna viruses where modified live vaccines are used to control the disease. in this study, we also identified a recombinant ibv isolate with two major parents and no fewer than 7 breakpoints distributed across the entire genome. although recombination among coronaviruses can contribute to genetic diversity of the virus and recombination has been shown to lead to the emergence of new viruses, it appears that monitoring mutations in the spike gene that accumulate over time is currently the best method of identifying potentially new genetic variants and ibv types capable of causing disease. identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi the replicase gene of avian coronavirus infectious bronchitis 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genetic diversity generated by recombination among avian coronavirus ibv origin and evolution of georgia 98 (ga98), a new serotype of avian infectious bronchitis virus full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination transmissibility of infectious bronchitis virus h120 vaccine strain among broilers under experimental conditions avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination genetic and phenotypic characterization of the california 99 (cal99) variant of ibv the population genetics and evolutionary epidemiology of rna viruses establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens molecular epidemiology of infectious bronchitis virus emergence of subtype strains of the arkansas serotype of infectious bronchitis virus in delmarva broiler chickens the general stochastic model of nucleotide substitution the neighbor-joining method: a new method for reconstructing phylogenetic trees mega4: molecular evolutionary genetics analysis (mega) software version 4.0 evolutionary insights into the ecology of coronaviruses evidence of natural recombination within the s1 gene of infectious bronchitis virus jalview version 2 -a multiple sequence alignment editor and analysis workbench coronavirus diversity, phylogeny and interspecies jumping maximum likelihood phylogenetic estimation from dna sequences with variable rates over sites: approximate methods molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia abbreviations: ibv, infectious bronchitis virus; nsps, nonstructural proteins; orf, open reading frame; pbs, phosphate buffered saline; rdrp, rna-dependent rna-polymerase; rt-pcr, reverse transcriptase-polymerase chain reaction; utr, untranslated region. this research was supported by usda, csrees award number 2007-35600-17786. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.04.006. key: cord-003148-o7y3wygc authors: shirvani, edris; paldurai, anandan; manoharan, vinoth k.; varghese, berin p.; samal, siba k. title: a recombinant newcastle disease virus (ndv) expressing s protein of infectious bronchitis virus (ibv) protects chickens against ibv and ndv date: 2018-08-10 journal: sci rep doi: 10.1038/s41598-018-30356-2 sha: doc_id: 3148 cord_uid: o7y3wygc infectious bronchitis virus (ibv) causes a highly contagious respiratory, reproductive and urogenital tract disease in chickens worldwide, resulting in substantial economic losses for the poultry industry. currently, live-attenuated ibv vaccines are used to control the disease. however, safety, attenuation and immunization outcomes of current vaccines are not guaranteed. several studies indicate that attenuated ibv vaccine strains contribute to the emergence of variant viruses in the field due to mutations and recombination. therefore, there is a need to develop a stable and safe ibv vaccine that will not create variant viruses. in this study, we generated recombinant newcastle disease viruses (rndvs) expressing the s1, s2 and s proteins of ibv using reverse genetics technology. our results showed that the rndv expressing the s protein of ibv provided better protection than the rndv expressing s1 or s2 protein of ibv, indicating that the s protein is the best protective antigen of ibv. immunization of 4-week-old spf chickens with the rndv expressing s protein elicited ibv-specific neutralizing antibodies and provided complete protection against virulent ibv and virulent ndv challenges. these results suggest that the rndv expressing the s protein of ibv is a safe and effective bivalent vaccine candidate for both ibv and ndv. generation of rndvs expressing s1, s2 or s protein of ibv. the expression cassettes containing the codon optimized s1, s2, s and non-codon optimized s genes of ibv were cloned into the cdna encoding the complete antigenome of ndv strain lasota, using the pmei site, between p and m genes (fig. 1) . the correct sequences of genes cloned into full length cdna of ndv were confirmed by nucleotide sequence analysis. infectious recombinant ndvs containing s1, s2 and s genes of ibv were recovered from all cdnas. the sequences of s1, s2 and s genes present in the rndvs were confirmed by rt-pcr. to evaluate genetic stability of rndv expressing codon optimized s protein, the viruses were passaged five times in 9-day-old embryonated specific pathogen free (spf) chicken eggs. the nucleotide sequence analysis of the s gene showed that the inserted orf were maintained without any adventitious mutations. evaluation of the expression of the s1, s2 and s proteins of ibv. the expression of codon optimized s2, and s proteins and non-codon optimized s protein of ibv strain mass-41 by rndv constructs was detected by western blot analysis in df-1 cells using a chicken polyclonal anti ibv serum ( fig. 2a -upper panel and b). as the expression of non-codon optimized s was not detected clearly in the first attempt ( fig. 2a) , we detected it in another attempt (fig. 2b) . the expression level of codon optimized s protein of ibv was significantly higher than that of the non-codon optimized s protein of ibv. for the codon optimized s protein of ibv expressed from rndv ( fig probably represent uncleaved s protein (s0) or polymeric forms of s protein. the ~95 kd band represents s2 or s1 subunit of cleaved s protein of ibv. in the case of rndv/ibv-s2 strain ( fig. 2a-lane 1) , there are two bands (~170-220 kda) on top, representing polymeric folded forms of s2 protein, a ~105 kda band and a ~95 kda band representing s2 subunit. the expression of s2 protein from a transcription cassette in which the signal peptide sequences of s protein was not fused with s2 gene was not detected (data not shown). lane 4 of fig. 2a and lane 3 of fig. 2b represent rndv as control. lane 5 of panel a represents non-infected df-1 cells. these results showed that codon optimized s and s2 proteins of ibv were expressed efficiently. the non-codon optimized s protein was also expressed from rndv, but not efficiently and not consistently. a monoclonal anti-ndv/hn antibody was used to detect a ~70 kda of hn protein of ndv in lysates, confirming similar level of ndv protein in each lane ( fig. 2a-lower panel) . we further evaluated incorporation of ibv s and s2 proteins into ndv virions. the rndvs expressing codon optimized s and s2 proteins and rndv expressing non-codon optimized s protein were inoculated into eggs, 3 days after inoculation, viral particles in infected allantoic fluid were partially purified and analyzed by western blot (fig. 2c-upper panel) . two bands (~170-220 kda) on top, representing s protein, a ~95 kda band and a ~60 kda band representing s2 or s1 subunit of cleaved s protein, were detected in purified particles of rndv expressing codon optimized s protein by western blot analysis (fig. 2c-lane 2) . the lane 4 of fig. 2c shows two bands (~170-220 kda) on top, representing polymeric folded of s2 protein, a ~105 kda band and a ~95 kda band representing s2 subunit. the lane 1 of fig. 2c represents purified rndv control and lane 3 of fig. 2c shows purified rndv expressing non-codon optimized s protein. these results suggested that the codon optimized s and s2 proteins of ibv expressed by rndvs were incorporated into rndv particles. a monoclonal anti-ndv/hn antibody was used to detect a ~70 kda of hn protein of ndv in partially purified virions, confirming similar level of ndv protein in each lane (fig. 2c-lower panel) . the expression of codon optimized s1 protein expressed from four individual rndv constructs were detected by western blot analysis in lysates (fig. 3a) and supernatant (fig. 3b ) of infected df-1 cells, using a chicken polyclonal anti ibv serum. the lanes 1-5 represent infected df-1 cell lysates of rndv, rndv/s1, rndv/ s1 + ibv-s-tm&ct, rndv/s1(cs−) + ndv-f-tm&ct and rndv/s1(cs+) + ndv-f-tm&ct, respectively. a ~130 kda band representing expression of s1 by rndv/s1 + ibv-s-tm&ct, rndv/s1(cs−) + ndv-f-tm&ct, and rndv/s1(cs+) + ndv-f-tm&ct in lysate of df-1 cells (fig. 3a -lanes 3-5) and rndv/s1 in infected df-1 cell supernatant (fig. 3b-lane 2) was observed. our attempts to detect the incorporation of the s1 protein into ndv envelope were not successful, due to the difficulties in the detection of very low level of s1 protein by western blot analysis (data not shown). our results showed that the s1 protein was expressed at very low level by all the rndvs based on western blot analysis. only the unmodified s1 protein was detected in the cell culture supernatant. growth characteristics of rndv constructs. the recovered rndvs were passaged in 9-day-old embryonated spf chicken eggs. all the viruses were able to replicate well in eggs (≥2 8 hau/ml). rndv/s1, rndv/ s1(cs+) + ndv-f-tm&ct, rndv/s2, rndv/codon optimized-s and rndv were evaluated in the presence of exogenous protease in df-1 cells (fig. 4) . compared to the parental virus, rndv expressing codon optimized s protein of ibv grew slightly less efficiently. the maximum titer of parental virus reached 10 7.5 tcid 50 /ml at 40 hours post infection, whereas the maximum titer of rndv expressing codon optimized s gene of ibv reached 10 7.2 tcid 50 /ml at 40 hours post infection. these results indicated that presence of s, s1 and s2 genes did not significantly affect the growth characteristics of rndv. the protective efficacy of rndvs expressing s1, s2 or s protein of ibv in chickens against a virulent ibv challenge. ibv protection experiment 1. to evaluate the protective efficacy of rndvs expressing s1, s2 or s protein of ibv, spf chicks were immunized at 1-day-old age with each virus via oculanasal (on) route. at three weeks post-immunization, chickens were challenged with virulent ibv strain mass-41. the severity scores of ibv clinical signs were recorded twice a day for 10 days post-challenge (fig. 5a ). compared to chickens immunized with parental rndv and chickens inoculated with pbs, chickens immunized with rndvs expressing figure 1 . schematic diagram of recombinant ndv constructs containing ibv genes. seven transcription cassettes including; 1-4) four versions of codon optimized s1 subunit of s gene of ibv strain mass-41; namely, (a) s1 subunit of s gene (1614 nt), (b) s1 subunit of s gene (1611 nt) fused with n-terminus of transmembrane and cytoplasmic tail of s gene (255 nt), (c) s1 subunit of s gene (1611 nt) containing five putative cleavage site residues of s gene fused with n-terminus of transmembrane and cytoplasmic tail of f gene of ndv (171 nt). in this construct, five c-terminus putative cleavage site residues of s1 gene (rrfrr) plus the first serine (s) residue of n-terminus of transmembrane and cytoplasmic tail of f gene of ndv provides six putative cleavage site residues of s protein of ibv strain mass-41 (rrfrr/s). (d) s1 gene (1593 nt) without cleavage site residues of s gene fused with n-terminus of transmembrane and cytoplasmic tail of f gene of ndv (171 nt), 5) the n-terminus of codon optimized s2 gene of ibv strain mass-41 (1878 nt) fused with c-terminus of signal peptide sequence of s gene (69 nt), 6) the codon-optimized s gene (3489 nt) and 7) the non-codon optimized s gene of ibv strain mass-41 (3489 nt) were flanked into individual plasmids containing cdna of lasota between p and m genes using pmei site. each transcription cassette contains the orf of foreign gene with the addition of pmei restriction enzyme site sequence, 15 nt of ndv utr, ge signal of ndv, one t nucleotide as intergenic sequence, gs signal of ndv, nucleotides for maintaining the rule of six and kozak sequence. scientific reports | (2018) 8:11951 | doi:10.1038/s41598-018-30356-2 codon optimized s, s1 or s2 protein of ibv showed significantly less severe of clinical signs (p < 0.05). among groups of chickens immunized with rndvs expressing codon optimized s1, s2 or s protein, the group immunized with rndv expressing codon optimized s protein showed the least severity of clinical signs (p < 0.05). in order to evaluate the efficacy of rndvs expressing s1, s2 or s protein of ibv in preventing shedding of virulent ibv challenge virus in immunized chickens, on day five post-challenge, tracheal swab samples were collected from chickens of each group and were evaluated for the viral load by rt-qpcr. our results did not show significant difference in virus shedding among groups of immunized chickens at day five post challenge (fig. 5b) . however, the results of the inoculation of the tracheal swab samples into 10-day-old embrynated chicken eggs showed that 14 out of 15 (93.3%) chickens vaccinated with rndv expressing codon optimized s protein of ibv and 0 out of 5 (0%) of non-infected chickens were shedding virus in trachea, respectively, whereas 15 out of 15 (100%) of chickens of all other groups were shedding virus in the trachea (data not shown). ibv protection experiment 2. to evaluate the protective efficacy of rndv expressing codon optimized s protein of ibv in adult chickens, spf chickens were immunized at 4-week-old age. the protective efficacy of rndv two bands (~170-220 kda) on top represent uncleaved s protein (s0) or polymeric forms of s2 or s1 protein (c-lane 2). the ~95 kda and the ~60 kda band represent s2 or s1 subunit of cleaved s protein (c-lane 2). the two bands (~170-220 kda) on top represent polymeric forms of s2 protein, the ~105 kda band and the ~95 kda expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with the world organization for animal health (oie) recommended dose (10 3.1 eid 50 ) of virulent ibv strain mass-41 at 3 week post-immunization 1 . the severity scores of ibv clinical signs were recorded twice a day for 10 days post-challenge (fig. 6a ). compared to chickens inoculated with pbs, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs (p < 0.05). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day 5 following challenge with a figure 3 . western blot analysis of rndv expressing s1 protein of ibv. the expression of codon optimized s1 protein of ibv expressed from four individual rndvs expressing four different expression cassettes of s1 protein were detected using western blot in cell lysates (a) and cell supernatant (b) of infected df-1 cells infected with rndvs, using a chicken polyclonal anti ibv serum. the lanes 1-5 represent cell lysates of rndv, rndv/s1, rndv/s1 + ibv-s-tm&ct, rndv/s1(cs−)+ndv-f-tm&ct, rndv/s1(cs+) + ndv-f-tm&ct, respectively. a ~130 kd band represent expression of s1 protein by rndv/s1 + ibv-s-tm&ct, rndv/ s1(cs−) + ndv-f-tm&ct and rndv/s1(cs+) + ndv-f-tm&ct in infected df-1 cell lysate (a lanes [3] [4] [5] and rndv/s1 in infected df-1 cell supernatant (b-lane 2). the full-length gel is presented in supplementary figure s1 . the severity scores of ibv clinical signs include; ocular discharge, nasal discharge and difficulty in breathing (0 = normal, 1 = presence of mild ocular discharge, mild nasal discharge and or sneezing 2 = presence of heavy ocular discharge and or heavy nasal discharge with mild tracheal rales and mouth breathing and or coughing 3 = heavy ocular discharge and heavy nasal discharge with sever tracheal rales and mouth breathing, gasping, dyspnea and or severe respiratory distress) were recorded twice a day for each chicken for 10 days after challenge. the severity scores represent as average scores of clinical signs measured for each chicken over 10 days. (b) relative viral load determined by rt-qpcr in tracheal swab samples at day five following virulent ibv challenge. the relative viral load expressed as mean reciprocal ± sem log 10. whereas chickens inoculated with pbs showed high levels of viral load in the trachea (p < 0.05). however, compared to chickens immunized with a commercial ibv vaccine, chickens immunized with rndv expressing codon optimized s showed slightly less viral load in the trachea (fig. 6b ). ibv protection experiment 3. to evaluate the protective efficacy of rndv expressing codon optimized s protein of ibv in adult chickens against a higher dose of virulent ibv challenge, spf chickens were immunized at 4-week-old age. the protective efficacy of rndv expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with 10 4.7 eid 50 virulent ibv strain mass-41 at 3 week post-immunization. the severity scores of ibv clinical signs were recorded twice a day for 8 days post-challenge (fig. 7a ). compared to chickens immunized with rndv and chickens inoculated with pbs, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs (p < 0.05). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at days 4 following challenge with virulent ibv, the tracheal swab samples collected from five chickens of each group were analyzed for the ibv specific lesions in chicken embryo. our results showed that 2 out of 5 (40%) chickens vaccinated with rndv expressing codon optimized s protein of ibv and 1 out of 5 (20%) chickens vaccinated with a commercial ibv vaccine were shedding virus in trachea, respectively, whereas 5 out of 5 (100%) of chickens immunized with parental rndv and 5 out of 5 (100%) of chickens inoculated with pbs were shedding virus in the trachea (fig. 7c) . the tracheal swab samples collected from five chickens of each group were also analyzed for the viral load by rt-qpcr. our results showed that chickens vaccinated with rndv expressing codon optimized s protein of ibv showed low levels of viral load in the trachea and chickens vaccinated with a commercial ibv vaccine showed very low levels of viral load in the trachea, whereas chickens inoculated with pbs and rndv showed high levels of viral load in the trachea. compared to chickens immunized with rndv expressing codon optimized s protein, chickens immunized with a commercial ibv vaccine showed less viral load in the trachea (p < 0.05) (fig. 7b ). ibv protection experiment 4. to evaluate the effect of the route of inoculation of virulent ib challenge virus on the outcomes of the protective efficacy of rndv expressing codon optimized s protein of ibv, spf chicks were immunized at 1-day-old age. the protective efficacy of rndv expressing codon optimized s gene of ibv was determined by challenging the immunized chickens with 10 4 eid 50 virulent ibv strain mass-41 by the intraocular route at 3 week post-immunization. this route of challenge has been specified in usda-cfr-9 for ibv 33 . the severity scores of ibv clinical signs were recorded twice a day for 10 days post-challenge. compared to chickens immunized with rndv and unvaccinated chickens, chickens immunized with rndv expressing codon optimized s protein of ibv and chickens immunized with a commercial live attenuated ibv vaccine showed significantly less severe clinical signs. however, compared to chickens immunized with commercial ibv vaccine, chickens immunized with rndv expressing s protein showed less severe clinical signs (p < 0.05) (fig. 8a) . in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at days 5 following challenge with virulent ibv, the tracheal swab samples collected from all chickens of each group were analyzed for the ibv specific lesions in chicken embryos. our results showed that 2 out of 10 (20%) chickens vaccinated with rndv expressing codon optimized s protein of ibv and 5 out of 10 (50%) chickens vaccinated with a commercial ibv vaccine were shedding virus in trachea, respectively, whereas 10 out of 10 (100%) of chickens immunized with parental rndv and 5 out of 5 (100%) of unvaccinated chickens, infected with ibv, were shedding virus in the trachea (fig. 8b) . the protective efficacy of rndvs against a highly virulent ndv challenge. to evaluate the protective efficacy of rndv expressing s gene of ibv against a virulent ndv strain, groups of five 1-day-old chicks were immunized with rndv, rndv expressing codon optimized s protein and pbs. three weeks after immunization, chickens were challenged with virulent ndv strain texas gb in our bsl-3 plus facility. our results showed that all chickens immunized with the rndv and rndv expressing codon optimized s gene of ibv survived after highly virulent ndv challenge, while all chickens in pbs group died at day 5 and 6 post-challenge (fig. 9a) . antibodies produced against ibv and ndv. hemagglutination inhibition (hi) assay using a standard protocol of oie was used to assess the level of antibodies mounted against ndv in serum samples of chickens 21 days after immunization. the results showed that hi titers of ndv was detected in serum samples of all chickens immunized with rndv and rndv expressing codon optimized s protein. there was no significant differences observed among hi titers against ndv in serum samples of chickens from groups immunized with rndv and rndv expressing s protein (fig. 9b ). virus neutralization assay was performed according to a standard protocol of oie to assess the level of neutralizing antibodies mounted against ibv strain mass-41 in serum samples of chickens at 21 days after immunization. the results showed that the neutralizing antibodies against ibv were detected in serum samples of chickens immunized with rndv expressing codon optimized s protein of ibv and with commercial live attenuated ibv vaccine (fig. 7d) . neutralizing antibodies against ibv were not detected in 1:8 dilution of a serum sample from a chicken immunized with empty rndv vector. this result showed that the rndv expressing codon optimized s protein of ibv induces neutralizing antibodies against ibv. this study was conducted to compare the protective efficacies of s1, s2, and s proteins of ibv using rndv as a vaccine vector. the s1, s2, and s genes of ibv strain mass-41 were individually inserted between the p and m genes of rndv strain lasota. this site was chosen because it has been identified as the optimal site for insertion of foreign genes into ndv genome 26,34-37. four different versions of ibv s1 gene were used to identify the version that is expressed at the highest level and incorporated into ndv particles. we were able to recover all the recombinant viruses and their growth characteristics were similar to rlasota. however, the recombinant viruses containing ibv s gene grew slightly slowly than the parental virus. the viruses were stable after passages in spf chicken embryos. western blot analysis showed that chicken codon optimized s2 and s proteins were expressed at much higher levels and were incorporated into ndv particles. whereas, all the four versions of s1 protein were detected at very low levels by western blot analysis. it is noteworthy that the unmodified s1 protein was detected in the infected cell culture supernatant, indicating that the modification of s1 protein probably caused retention of the protein in the cell. these results suggest that the s2 protein acts as a chaperone to assist in the folding of the s1 protein. the s1 protein is folded incorrectly in the absence of s2 protein and the new structure probably causes loss of some conformational epitopes for ibv antibodies. in the first ibv protection experiment, we found that 1-day-old chicks immunized with rndv expressing the s protein of ibv conferred better protection from disease compared to 1-day-old chicks immunized with rndvs expressing either s1 or s2 protein of ibv. our results showed that the s protein, which contains both s1 and s2 proteins, is the best protective antigen of ibv. the s2 protein lacks major neutralizing epitopes which are present in the s1 protein, hence it is not an effective antigen. the s1 protein contains major neutralizing epitopes, but it losses some conformational epitopes when expressed separately. although in the first study we showed that rndv expressing s protein provided enhanced protection, it could not reduce virus shedding, indicating that we needed to determine whether the elimination of virus shedding would probably require a much higher level of immune response than that is induced by rndv expressing s protein or would require the optimization of the ibv protection study. in this study, our results support previous reports that rndv vectored ibv vaccines prevent disease but do not stop virus shedding 23, 24 . these results also support the recent report that a spike ectodomain subunit vaccine protects chickens against ibv 38 . in the second ibv protection experiment, we investigated whether age at immunization influences the outcome of ibv challenge. our results showed that a single immunization of 4-week-old chickens with rndv expressing s protein completely protected chickens against ibv challenge based on disease and viral load in tracheas. indeed, the level of protection conferred by rndv expressing s protein was similar to that of a commercial ibv vaccine. however chickens immunized with either commercial live attenuated ibv vaccine or rndv expressing ibv s protein showed very low levels of tracheal viral load. this showed that protection was greater when the chickens were immunized at an age when their immune system is relatively well developed. in the third ibv protection experiment, we showed that rndv expressing ibv s protein protects adult chickens against a higher dose of virulent ibv challenge. however, compared to standard challenge dose of virulent ibv, a higher challenge dose of virulent ibv caused higher levels of tracheal viral load in adult chickens immunized with rndv expressing ibv s protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated ibv vaccine. our result showed that although both the age of immunization and dose of challenge virus affect the results of ibv challenge, the influence of the age of immunization is greater than the effect of the dose of challenge virus. our results also showed that when we challenged adult immunized chickens with standard challenge dose of virulent ibv, rndv expressing s protein showed slightly better protection than a commercial live ibv vaccine, based on disease and viral shedding in trachea, but when the adult immunized chickens were challenged with a higher dose of virulent ibv, commercial live ibv vaccine showed slightly better protection than rndv expressing s protein. hence to compare the efficacy of rndv expressing s protein of ibv with the efficacy of live attenuated ibv vaccine, a large ibv protection study using commercial chickens is needed. in the fourth ibv protection experiment, we showed that rndv expressing ibv s protein protected young chickens against virulent ibv challenge by the intraocular route. the route of challenge has been recommended by usda-cfr-9. our results showed that compared to infection of chickens with virulent ibv by the oculanasal route, infection of chickens with virulent ibv by the intraocular route, caused much lower levels of tracheal viral load in young chickens immunized with rndv expressing ibv s protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated ibv vaccine. our results showed that the route of the challenge virus inoculation affected the results of the tracheal virus shedding in young chickens immunized by rndv expressing s protein following ibv challenge; however, it did not affect the outcomes of the severity of clinical signs. although our studies showed that the rndv expressing the s protein and commercial live ibv vaccine provided comparable protection, rndv expressing the s protein has several advantages over live ibv vaccines in controlling ib in the field. (i) ndv vectored ibv vaccine is highly safe in 1-day-old chicks, (ii) it will not create new vaccine derived variant viruses, which is a major concern in using live modified ibv vaccines, (iii) a single vaccine can be used to control both ndv and ibv, (iv) we believe that the level of immunity induced by the ndv vectored vaccine against ibv is probably sufficient to completely stop ibv infection in field condition, and (v) the immune response of ndv vectored vaccine can be enhanced by prime-boost vaccination strategy. in summary, we have shown that although the s1 and s2 proteins of ibv are known to contain virus neutralizing epitopes, the presence of the whole s protein is necessary for eliciting a strong protective immune response. the s protein is the antigen of choice for any vectored ibv vaccine. ndv is an attractive vaccine vector for ibv, because it can be used as a bivalent vaccine. our results suggest that a recombinant ndv vectored ibv vaccine is the vaccine of choice for controlling ibv infection in the field. cells were obtained from the american type culture collection (atcc, manassas, va). they were grown in dulbecco's minimal essential medium (dmem) containing 10% fetal bovine serum (fbs). the recombinant avirulent ndv strain lasota was generated previously in our laboratory using reverse genetics 39 . the rndv and rndvs expressing chicken codon optimized s1, s2 and s genes and non-codon optimized s gene of ibv strain mass-41 were grown in 9-day-old embryonated spf chicken eggs at 37 °c. the virulent ibv strain mass-41 was propagated in 10-day-old spf embryonated chicken eggs and harvested five days after infection. the titer of virus in harvested allantoic fluid was determined by 50% embryo infectious dose (eid 50 ) method. briefly, ten-fold serial dilutions of ibv strain mass-41 was inoculated into 10-day-old embryonated spf chicken eggs. seven days after inoculation, infected embryos were examined for ibv specific lesions such as stunting or curling. the titer of virus was calculated using reed and muench method 40 .the modified vaccinia virus strain ankara expressing t7 rna polymerase (mva-t7) was propagatd in monolayer primary chicken embryo fibroblast cells. generation of rndvs containing s1, s2 or s gene of ibv. a plasmid containing full-length antigenomic cdna of ndv strain lasota has been constructed previously 39 . in order to develop an effective ibv vaccine the maximum neutralizing epitopes with correct conformation are needed to be displayed. most neutralizing epitopes are located in the s protein. in this study seven transcription cassettes containing s, s1 or s2 genes of ibv were constructed to identify the best protective antigen for the development of ndv vectored ibv vaccines. the s, s1 and s2 genes were chicken codon optimized for higher level of expression in chickens. the following transcription cassettes were designed: (i) a transcription cassette containing the s gene of ibv strain mass-41 (3489 nt) was designed to determine whether the expression of the whole s gene from ndv will lead to display the maximum neutralizing epitopes in correct conformation, (ii) a transcription cassette containing the s2 subunit of s gene (1878 nt) of ibv fused with c-terminus of signal peptide sequence of s gene (69 nt) was constructed for transport of the protein from the cell (iii) a transcription cassette containing the s1 subunit of s gene (1614 nt) was designed to determine the protective efficacy of s1 protein, (iv) a transcription cassette containing the s1 gene (1611 nt) fused with n-terminus of transmembrane and cytoplasmic tail of s gene (255 nt) was designed for incorporation into ndv envelop, (v) a transcription cassette containing the s1 subunit of s gene without s1 protein cleavage site residues (1593 nt) fused with n-terminus of transmembrane and cytoplasmic tail of ndv f gene (171nt) was designed for incorporation of the s1 protein into envelope of ndv. (vi) a transcription cassette containing the s1 subunit of s gene containing s1 protein cleavage site residues (1611 nt) fused with n-terminus of transmembrane and cytoplasmic tail of ndv f gene (171 nt) was designed to incorporate the s1 protein into ndv envelope and also to know whether adding the cleavage site residues has any effect on the fusion of two scientific reports | (2018) 8:11951 | doi:10.1038/s41598-018-30356-2 proteins, and (vii) a transcription cassette containing the non-codon optimized s gene (3489 nt) was constructed to compare the level of protein expression between the codon optimized and non-codon optimized s genes. ndv genome contains six genes: nucleocapsid(n), phosphoprotein(p), matrix(m), fusion(f), hemagglutinin-neuraminidase(hn) and large(l). the genes are ordered 3′-n-p-m-f-hn-l-5′. the beginning and the end of each gene contain conserved transcriptional sequences known as the gene-start (gs) and gene-end (ge), respectively. between the genes, there are gene junctions 26 . any of the gene junctions is a potential insertion site for the transcription cassette of a foreign gene. however, we and others have found that the intergenic region between the p and m genes is a good site for expression of most foreign genes 26, [34] [35] [36] [37] . the transcription cassettes containing ibv genes contained pmei restriction enzyme sequence, 15 nt of untranslated region (utr) of ndv, ndv ge signal, one t nucleotide as intergenic sequence, ndv gs signal, extra nucleotides to maintain the rule of six 26, 41 , kozak sequence at the upstream of foreign gene orfs and pmei restriction enzyme sequence at downstream of foreign gene orf. the transcription cassettes of codon optimized and non-codon optimized s gene were digested from two commercially synthesized (genscript; puc57-ibv-mass-41-s syn) plasmids containing codon optimized (genscript; optimization on gallus gallus codons using optimumgene tm pso algorithm) and non-codon optimized s gene of ibv strain mass-41(genbank accession no. ay851295. 1), respectively. the transcription cassettes of codon optimized s1 and s2 genes were amplified from the commercially synthesized plasmid containing codon optimized s gene of ibv strain mass-41 and cloned into individual shuttle vectors (pgem ® -t easy vector, promega corporation). then the flanking dna of transcription cassettes were digested from shuttle vectors. the transcription cassettes derived from shuttle vectors were cloned into complete individual plasmids containing cdna of rlasota at p and m gene junction using pmei site (fig. 1) . the correct sequences of the foreign genes were confirmed by nucleotide sequence analysis. rndvs containing the ibv genes were recovered by reverse genetics as described previously 39 .briefly, each full length cdna was co-transfected with three expression plasmids containing n, p or l gene of ndv strain lasota into mva-t7 infected hep-2 cells. three days post-transfection, 200 µl of supernatant of transfected cells were inoculated in 9-11 day-old spf embryonated chicken eggs. after three days, haemaglutination test was used to detect infected allantoic fluids collected from eggs. rndvs containing s1 gene, s1 gene fused with transmembrane and cytoplasmic tail of ibv s gene, s1 gene containing cleavage site residues of s gene of ibv fused with transmembrane and cytoplasmic tail of ndv f gene, s1 gene without cleavage site residues of s gene fused with transmembrane and cytoplasmic tail of ndv f gene, s2 gene, codon optimized s gene and non-codon optimized s gene were named rndv/s1, rndv/s1 + ibv-s-tm&ct, rndv/s1(cs+) + ndv-f-tm&ct, rndv/s1(cs−) + ndv-f-tm&ct, rndv/s2, rndv/codon optimized-s and rndv/non-codon optimized-s, respectively. the ibv genes were amplified from the rndv constructs by rt-pcr. expression of s1, s2 and s proteins of ibv. confluent monolayers of df-1 cells were infected at a multiplicity of infection (moi) of 0.01-0.1 with rndv strain lasota, rndv/s1, rndv/s1 + ibv-s-tm&ct, rndv/ s1(cs+) + ndv-f-tm&ct, rndv/s1(cs−) + ndv-f-tm&ct, rndv/s2, rndv/codon optimized-s or rndv/ non-codon optimized-s. df-1 cells were harvested 30 hours post-infection, lysed and analyzed by western blot. a polyclonal chicken anti-ibv strain mass-41 was used to detect the expression of s1, s2 and s proteins of ibv. to determine the incorporation of ibv proteins into ndv envelope, rndv, rndv/s2, rndv/codon optimized-s and rndv/non-codon optimized-s were inoculated into 9-day-old embryonated spf chicken eggs. three days after incubation, recombinant viral particles from infected allantoic fluids were partially purified by sucrose density gradient centrifugation and analyzed by western blot analysis. a monoclonal anti-ndv/hn antibody also was used to detect hn protein of ndv in lysates and purified virions by one more western blot analysis. growth characteristics of rndv constructs. in order to determine the growth kinetics of rndvs expressing s1, s2 or s protein of ibv, confluent monolayers of df-1 cells in 6-well tissue culture plates were infected at a moi of 0.1 with rndv, rndv/s1(cs+) + ndv-f-tm&ct, rndv/s2 and rndv/codon optimized-s and adsorbed for 90 minutes at 37 °c. after adsorption, cells were washed with pbs, then incubated with dmem containing 2% fbs and 10% fresh spf chicken egg allantoic fluid at 37 °c in presence of 5% co 2 . aliquots of 200 µl of supernatant from infected cells were collected and replaced with fresh dmem including fbs at intervals of 8 hours until 64 hours post-infection. the titer of virus in the harvested samples was determined by tcid 50 method in df-1 cells in 96-well tissue culture plates. the protective efficacy of rndvs expressing s1, s2 and s protein of ibv against virulent ibv challenge. based on the level of expression of s1, s2 and s proteins of ibv from rndvs, rndv/ s1(cs+) + ndv-f-tm&ct, rndv/s2, and rndv/codon optimized-s viruses were selected for animal study to evaluate their protective efficacy against virulent ibv challenge. ibv protection experiment 1. in this study, the protective efficacy of rndvs expressing s1, s2 or s protein of ibv strain mass-41 were evaluated in 1-day-old spf chicks. briefly, a total of eighty 1-day-old chicks were divided into five groups of fifteen each and one group of five. chicks of the first four groups were inoculated with 10 7 eid 50 of rndv, rndv/s1(cs+) + ndv-f-tm&ct, rndv/s2 and rndv/codon optimized-s strains via oculonasal route. the fifteen chicks of group five and five chicks of group six were inoculated with pbs. three weeks after immunization, all immunized chickens, were challenged with 10 3.1 eid 50 of virulent ibv strain mass-41. this challenge virus dose was determined by an experimental chicken infection study. the severity scores of clinical signs of ibv including, nasal discharge, ocular discharge and difficulty in breathing (0 = normal, 1 = presence of mild ocular discharge, mild nasal discharge and or sneezing 2 = presence of heavy ocular discharge and or heavy nasal discharge with mild tracheal rales and mouth breathing and or coughing 3 = heavy ocular discharge and heavy nasal discharge with sever tracheal rales and mouth breathing, gasping, dyspnea and or severe scientific reports | (2018) 8:11951 | doi:10.1038/s41598-018-30356-2 respiratory distress) were recorded twice a day for 10 days post-challenge. in order to evaluate protective efficacy of rndvs expressing s1, s2 and s genes of ibv in preventing shedding of virulent ibv in immunized chickens, at day five post-challenge, tracheal swab samples were collected from fifteen birds of each group and placed in 1.5 ml serum free dmem with 10 x antibiotics. the swab samples were analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. ibv protection experiment 2. in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in 4-week-old spf chickens against the oie recommended dose of virulent ibv challenge 1 . a total of twenty 4-week-old spf chickens were divided into four groups of five each. five chickens of groups one and two were inoculated with 10 7 eid 50 of rndv and rndv/codon optimized-s, respectively, via oculanasal route. five chickens of group three were inoculated with 10 recommended doses of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of group four were inoculated with pbs. three weeks after immunization, chickens of all groups were challenged with 10 3.1 eid 50 of virulent ibv strain mass-41 by the oculonasal route. the severity scores of clinical signs of ibv, described in ibv protection experiment 1, were recorded for 10 days post-challenge. in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day 5 post-challenge, tracheal swab samples were collected from twenty chickens and placed in 1.5 ml serum free dmem with 10 x antibiotic. the swab samples were analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. ibv protection experiment 3. in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in 4-week-old spf chickens against a higher dose of virulent ibv challenge. a total of thirty two 4-week-old spf chickens were divided into four groups of eight each. eight chickens of group one and two were inoculated with 10 7 eid 50 of rndv and rndv/codon optimized-s, respectively, via oculanasal route. eight chickens of group three were inoculated with 10 recommended doses of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of group four were inoculated with pbs. three weeks after immunization, chickens of all groups were challenged with 10 4.7 eid 50 of virulent ibv strain mass-41 by the oculonasal route. the severity scores of clinical signs of ibv, described in ibv protection experiment 1, were recorded for 8 days post-challenge. at day 4 post-challenge, three chickens from each group were euthanized for tracheal ciliostasis analysis (data not shown). in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, tracheal swab samples were collected from five chickens from each group and placed in 1.5 ml serum free dmem with 10 x antibiotic. each fluid was tested for ibv specific lesions on chicken embryo by inoculation (0.1 ml) of one 10-day-old embryonated spf chicken egg. the swab samples were also analyzed for quantification of viral rna using an ibv-n gene-specific rt-qpcr. the swab samples collected from two non-vaccinated spf chickens involved in another ibv protection study also were used as control. ibv protection experiment 4. in this study, the protective efficacy of rndv expressing codon optimized s protein of ibv was evaluated in 1-day-old spf chicks against virulent ib challenge virus infected by the intraocular route. intraocular route was used, because this route of ibv challenge has been specified by the usda-cfr-9 33 . a total of forty five 1-day-old spf chickens were divided into three groups of ten each and three groups of five each. ten chickens of group one and two were inoculated with 10 7 eid 50 of rndv, rndv/ codon optimized-s, respectively, via oculanasal route. ten chickens of group three were inoculated with one recommended dose of a commercial live attenuated mass-type ibv vaccine via oculanasal route and chickens of groups four to six were left non-vaccinated. three weeks after immunization, chickens of all groups one to four were challenged with 10 4 eid 50 of virulent ibv strain mass-41 by the intraocular route, chickens of group five were challenged with 10 4 eid 50 of virulent ibv strain mass-41 by the oculanasal route, and chickens of group six were left non-infected. the severity scores of clinical signs of ibv, described in ibv protection experiment 1, were recorded for 10 days post-challenge. in order to evaluate the efficacy of rndv expressing s protein of ibv in preventing shedding of virulent ibv in immunized chickens, at day 5 post-challenge, tracheal swab samples were collected from all chickens of each group and placed in 3 ml serum free dmem with 10 x antibiotic. each fluid was tested for ibv specific lesions on chicken embryo by inoculation with 0.2 ml to each of five 10-day-old embryonated spf chicken egg. the sample was considered positive for virus shedding, if any of the five embryos showed ibv lesions. we performed all experiments involving virulent ibv in our usda approved biosafety level-2 and biosafety level-2 plus facilities following the guidelines and approval of the animal care and use committee (iacuc), university of maryland. the protective efficacy of rndv expressing s protein of ibv against virulent ndv challenge. the protective efficacy of rndv expressing s protein of ibv strain mass-41 was evaluated against a virulent ndv strain gb texas challenge in our biosafety level 3 (bsl-3) plus facility. briefly, a total of fifteen 1-day-old chicks were divided into three groups of five each. chicks of two groups were inoculated with 10 7 eid 50 of rndv and rndv/ibv-codon optimized-s via oculonasal route. the five chickens of group three were inoculated with pbs. three weeks after immunization, blood samples of all birds were collected for ndv antibody response analysis and challenged with one hundred 50% chicken lethal dose (cld 50 ) of the highly virulent ndv strain gb texas via oculonasal route. the chickens were observed daily for 10 days after challenge for mortality with clinical signs of disease (neurological signs included torticollis, paralysis, and prostration). we performed the experiment involving virulent ndv in our usda approved biosafety level-3 plus facility following the guidelines and approval of the animal care and use committee (iacuc), university of maryland. scientific reports | (2018) 8:11951 | doi:10.1038/s41598-018-30356-2 serological analysis. the level of antibodies induced against ndv and ibv were evaluated. the serum samples were collected three weeks post-immunization. hemagglutination inhibition (hi) assay using a standard protocol oie was used to assess the level of antibody titer mounted against ndv in chickens immunized by rndvs 27 . the virus neutralization assay according to oie was used to measure the level of neutralizing antibodies mounted against ibv 1 . briefly, serum samples of three birds from the group immunized with rndv expressing codon optimized s protein of ibv and serum samples of three birds from the group immunized with commercial ibv vaccine group were incubated at 56 °c for 30 minutes. one hundred eid 50 of ibv strain mass-41 was mixed with 2 fold dilutions of antiserum and incubated for 1 hour at 37 °c. one hundred µl of each serum and virus mixture was inoculated into three 10-day-old embryonated spf chicken eggs. to confirm that at least 100 eid 50 of virus was inoculated into each egg, three eggs were inoculated with 100 µl of pbs containing 100 eid 50 of ibv. three eggs were inoculated with 100 µl of pbs as negative control. three eggs were inoculated with a mixture of 100 eid 50 of ibv and a dilution of 1:8 of a randomly selected serum sample collected from a bird immunized with rndv strain lasota as vector control. the eggs were incubated at 37 °c and were observed daily for dead chicken embryos for 7 days post inoculation. the serum titers were calculated according to the method of reed and muench 40 , based on mortality and ibv specific lesions on chicken embryos. quantitative reverse transcription-polymerase chain reaction (rt-qpcr). rna was extracted using trizol reagent (invitrogen) from tracheal swab samples collected from chickens. the first strand cdna was synthesized using thermo scientific revertaid reverse transcriptase (rt). sybr green rt-qpcr was performed using a specific primer pair set: (a) n gene -296 forward primer: 5′ gaccagccgctaacctgaat 3′ and (b) n gene -445 reverse primer: 5′ gtcctccgtctgaaaaccgt 3′ amplifying 150 nt of n gene of ibv strain mass-41. pcrs were performed using a bio-rad cfx96 cycler. each 20 µl reaction was carried out using 5 µl of cdna, 10 µl of itaq universal sybr green supermix (bio-rad), 2 µl of forward and reverse primers and 3 µl of nuclease free water. forty cycles of pcr at 95 °c for 10 s (denaturation), 58 °c for 20 s (annealing), and 72 °c for 30 s (elongation) followed by melting curve analysis that consisted of 95 °c for 5 s and 65 °c for 60 s. a serial 10 fold dilution of cdna synthesized from extracted rna of allantoic fluid stock of a virulent ibv strain mass-41 with 10 7.5 eid 50 /ml was used to establish the standard curve. the cdna synthesized from extracted rna of allantoic fluid stock of a virulent ibv strain mass-41 and the cdna synthesized from extracted rna of swab sample solution were served as positive and negative controls, respectively. melting point analysis was used to confirm the specificity of the test. statistical analysis. data were analyzed among groups by one-way-anova test. the student t-test was used to compare two groups. to avoid bias, all animal experiments were designed as blinded studies. avian infectious bronchitis. version adopted by the world assembly of delegates of the oie in may the long view: 40 years of infectious bronchitis research infectious bronchitis coronavirus avian infectious bronchitis virus fields virology mechanisms of coronavirus cell entry mediated by the viral spike protein identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus the neutralization epitopes on the spike protein of infectious bronchitis virus and their antig enic variation mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism factors influencing the outcome of infectious bronchitis vaccination and challenge experiments emergence of subtype strains of the arkansas serotype of infectious bronchitis virus in delmarva broiler chickens current status of veterinary vaccines progress and challenges toward the development of vaccines against avian infectious bronchitis avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination attenuated vaccines can recombine to form virulent field viruses embryo vaccination of chickens with infectious bronchitis virus: histologic and ultrastructural lesion response and immunologic response to vaccination safety and efficacy of an infectious bronchitis virus used for chicken embryo vaccination cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/98. avian diseases evaluation of recombinant fowl pox virus expressing infectious bronchitis virus s1 gene and chicken interferon-γ gene for immune protection against heterologous strains a recombinant fowl adenovirus expressing the s1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus infectious bronchitis virus s2 expressed from recombinant virus confers broad protection against challenge recombinant newcastle disease virus expressing the infectious bronchitis virus s1 gene protects chickens against newcastle disease virus and infectious bronchitis virus challenge construction and immunogenicity of a recombinant fowl pox vaccine co-expressing s1 glycoprotein of infectious bronchitis virus and chicken il-18 newcastle disease and related avian paramyxoviruses newcastle disease. version adopted by the world assembly of delegates of the oie in newcastle disease virus as a vaccine vector for development of human and veterinary vaccines immunization of chickens with newcastle disease virus expressing h5 hemagglutinin protects against highly pathogenic h5n1 avian influenza viruses vaccination with newcastle disease virus vectored vaccine protects chickens against highly pathogenic h7 avian influenza virus recombinant newcastle disease virus expressing h9 ha protects chickens against heterologous avian influenza h9n2 virus challenge a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens code of federal regulations, standard requirements for ibv vaccines p and m gene junction is the optimal insertion site in newcastle disease virus vaccine vector for foreign gene expression avian paramyxovirus type-3 as a vaccine vector: identification of a genome location for high level expression of a foreign gene optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses recombinant newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication protection against infectious bronchitis virus by spike ectodomain subunit vaccine high-level expression of a foreign gene from the most 39-proximal locus of a recombinant newcastle disease virus a simple method of estimating fifty percent endpoints the rule of six, a basic feature for efficient replication of sendai virus defective interfering rna we would like to thank dr. laura crespi sanglas and all our laboratory members for their excellent technical assistance. we also thank girmay gebreluul and johanna lavigne for their animal study assistance. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-30356-2.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-257999-apg4uhhq authors: ababneh, mustafa; dalab, abd elhafeed; alsaad, saad; al-zghoul, mohammad title: presence of infectious bronchitis virus strain ck/ch/ldl/97i in the middle east date: 2012-04-11 journal: isrn vet sci doi: 10.5402/2012/201721 sha: doc_id: 257999 cord_uid: apg4uhhq infectious bronchitis virus (ibv) is a very dynamic and evolving virus, causing major economic losses to the global poultry industry. in early 2011, respiratory disease outbreaks were investigated in iraq, jordan, and saudi arabia. five ibv isolates (joa2, joa4, saudi-1, saudi-2, and iraqi ibv) were detected by diagnostic-nested nucleocapsid rt-pcr. strain identification was characterised by sequencing and phylogenetic analysis of the amplified hypervariable region of the spike 1 (s1) gene. these five ibv isolates were found to be of the ibv strain ck/ch/ldl/97i. nucleotide identity between these five ibv isolates ranged from 96.9% to 99.7%, and between these isolates and the ck/ch/ldl/97i strain in the range of 96.6–99.1%. the sequenced fragment of the s1 gene of the ck/ch/ldl/97i strain had less than 80% nucleotide identity to the ibv vaccine strains commonly used in the middle east (m41 and h120). the presence of these ck/ch/ldl/97i-like strains may account for vaccination failure against ibv, since all ibv isolates were from vaccinated chickens. in this paper, we documented for the first time the presence of ibv strain ck/ch/ldl/97i in the middle east. this strain is known to have originated in china and taiwan. infectious bronchitis virus (ibv) is a positive sense singlestranded rna virus, belonging to the genus coronavirus, group 3 (gamma coronaviruses) [1] . infectious bronchitis virus is not only an important pathogen of the respiratory system, but also can be nephropathogenic and cause infection of the reproductive system [2] [3] [4] . ibv was first recognised as avian respiratory pathogen in 1930, after that many ibv vaccines were introduced to tackle this problem (h52, h120, m41, 4/91(793/b), and other strains). recently, different ibv variants have emerged causing nephropathogenic and reproductive problems which require a dramatic change in vaccination programmes [5, 6] . ibv strain ck/ch/ldl/97i was first reported in china in 1995 [7] . this strain was first implicated in proventriculitis in chickens [8] , but in recent studies, this strain has been isolated from the trachea of infected chickens [9] . ibv ck/ch/ldl/97i strain accounts for 3.2% of total ibv strains found in china in the last 15 years [9] . the spike glycoprotein gene is the most variable gene in the ibv genome [10] and is composed of s1 and s2 subunits. spike subunit 1 (s1) is about 1644 nt in length. the s1 protein is highly variable; it can differ from 20% to 25% and even up to 50% in the amino acid sequence among ibv serotypes [11] . this variability makes the s1 gene an ideal target in molecular assays to type ibv strains by rt-pcr and sequencing. the level of homology of the s1 subunit or part of it can predict cross-protection, that is, the higher the homology, the higher the chance of cross-protection [12, 13] , but this rule is not always fulfilled [3] . heterologous protection against the ck/ch/ldl/97i strain has not been achieved, since the nucleotide and amino acid homology of the ck/ch/ldl/97i s1 gene is only around 79% compared to the ibv vaccine strains. only homologous protection has been achieved against ck/ch/ldl/97i [7] . in the middle east, the common circulating ibv strains are mass serotype vaccine isolates, h120 serotype vaccine isolates, d274 [14] [15] , along with other unique strains like is/885/00 [3] and the egypt/beni-seuf/01 strain found in egypt. by the end of 2009, variant 2 and variant 2-like strains appeared in jordan (unpublished data) and in northern iraq (sul/01/09) [16] . in the early months of 2011 we noticed ibv outbreaks in spite of massive vaccination, this was due to the presence of a new strain of ibv. in this study, we documented the presence of the ibv strain ck/ch/ldl/97i for the first time in three middle eastern countries. samples and viral rna extraction. trachea, kidney, ovarian tissues, and cecal tonsils samples were collected from suspected ibv outbreaks in iraq, jordan, and saudi arabia. these samples were stored at −70 • c in rnalater solution (qiagen, germany) until rna extraction was performed. homogenised tissues were subjected to viral rna extraction using the viral gene-spin viral dna/rna extraction kit (intron biotechnology, korea). viral rna extraction was performed according to the manufacturer's instructions. viral rna was stored at −70 • c until analysis by rt-pcr. the reversetranscription (rt) step was performed using an rt system (promega, usa). briefly, viral rna was denatured at 70 • c for 10 minutes, followed by the addition of a reaction mix including 4 µl mgcl 2 , 2 µl reverse transcription 10x buffer, 2 µl dntp mixture (10 mm), 0.5 µl random primers, 0.75 µl amv reverse transcriptase enzyme, 1 µg rna, and nuclease-free water to a final volume of 20 µl. then, the reaction was incubated at 42 • c for 60 minutes, then 94 • c for 5 minutes. the cdna was diluted up to 100 µl with nuclease-free water for pcr amplification. two rt-pcr assays were used. first, a diagnostic-nested rt-pcr assay based on the amplification of the nucleocapsid (n) gene. ibv-specific oligonucleotides for the n gene were obtained according to a published primers sequences [17] ; the ibv primers sequences are presented in table 1 . the diagnostic n gene amplification was done in a final volume of 25 µl gotaq green master mix (promega, usa) in which the final concentration of the n gene primers was 0.5 mmol. rt-pcr amplification was performed with a thermal profile of 94 • c for 45 sec, 60 • c (first step) and 53 • c (second step) for 1 min and 72 • c for 2 min for 40 cycles. next, a phylogenetic rt-pcr was performed using nested spike gene primers according to published protocols [4, 18] . the amplification profile was the same as that used for n gene-nested rt-pcr, but the annealing temperature for both rt-pcr steps was 50 • c. amplification was performed using the genepro thermal cycler (bioer, china). nested s1 gene rt-pcr products were sequenced using an abi prism 310 genetic analyser at the princess haya biotechnology centre (jordan university of science and technology, jordan). sequences were aligned using bioedit (7.0.5.3) and muscle (3.7) software. aligned sequences were used for phylogenetic analysis. maximum likelihood (ml) phylogenetic analysis with bootstrap values for n = 100 replicates was performed using the phyml phylogenetic interface [19] . the nucleotide sequence identities were prepared using clustal w in the megalign programme of the lasergene software. five ibv isolates were obtained in early 2011. two ibv isolates were from jordan (joa2 and joa4), two were from saudi arabia (saudi-1 and saudi-2), and one isolate was from iraq (iraqi ibv). the jordanian isolates were isolated from layer farms at their peak of egg production, the saudi strains were obtained from broilers and the iraqi ibv isolate was obtained from a breeder farm as shown in table 2 . according to the clinical and gross examination coupled with the rt-pcr results from the different tissues (trachea, kidney, ovarian tissues, and cecal tonsils), these isolates had an extensive tissue tropism, different than the ck/ch/ldl/97i strain, which has tropism only for the respiratory system. the clinical signs ranged from respiratory to reproductive symptoms; two isolates (joa2, iraqi ibv) were implicated in kidney pathology in affected birds. rt-pcr products of the diagnostic n gene assay were detected (380 bp) and for the phylogenetic s1 gene (392 bp). direct-automated sequencing of the second rt-pcr product of nested s1 (sx3 and sx4 primers) was performed. the reference ibv strains included in the s1 sequence analysis from the united states were m41, connecticut, del072, ark/15c/96, and beaudette. the european strains were 4/91, d3896, b1648, and h120. the australian strains were n1/62 and n1/88. the chinese strains were ibv lx4, qx ibv, j2, q1, ck/ch/ldl/97i, and ck/ch/scya/10i. middle eastern ibv strains were is/1201, is/1366, is/1464, is/885, variant 1, variant 2, and sul/01/09 as shown in table 3 . all five ibv isolates (joa2, joa4, saudi-1, saudi-2, and iraqi ibv) were found to be of the ibv strain ck/ch/ldl/97i and therefore they are ck/ch/ldl/97ilike strains. the ck/ch/ldl/97i strain is known to be endemic in china and taiwan. the nucleotide identity between these five ibv isolates ranged from 96.9 to 99.7% with the saudi-2 isolate being the most divergent. the identity of relatedness between the isolated ibv strains, ck/ ch/ldl/97i, and the related strain ck/ch/scya/10i [20] is shown in identity to the ck/ch/ldl/97i and ck/ch/scya/10i strains, respectively. a phylogenetic tree was constructed based on the alignment of the partial s1 sequence. the five ibv isolates were aligned with other reference and related ibv strains as shown in figure 1 . it showed that all five ibv isolates were grouped with ck/ch/ldl/97i, ck/ch/scya/10i, j2, and q1 (both j2 and q1 were isolated from the proventriculus) [8] in one group. the nucleotide identity of these five isolates to the ibv vaccine strains in the three countries (m41, h120, and 4/91) were in the range of 78-82.1%, to variant 1 ibv strains in the range of 78.9-81.5%, to variant 2 ibv strains in the range of 81.7-83.6%, and to the is/885 and sul/01/09 strains in the range of 78.3-80.9% (data not shown). also, these ibv isolates represent a group distinct from the lx4 ibv type and from qxibv and is/1201 (a middle eastern strain) related to it. these strains share only 79% nucleotide sequence identity. the lowest nucleotide identity was seen with the del072 strain (58%) and with the n1/88 strains (63%) from australia. in this study, the ck/ch/ldl/97i-like strains were isolated from three different middle eastern countries (jordan, saudi arabia, and iraq) in 2011. the ck/ch/ldl/97i-like strains were isolated from broilers in saudi arabia, layers in jordan, and from breeders in iraq. the ck/ch/ldl/97i-like strain was first isolated in china in 1995 [7] . this strain was isolated from the tracheas of infected chickens [9] . the ibv ck/ch/ldl/97i strains showed slight variability in the partial s1 sequence among ibv isolates from these three countries. the mode transmission of this strain from china to the middle east is not clear; one reason might be that chinese poultry vaccines and products have recently been registered and used in the middle east. all isolates came from flocks vaccinated with masstype h120, and 4/91 vaccines, which implies insufficient protection against these isolates. the poor relationship in the partial s1 sequence between the five ibv isolates and the vaccine strains (average nucleotide identity of 80%) could explain the failure of the mass 41, h120, and 4/91 vaccination programmes to control ibv in these flocks [5] . there is no solution to tackle infection coming from these five ibv isolates except to make a homologous vaccine against this strain. homologous vaccine was made for the ibv, ck/ch/ldl/97i strain found in china, for ibv qx strain and a nephropathogenic ibv strain in korea [7, 21, 22] . in summary, this is the first paper indicting the presence of the ibv ck/ch/ldl/97i strain outside mainland china and taiwan (in three middle eastern countries). in the near future, we expect that this strain might represent a serious problem for the poultry industry and there will be an urgent need to develop a homologous vaccine against this strain. severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus pathogenicity and antigenicity of a new variant of korean nephropathogenic infectious bronchitis virus identification of a novel nephropathogenic infectious bronchitis virus in israel a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes phylogenetic analysis of infectious bronchitis coronaviruses newly isolated in china, and pathogenicity and evaluation of protection induced by massachusetts serotype h120 vaccine against qx-like strains evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/97i strain of infectious bronchitis coronavirus characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens a 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china emergence of a group 3 coronavirus through recombination variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs infectious bronchitis virus s1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo infectious bronchitis virus in jordanian chickens: seroprevalence and detection molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east molecular epizootiology of infectious bronchitis 6 isrn veterinary science virus in sweden indicating the involvement of a vaccine strain efficacy of live infectious bronchitis vaccines against a novel european genotype, italy 02 phylogeny.fr: robust phylogenetic analysis for the non-specialist genetic analysis revealed lx4 genotype strains of avian infectious bronchitis virus became predominant in recent years in sichuan area, china characterization of a novel live attenuated infectious bronchitis virus vaccine candidate derived from a korean nephropathogenic strain efficacy and safety of an attenuated live qx-like infectious bronchitis virus strain as a vaccine for chickens this work was funded by a grant from the deanship of research at jordan university of science and technology. the authors would like to thank hazar shawash from the jordan bio-industries center (jovac) for her technical support. key: cord-256859-7ixegm72 authors: liu, s. w.; zhang, q. x.; chen, j. d.; han, z. x.; liu, x.; feng, l.; shao, y. h.; rong, j. g.; kong, x. g.; tong, g. z. title: genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between 1995 and 2004 date: 2006-01-09 journal: arch virol doi: 10.1007/s00705-005-0695-6 sha: doc_id: 256859 cord_uid: 7ixegm72 twenty-six avian infectious bronchitis (ib) viruses (ibv) were isolated from outbreaks in chickens in china between 1995 and 2004. they were characterized by comparison with twenty-six chinese reference strains and five other ibv strains. chinese ibvs, which were mainly nephropathogenic, were placed into seven genotypes. fourteen chinese ibv isolates were placed in genotype i, having small evolutionary distances from each other. genotype ii included 6 strains that were isolated in the 1990s in china. genotype iii consisted of eight chinese isolates that showed close relationship with korean ibv isolates. another eight ibv isolates clustered in genotype iv and showed larger evolutionary distances. the massachusetts serotype was present in china in 1990s and was in a separate genotype. two isolates, hn99 and ck/ch/lhn/00i, which might be a reisolation of vaccine strains, clustered into genotype vi. four chinese ibv isolates formed another genotype and showed larger evolutionary distances from other chinese ibv genotypes (genotype vii). ibvs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%. the results showed that ibvs in china came from genetic changes both in ibv populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. ibvs showing various genetic differences are cocirculating in china. infectious bronchitis virus (ibv) is a highly infectious and contagious pathogen of chickens worldwide [11] . the primary tissue of ibv infection is the respiratory tract, though some isolates replicate in the kidney and oviduct, resulting in nephritis and reduced egg production. generally, infectious bronchitis (ib) has been controlled with serotype-specific vaccines, but outbreaks of ib still occur, because vaccines offer little cross-protection between serologically distinct viruses [19] . a high mutation frequency and rna recombination leads to the emergence of new viruses capable of causing disease in vaccinated chickens [31, 41, 35] . although many countries share some common antigenic types, ibv strains within a geographic region are unique and distinct [1, 4, 15-17, 21, 47] . the identification of the circulating ibv field strains is extremely important for the selection of vaccine strains for the corresponding geographical region. ibv is the type species of the genus coronavirus in the family coronaviridae, order nidovirales [11] . it is a pleomorphic enveloped virus and has a singlestranded rna genome, approximately 27 kb in length, of positive polarity that specifies the production of three major structural proteins: the phosphorylated nucleocapsid (n) protein, the membrane (m) glycoprotein, and the spike (s) glycoprotein. the s glycoprotein of ibv, located on the outside of all virions, is responsible for fusion (virus envelope to cell membrane and cell membrane to cell membrane) and is translated as a precursor protein (s 0 ), then cleaved into a carboxy-terminal s2 subunit (approximately 625 amino acid residues), which anchors s in the virus envelope, and an amino-terminal s1 subunit (approximately 520 residues), believed to largely form the distal bulbous part of s [3, 7] . the s1 subunit of spike glycoprotein of ibv is responsible for inducing neutralizing and serotype-specific antibodies in chickens, and mutations in the antigenically important spike glycoprotein s1 subunit leads to the emergence and proliferation of variant serotypes [34] associated with disease outbreaks. serotypic evolution in ibv is associated primarily with the sequences of the s1 glycoprotein, and the genetic diversity of ibv is mainly monitored by analysis of the s1 gene [2, 9, 10, 23, 27, 32, 42] . ibv strains have been isolated and identified since 1982 in china. the outbreaks of ib have been ongoing, and ib continues to be an economically important disease to the poultry industry, although vaccines based on massachusetts (mass) strains such as h120 and h52 have been used for many years. however, the epidemiological analysis of ibv isolates in china has not been thorough except for with a few strains [31, 28, 46] . the relationships between chinese ibv isolates and foreign ibv isolates, especially korean, taiwanese and japanese ibv isolates, are not known. the focus of this study was to determine the molecular typing of the spike glycoprotein s1 subunit of ibv isolated between the years 1995 and 2004 in china. this will determine the ibv type(s) which are necessary for understanding the epidemiology and evolution of ibvs, as well as isolation of the virus, which is important for improved vaccination. twenty-six field ibvs were isolated from kidney, preventriculus, or oviduct of ib-suspected broilers or layers using specified pathogen-free (spf) embryonated eggs between 1995 (table 1) were pooled and 10% w/v tissue suspensions were made in 0.1% phosphate-buffered saline containing 100 u penicillin and 100 µg streptomycin/ml. after 12 h at 4 • c, 200 µl supernatant from the suspensions was inoculated into the allantonic cavity of 9-to 11-day-old embryos. three to 5 eggs were used for each sample. the inoculated eggs were incubated at 37 • c and candled daily. two to 7 blind passages were performed until the characteristic embryo changes such as the dwarfing, stunting, or curling of embryos were observed between 2 and 7 days after inoculation [12] . all allantoic fluids were harvested and tested for the presence of ibv using electron microscopy. samples of allantoic fluids were submitted for electron microscopy. briefly, after low-speed centrifugation at 1500 g for 30 min (allegra tm 21r centrifuge; beckman), the supernatant of the 1.5 ml allantoic fluids were centrifuged at 12 000 g for 30 min. the resulting pellet was resuspended in a minimal volume of deionized water and examined by negative contrast electron microscope (jem-1200, ex). genomic rna was extracted from virus-inoculated allantoic fluid with trizol reagent (invitrogen) following the manufacturer's instructions. the first-strand cdna was synthesized according the procedures of a previous report [31] using s1oligo3 [25] and genomic antisense ibv-212 oligonucleotide, 5 -atacaaaatctgccataa-3 . ibv-212 was designed based on a comparison and alignment of the genbank sequences of several known chinese ibv strains and situated in the downstream of s1oligo3 which had 5 nt overlapped between them. the pcr profiles involved an initial denaturation for 5 min at 95 • c followed by 30 cycles of denaturation at 94 • c for 1 min, annealing at 50 • c for 1 min, and polymerization at 72 • c for 2 min. the final polymerization step was performed at 72 • c for 10 min. owing to genetic variations among ibv isolates, it is difficult to design pcr primers that can be used to detect all ibv isolates. therefore, three genome-sense oligonucleotides, s1oligo5 [25] , s1uni2 [1] , or ibv-87, 5 -tattgattagagatgttggg-3 , which was selected from conserved areas by aligning several known chinese ibv sequences from genbank, were used with s1oligo3 [25] or ibv-212 as antisense primer ( table 2 ). the pcr products were analyzed on a 1.0% agarose gel and were sequenced directly. in addition, pcr products were also sequenced after cloning into the pmd18-t vector (takara). each region was sequenced at least three times from two pcr products from different rt reactions. the nucleotide and amino acid sequences of the s1 protein gene of the twenty-six ibv isolates were assembled, aligned, and compared with reference ibv strains using the megalign program in dnastar. phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences of the s1 protein gene was performed by the clustal v method using dnastar software [18] . thirty-one reference strains were selected for molecular his-arg-arg-arg-arg dq167137 ck/ch/lgd/96i s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167136 ck/ch/ldl/97i ibv-87 + s1oligo3 arg-arg-thr-gly-arg dq068701 ck/ch/lln/98i s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167145 ck/ch/ldl/98i ibv-87 + s1oligo3 arg-arg-thr-gly-arg dq167132 ck/ch/lhlj/99i s1oligo5 + ibv-212 ibv-87 + s1oligo3 arg-arg-thr-gly-arg dq167130 ck/ch/lxj/02i s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167152 ck/ch/lhlj/02i s1oligo5 + s1oligo3 his-arg-arg-arg-arg dq167138 ck/ch/lshh/03i s1uni2 + s1oligo3 his-arg-his-arg-arg dq167149 ck/ch/lshh/03ii s1uni2 + s1oligo3 his-arg-his-arg-arg dq167150 ck/ch/lgd/03i s1oligo5 + ibv-212 arg-arg-phe-arg-arg dq167133 ck/ch/lah/03i s1oligo5 + ibv-212 arg-arg-his-ser-arg dq167128 ck/ch/lsd/03i s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167148 ck/ch/ljl/04i s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167144 ck/ch/lhlj/04v s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167139 ck/ch/ldl/04ii s1oligo5 + ibv-212 arg-arg-tyr-arg-arg dq167131 ck/ch/lgd/04ii s1oligo5 + ibv-212 arg-arg-phe-arg-arg dq167134 ck/ch/lgd/04iii s1oligo5 + ibv-212 arg-arg-leu-arg-arg dq167135 ck/ch/lhlj/04xi s1oligo5 + ibv-212 his-arg-arg-arg-arg dq167140 a oligonucleotides used for amplifying s1 protein gene b arg arginine, phe phenylalanine, his histidine, thr threonine, gly glycine, ser serine, tyr tyrosine, leu leucine analysis. of these, twenty-six were chinese ibv strains from the genbank database, and they represented most of the chinese ibv field isolates available through genbank or other publications. a total of fifty-two chinese ibv field isolates, including our twenty-six isolates, were chosen to give a representation based on geographic distribution, year of isolation, and phylogenetic position. in addition, two ibv strains, 3051/02 and t07/02, representing tw i and tw ii ibv isolates in taiwan [20] , were selected. a korean ibv isolate, k069-01, was also selected. this ibv strain belonged to genotype iii of korean ibv strains, and this genotype was a major type of ibv in korea. jp8127, a japanese ibv strain, was also selected, and its s1 protein gene was compared with chinese ibv isolates. ibv strains from the above 3 geographically different areas were selected because we were interested in knowing whether the ibv isolates in china were introduced from neighboring countries and continents or whether they arose by mutations of circulating chinese ibv strains. furthermore, the s1 protein gene of the h120 vaccine strain was selected and compared in this study because the vaccine was widely used for many years on poultry farms in china. the entire coding region of the s1 protein gene of these strains was chosen for analysis. the fifty-seven ibv strains, including our twenty-six isolates, were molecularly analyzed. the twenty-six ibv isolates in this study and their accession numbers are listed in table 2 . the ibv reference strains and their accession numbers are listed in table 3 . twenty-six ibv strains were isolated from flocks that were suspected of ibv infection. the isolates were from flocks in different parts of china (fig. 1 ) that showed clinical ib and had 5 to 60% mortality. the nephritis observed in all flocks was characterized by enlarged and pale kidneys, frequently with urate deposits in the tubules, and severe dehydration and weight loss. typical signs, including dwarfing and death of the embryo, were observed in different passages when each isolate was inoculated into embryos (table 1) . diagnoses based on electron microscopy examination showed all isolates had typical coronavirus morphology and were free of other agents such as newcastle disease virus (ndv) (results not shown). to assess the genetic relatedness among the ibv strains, a phylogenetic tree was performed with s1 protein genes. the results are shown in fig. 2 . the fiftyseven ibv strains were separated into seven genotypes (i to vii) by phylogenetic analysis of the s1 protein genes (fig. 2) . genotype i consisted of fourteen chinese strains having small evolutionary distances from each other as shown in the rooted tree (fig. 2) . genotype ii included 6 strains that were isolated in the 1990s in china. most of the chinese ibv isolates included in genotype iii were also isolated in the 1990s, except tl/ch/ldt3/03 and ck/ch/lgd/03i, which were both isolated in guangdong province in 2003 from teal [29] and layer hens, respectively. the korean ibv isolate, k069-01, which belonged to genotype iii of korean ibv strains [27] , was closely related to those isolates in genotype iii. six of eight ibv isolates displayed in genotype iv were isolated after 2000, and most of them came from southern china. furthermore, isolates included in genotype iv showed larger evolutionary distances (fig. 2 ). ten chinese ibv isolates formed the genotype v in which h120 was included, and none of our twenty-six isolates were grouped under this genotype. the isolates hn99 and ck/ch/lhn/00i, both isolated in henan province in 1999 and 2000, respectively, together with a japanese isolate, jp8127, were grouped into genotype vi. our three ibv isolates recovered in dalian, china, between 1997 and 2001, were grouped into genotype vii. a chinese ibv isolate, j2, which was isolated from the proventricular tissues of infected chickens [46] , was also placed in genotype vii. two ibv isolates, 3051/02 and t07/02, belonging to tw i and tw ii, formed a unique genotype. phylogenetic relationships, based on the sequence of the s1 subunit of the s protein gene, of our twenty-six isolates and thirty-one reference strains (the first 1669 nt, starting at the aug translation initiation codon, of the s protein genes) using the megalign program dnastar with the clustal v method [18] . our ibv isolates are in bold type the spike glycoprotein of ibv is translated as a precursor protein (s0) and then cleaved into two subunits s1 and s2 [9, 24] . cleavage site motifs of the fifty-seven ibv strains are listed in table 2 and table 3 , and twelve different cleavage site sequences were observed. the most common cleavage recognition site observed (24 of 57 viruses) was arg-arg-phe-arg-arg. viruses with this cleavage recognition site are the h120 vaccine strain, one korean strain, k069-01, taiwan isolates 3015/ 02 and t07/02, ten chinese mass-type isolates, and ten other chinese isolates included in genotype iii (six strains) and iv (four strains). the second most common site was his-arg-arg-arg-arg. viruses with this cleavage recognition site include twelve isolates in genotype i and three in genotype ii. this recognition site was unique for virus isolates in china. the third most common site was arg-arg-thr-gly-arg. viruses with this cleavage recognition site were placed in genotype vii, which included our three isolates (ck/ch/ldl/97i, ck/ch/ldl/98i, and ck/ch/ldl/01i) and isolate j2. this cleavage recognition site was also unique to viruses in china. the jx/99/01, ck/ch/lah/99i, and sc021202 viruses had a cleavage recognition site, arg-arg-his-arg-arg, as did d1466 [22] . chinese ibv isolates hn99 and ck/ch/lhn/00i, which were grouped in genotype vi, had a cleavage recognition site, arg-arg-ser-arg-arg, which was the most common site reported by jackwood [22] , who had compared the cleavage recognition sites of fifty-five ibv isolates to determine if the site sequence correlates with host cell range, serotype, geographic origin, and pathogenicity. the ck/ch/lshh/03i and ck/ch/lshh/03ii viruses had a unique cleavage recognition site, his-arg-his-arg-arg, as did isolates bj and bjy, arg-arg-thr-arg-arg, ck/ch/lah/03i, arg-arg-his-ser-arg, ck/ch/ldl/04ii, arg-arg-tyr-arg-arg, ck/ch/lgd/04iii, arg-arg-leu-arg-arg, bjs, his-arg-thr-lys-arg, and japanese strain, jp8127, arg-arg-phe-lys-arg. the complete nucleotide and predicted amino acid sequences of the s1 protein of the fifty-seven ibv strains were determined and compared. except for isolates in genotype v, which included the mass-type strains, none of the chinese ibv isolates examined in this study shared more than 83% amino acid similarity in the s1 protein with the h120 vaccine strain. the s1 protein genes, which varied from 0.2 to 26.7% among the strains examined, indicated that point mutations, deletions, and insertions contribute to the evolution of ibv. ibvs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%, with the exceptions of isolate bjs (genotype ii) and isolates in genotype iv, bjq (genotype iii) and isolates in genotype v, isolates between genotypes i and iii, which showed amino acid similarities of 91-94.5%, 90.5-92%, and 93.2-95.6%, respectively. the overall predicted amino acid sequence comparisons of the entire s1 protein of fifty-seven ibv strains reflected that most of the sequence variations were concentrated in three regions. the first included residues 50-87, corresponding to the s1 protein of the h120 vaccine strain, in which the hypervariable region 1 (hvr1) is located [8, 37, 42] . the second contained amino acid sequences between residues 114-140, which encompasses the hypervariable region 2 (hvr2) [8, 37, 42] . the last included residues 273-293, in which the hypervariable region 3 (hvr3) a amino acid abbreviations: s serine, d aspartic acid, g glycine, a alanine, n asparagine, h histidine, e glutamic acid, t threonine, y tyrosine, p proline, q glutamine, v valine, i isoleucine, k lysine, r arginine, m methionine; b positions of residues in deduced amino acid sequences of the s1 protein of the h120 vaccine strain; c positions of residues in deduced amino acid sequences of the s1 protein of the h120 vaccine strain between which the residue(s) of other ibvs was (were) inserted; d missing amino acid residues is present [39] . furthermore, almost all of the chinese ibv isolates contained deletions and insertions except for those of the mass-type ibv, which were included in genotype v in this study, and had amino acid sequences similar to those of the h120 strain ( table 4 ). the deletions and insertions, which occurred in the predicted amino acid sequences of the s1 proteins of fifty-seven ibv strains in this study, were correlated with the genotypes of s1 protein genes, as shown in table 4 . in addition, the korean strain, k069-01, shared most of the motifs of deletions and insertions with chinese ibv field isolates in genotype iii. in 1962, winterfield and hitchner reported a nephrosis condition associated with ib in the united states, and cumming reported an ib outbreak causing severe kidney lesion in chickens in australia [13, 43] . since this time, various nephropathogenic strains of ibv have been identified throughout the world [33] . in china, ib with nephritis was first reported in 1982 and several nephropathogenic ibv strains have been isolated in different parts of china since then [31, 32, 44] . of the twenty-six ibv isolates in this study, one was isolated from atrophic oviduct of a diseased layer hen, six from swollen proventricular tissues of infected chickens, and the rest from swollen kidneys of ib-suspected chickens. although seven ibv strains were isolated from tissues other than kidney, the gross lesions of kidney in these diseased chickens were also obvious. based on the fact that these ibv strains were isolated from 1995 to 2004 in china, we considered that ib was prevalent all the while in china, although vaccines based on mass-type strains such as h120 and h52 have been used for many years on poultry farms, and nephropathogenic ibv was the major type of ibv circulating in china. although the genetic basis of ibv pathogenicity is not known, the s1 protein gene of ibv has serotype-specific and neutralization-specific epitopes, and serotypic evolution and the genetic diversity of ibv is mainly monitored by analysis of the s1 gene [2, 9, 10, 23, 27, 32, 42] . in the present study, phylogenetic analysis of s1 genes showed that chinese ibv isolates were grouped into seven genotypes (fig. 2) . ibvs isolated ten years ago were included in the same genotype with the strains isolated recently (for example, ck/ch/lsc/95i and ck/ch/ lgd/03i in genotype iii), indicating that this genotype may be indigenous and has been prevalent in china for at least ten years. serotype differences among the genetically distinct ibvs generally correlated with variations in the hvr of the s1 protein gene [4, 8] and differences of as little as 5% between s1 sequences of ibv could result in poor cross-protection offered by currently used vaccines [19] . the low identities (<83%) of amino acid sequences between chinese ibv isolates and h120, except for those of the mass-type ibv, which were included in genotype v in this study, may account for the prevalence of the viruses during the past ten years in spite of the extensive use of mass-type vaccines in the field in china. hence, developing vaccines from local strains is necessary for ibv control in china. although the number of basic residues around the spike glycoprotein cleavage recognition site of ibv does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with the envelope glycoproteins of orthomyxoviruses and paramyxoviruses, the sequences of cleavage recognition sites was correlated with geographic distribution of the viruses [22] . nine spike glycoprotein cleavage recognition site sequences were found in viruses of genotypes i to iv, in which six were unique to isolates in china, indicating genetically distinct evolution from viruses in other countries by cleavage recognition site analysis. however, a korean ibv strain, k069-01, shared the same cleavage recognition site sequence, arg-arg-phe-arg-arg, with ten chinese isolates included in subgenotypes iii (six strains) and iv (four strains), ten chinese mass-type isolates (genotype v), and two taiwan isolates, 3015/02 and t07/02. furthermore, k069-01 and chinese isolates in genotype iii shared more than 90% amino acid identities and they were also grouped into the same genotype (fig. 2) . k069-01 was clustered into genotype iii of korean ibv strains, which was the major type of ibv circulating in korea, and isolates in this genotype induced 50% mortality in 1-day-old chicks as well as severe renal urate deposition on the kidneys [40] . this was similar to chinese isolates in genotype i [29, 31] . based on these facts, ibvs between chinese genotype iii and korean genotype iii had a close relationship, as did ndv [26] , owing to the increased trade of agricultural products including poultry between two countries. unlike k069-01, isolates 3051/02 and t07/02, which represented tw i and tw ii strains, respectively [20] , were clustered into a separate branch that was separated from the chinese genotypes, indicating that they had different origins. ten chinese ibv strains were classified into the mass serotype (genotype v by phylogenetic analysis). as in china, mass-type ibvs were also present in other asian countries, such as korea [27, 40] , japan [32, 38] , and taiwan [20, 30] , although mass-type vaccines were commonly used in these countries or continents. however, chinese mass-type strains were all isolated in the 1990s and were not the major ibv type circulating in recent years in china. molecular studies have shown that a new serotype or variant can emerge as a result of only a few changes in the amino acid composition in the s1 part of the virus spike protein, with the majority of the virus genome remaining unchanged [6] . this could be due to immunologic pressure caused by the widespread use of vaccines, to recombination as a consequence of mixed infections, or to the decrease of dominant serotypes as a result of vaccination, allowing other field strains to emerge. to this study, the mass-type viruses may have come from the vaccine strains by point mutation, although the possibility that some of them were reisolations of vaccine strains cannot be excluded. two strains, hn99 and ck/ch/lhn/00i, both isolated in henan province in china, together with a japanese strain, jp8127, constituted a "novel" genotype vi (fig. 2) . these two chinese isolates did not show close similarity to any of the s1 protein sequences of other chinese ibv isolates available through genbank or publications. interestingly, blast searches revealed significant similarity (99%) of s1 protein genes between isolate hn99 and a vaccine strain, jaas (ay839140), which was from australia and used in china to control ibv. the isolate ck/ch/lhn/00i shared 99% similarity in the s1 protein gene with another ibv vaccine strain, jilin (ay839144), which was also used in china. when ck/ch/lhn/00i was inoculated experimentally into 15-day-old spf chickens, no disease signs were apparent (s. liu et al., unpub. data) . the spreading of a virus from one area or country to another could be due, at least in part, to its improper introduction by the trading of birds or by the use of attenuated vaccines. to our knowledge, no other ibv strains related to hn99 or ck/ch/lhn00i were isolated in recent years in china, and considering the pathogenicity and genetically close relationship between the two isolates and the corresponding vaccine strains, we speculated that the two isolates would be reisolations of vaccine strains. isolate j2, which was very similar to q1 and t3, was genetically distinct from most of the chinese ibv isolates [45] . in this study, our three isolates (ck/ch/ldl/97i, ck/ch/ldl/98i, and ck/ch/ldl/01i) were clustered into the same group (genotype vii) with j2. similar to j2, ck/ch/ldl/97i and ck/ch/ldl/98i were isolated in swollen proventriculars tissues of infected chickens, whereas ck/ch/ldl/01i was isolated from atrophic oviduct of a diseased layer hen. it was found that the gross lesions of the kidney in these diseased chickens were also obvious, as with isolate 2992/02 [20] . 2992/02 was isolated in taiwan and was very similar to the j2 strain by comparison of s1 protein genes [20] . the diversity of the pathogenicity of ibv strains was expected; although the primary tissue of ibv infection is the respiratory tract, some isolates can grow in nonrespiratory organs such as the kidney, the female reproductive tract, intestine, and spleen of chickens [2, 14, 33, 36] . with the exception of the massachusetts strain, a very interesting aspect of ibv epidemiology, as far as it is possible to know, is the presence and the spreading of the various ibv serotypes in different continents. about 20 emergent serotypes in north america did not spread to other continents. similarly, the european, australian, and asiatic serotypes apparently did not spread elsewhere. in china, ibv epidemiology is more complicated. besides genotypes i to iv and vii, the mass-type ibv and ibv closely related to australian classical strains were also present, indicating ibvs showing various genetic differences were cocirculating in china. molecular analysis of the 793/b serotype infectious bronchitis virus in great britain emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in india completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus infectious bronchitis virus: evidence for recombination within the massachusetts serotype coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce 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characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia rapid detection and identification of avian infectious bronchitis virus author's address: dr. xiangang kong we would like to thank dr. j. j. giambrone, department of poultry science,auburn university, auburn, u.s.a., for his helpful comments in reviewing the manuscript. key: cord-010608-eaa2znom authors: butt, salman l.; erwood, eric c.; zhang, jian; sellers, holly s.; young, kelsey; lahmers, kevin k.; stanton, james b. title: real-time, minion-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples date: 2020-03-05 journal: j vet diagn invest doi: 10.1177/1040638720910107 sha: doc_id: 10608 cord_uid: eaa2znom infectious bronchitis (ib) causes significant economic losses in the global poultry industry. control of ib is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (ibv), which has led to the emergence of several serotypes that lack complete serologic cross-protection. although serotyping requires immunologic characterization, genotyping is an efficient means to identify ibvs detected in samples. sanger sequencing of the s1 subunit of the spike gene is currently used to genotype ibv; however, the universal s1 pcr was created to work from cultured ibv, and it is inefficient at detecting multiple viruses in a single sample. we describe herein a minion-based, amplicon-based sequencing (ampseq) method that genetically categorized ibv from clinical samples, including samples with multiple ibvs. total rna was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and pcr amplified using modified s1-targeted primers. amplicons were barcoded to allow for pooling of samples, processed per manufacturer’s instructions into a 1d minion sequencing library, and then sequenced on the minion. the ampseq method detected ibv in 13 of 14 ibv-positive samples. ampseq accurately detected and genotyped both ibv lineages in 3 of 5 samples containing 2 ibv lineages. additionally, 1 sample contained 3 ibv lineages, and ampseq accurately detected 2 of the 3 lineages. strain identification, including detection of different ibvs from the same lineage, was also possible with this ampseq method. our results demonstrate the feasibility of using minion-based ampseq for rapid and accurate identification and lineage typing of ibv from oral swab samples. infectious bronchitis (ib), which is caused by infectious bronchitis virus (ibv; avian coronavirus), is one of the most important diseases of poultry, causing severe economic losses worldwide. 8 clinical signs of disease are diverse and include respiratory distress, severe ocular discharge, poor body weight gain, decreased egg production, flushing (renal disease), and occasionally mortality in chickens. 7 ib is often complicated by secondary bacterial (e.g., escherichia coli, mycoplasma sp.) and viral infections (e.g., influenza a virus, newcastle disease virus, infectious laryngotracheitis virus). 43 lack of cross-protection among ibv serotypes is a challenge to controlling ib 15 ; therefore, control of ib relies heavily on serotype-specific live attenuated vaccines. 8 collectively, the presence of multiple ibvs in a single sample, emergence of variant ibvs, and high genetic diversity of ibv can complicate the diagnosis of ib and illustrate the need for enhanced testing. 15 ibv is an enveloped, pleomorphic gammacoronavirus with an unsegmented, single-stranded, positive-sense, 26-27.8-kb, rna genome that encodes the nonstructural polyproteins, 1a and 1b, and several structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n). 38, 41 in addition, 2 accessory genes, expressing 3a, 3b and 5a, 5b, respectively, have also been described. 6, 14, 38 the s protein is highly glycosylated, and post-translational cleavage leads to 2 subunits: s1 and s2. 10, 48 besides acting as the viral attachment protein, the s1 protein is a major target of neutralizing antibodies. 7 as with many attachment proteins that are targets of virusneutralizing antibodies, the s1 subunit is highly diverse with almost 50% of the amino acids differing among ibv serotypes. 2, 21, 38 such variation leads to important biological differences between ibv serotypes and the emergence of novel variants. more than 60 serotypes of ibv have been reported, but the most common serotypes of ibv in north america are arkansas, connecticut (conn), delaware 072, georgia 08 (ga08), georgia 98 (ga98), and massachusetts (mass). 16 this genetic diversity leads to the emergence of new serotypes and a lack of complete cross-serotype protection by vaccines. 29 the correlation of ibv genotypes and serotypes of ibv has been reported 45 ; therefore, accurate genotypic identification of ibv is an important step to identify ibv in clinical respiratory cases, ensure selection of proper ibv vaccines for use in vaccination programs, and to better understand the epidemiology of this global virus. one comprehensive classification scheme for ibv uses s1 gene sequence-based phylogeny of ibv, and identified 6 genotypes (i-vi), 32 subgenotypic lineages, and a number of inter-lineage recombinants in global strains of ibv. 44 among the 6 genotypes, genotype i (gi) is the most diverse group of viruses, with 27 unique lineages. 44 as such, sequencing of the s1 subunit provides important information regarding the classification of ibv within samples. the genetic classification of ibv has relied on genotypespecific, reverse-transcription real-time pcr (rt-rtpcr) assays, serotype-specific s1 rt-rtpcr, 32, 40 and/or pan-ibv s1 rt-pcr assays coupled with sanger sequencing. 8, 31, 49 genotype-specific rt-rtpcr assays are limited to short fragments, which may miss important changes in the s1 gene (~1.6 kb) 44 outside the short target. additionally, given that the target is short, the primers lie within the variable regions of the s1 gene and may require a different assay for each genotype. in contrast to the genotype-specific primers, the pan-ibv s1 primers 1, 17, 22, 23, 25, 33 only require one reaction to determine the presence of ibv; however, they have a relatively low sensitivity and are typically used only on egg-cultured virus, which adds an additional, time-consuming step, and many diagnostic laboratories do not have specific pathogen-free embryos readily available. additionally, genotyping the pan-ibv results requires cloning of pcr products to detect multiple ibv subpopulations in the sample, 30 which is inefficient when potentially dealing with multiple (3 or 4) ibv types (i.e., "type" denoting genotype, lineage, and/or sublineage classification of an ibv). as such, the development of a pan-ibv sequencing method to rapidly determine the genotype(s) in samples would aid in lineage typing and tracking of ibv circulating in poultry flocks. third-generation sequencing technology has been used for the detection of viral nucleic acids and sequencing ultralong dna molecules. 34 the minion nanopore sequencer (oxford nanopore technologies [ont], oxford, uk), a new dna sequencing technology that allows for rapid, in-house, real-time detection and differentiation of ibv lineages, may be cost-effective and useful in the field. 19 amplicon-based sequencing (ampseq) has also been used to amplify specific regions of newcastle disease, 3, 13 infectious laryngotracheitis, 39 zika, 34 ebola, 35 and influenza a viruses, 46 by simple rt-pcr and then sequencing on the minion device. real-time data analysis, the lack of significant start-up cost investment or maintenance expenses, simultaneous and sequential multiplexing unique to minion, and the ability to sequence long dna molecules so that primers are in conserved regions while the product contains the variable region are the features that make the use of this technology highly feasible in disease diagnosis. accurate identification of ibv genotypes from samples, including the detection of multiple ibv types from a single sample, is crucial to respiratory disease diagnosis, selection of appropriate ibv vaccines, and epidemiologic studies. therefore, our aim was to create a single, amplicon-based protocol to sequence the ibv s1 gene and develop a sequence analysis workflow to identify ibv types from clinical swab samples. our method provides a useful assay for ibv and a model for the development of future ampseq assays. clinical swab samples (n = 15) were obtained from samples submitted to the poultry diagnostic & research center, university of georgia (athens, ga; table 1 ). trachea and choanal cleft palate swabs were collected from commercial chickens at 5 d of age (also corresponds to 5 d post spray vaccination in the hatchery with a commercial ibv ga08 vaccine and 5 d post placement in the chicken house). briefly, the birds were swabbed with sterile polyester swabs (puritan, guilford, me), and swabs were placed in 500 µl of phosphate-buffered saline without calcium and magnesium (cellgro; corning, manassas, va). swabs in tubes were stored on ice immediately following collection through delivery to the lab. total rna was extracted from each of the swab samples (qiaamp viral rna mini kit; qiagen, hilden, germany) as per the manufacturer's instructions, aliquoted, and stored at −80°c until further use. these samples were tested previously for ibv and the ga08 serotype of ibv. briefly, a pan-ibv reverse-transcription quantitative pcr (rt-qpcr) assay 4 was used to detect ibv in general, and then an ibv strain-specific rt-rtpcr assay (in-house validated set of primers) was used to detect ga08 in the samples (agpath-id one-step rt-pcr kit; applied biosystems 7500 fast; thermo fisher scientific, waltham, ma). for minion complementary dna (cdna) synthesis, rna was extracted from 500 µl of each clinical sample (trizol-ls; thermo fisher scientific) per the manufacturer's directions. a reaction mixture of 8 µl of total rna, 1 µl of random primers, and 1 µl of dntps was incubated at 65°c for 5 min, chilled on ice for at least 1 min, followed by addition of 10 µl of cdna synthesis mix including superscript iii reverse transcriptase (thermo fisher scientific) according to the manufacturer's instructions. the reaction was incubated at 25°c for 10 min, then at 50°c for 50 min for cdna synthesis. the reaction was terminated at 85°c for 5 min, and then chilled on ice. to remove residual rna, the cdna solution was incubated with rnase h at 37°c for 20 min. a universal s1 primer set, 27 tailed with the minion universal adapter sequence of 22 nucleotides (underlined, table 2 ) to allow barcoding of amplicons, was used for targeted amplification of the ibv s1 gene for ibv. the pcr reaction mixture (expand high fidelity pcr system; roche diagnostics, mannheim, germany) was composed of 10 µl of cdna, 1 µl of 10 µm forward primer, 1 µl of 10 µm reverse primer, 2.5 µl of 10× expand high fidelity buffer, 1 µl of expand high fidelity enzyme mix, and 1 µl of 10 mm dntps, to a final volume to 25 µl with 9 µl of nuclease-free water. the following thermocycling conditions were used for amplicon synthesis: denaturation at 95°c for 2 min; 30 cycles of 94°c for 1 min, 45°c for 2 min, and 72°c for 2 min; and a final extension at 72°c for 10 min. pcr products were electrophoresed in 1.5% agarose with sybr safe dna gel stain (invitrogen, carlsbad, ca) for visual evaluation. amplified dna was purified (agencourt ampure xp beads; beckman coulter, brea, ca) at 1.6:1 (volumetric bead:dna) and quantified (dsdna high sensitivity assay kit; qubit 3.0 fluorimeter; biotium, fremont, ca). the amplicons obtained from the tailed s1 primer set were then used to prepare minion-compatible dna libraries. briefly, each of the amplicons was diluted to 0.5 nm and amplified using barcoding primers (1d pcr barcoding amplicon kit; ont) and longamp taq 2× master mix (new england biolabs, ipswich, ma) with the following conditions: 95°c for 3 min; 15 cycles of 95°c for 15 s, 62°c for 15 s, and 65°c for 80 s; and a final extension at 65°c for 80 s. the barcoded amplicons were bead purified (1:1.4, bead:solution), pooled (run 1: pool of 10 samples; run 2: pool of 5 samples) into a single tube, end prepped (neb-next ultra end repair/da-tailing module, new england biolabs), bead purified (1:1, bead:solution), and ligated to the sequencing adapters (ligation sequencing kit 1d, catalog sqk-lsk108; ont), all per ont directions. final dna libraries were bead purified (0.4:1, bead:solution), eluted in 15 µl of elution buffer, and sequenced with the minion sequencer (ont). a new flow cell (flo-min106 r9.4; ont), stored at 4°c prior to use, was allowed to equilibrate to room temperature for 10 min and then primed with running buffer as per the manufacturer's instructions. the pooled dna libraries were prepared by combining 12 µl of the library pool with 2.5 µl of nuclease-free water, 35 µl of running buffer with fuel, and 25.5 µl of library loading beads. after the minion platform quality-control run, the dna library was loaded into the minion flow cell via the spoton port. the standard 48-h 1d sequencing protocol was initiated using the minknow software v.5.12. for a more rapid run, run 1 sequencing was allowed to continue for 2 h until 42,940 reads were obtained. for a deeper run, run 2 was allowed to continue for ~6 h until ~156,000 reads were obtained. first, a lineage-typing database, containing 32 ibv s1 gene sequences (1 sequence from each of 32 lineages in the 6 genotypes) 44 and the chicken genome (gcf_000002315.4_gal-lus_gallus-5.0), was constructed (hereafter, ibv-lineage-typing database). a second database was constructed with all of the avian coronavirus s1 sequences (n = 7,328) available in ncbi (as of 2017.09.08) and the chicken genome (hereafter, all-ibv database). prior to database construction, all sequences were dustmasked (ncbi c++ toolkit, https:// ncbi.github.io/cxx-toolkit/), and each ibv sequence was assigned a unique taxonomy id as a species hierarchically under the genus gammacoronavirus to allow easy sorting. the local blast (https://blast.ncbi.nlm.nih.gov/) databases were compiled using default settings. the ibv-lineage-typing database was used to assign ibv lineages to each read, as appropriate. the all-ibv database was used to assign a taxonomic id to each of the ibv reads from within ibv lineage read clusters. the ibv-lineage-typing database, which contains only one sequence per lineage, is required because centrifuge (computational tool for taxonomic classification of individual reads) divides the score for any given read by the number of hits that have an equal score. given that some lineages are overrepresented in genbank (e.g., mass strains), this results in the scores of those reads (e.g., mass reads) being divided by a large number, effectively reducing the score for any single alignment. however, because there is only one read per lineage in the ibv-lineage-typing database, it has insufficient diversity to determine if there is more than one strain of the same lineage present in a sample. thus, the all-ibv database provides the diversity required for that analysis. a schematic diagram of the workflow of minion data analysis is presented in figure 1 . for nanopore sequencing data, pre-processing steps were performed to prepare data for downstream analysis. briefly, nanopore reads (fast5) were basecalled using albacore v.2.02 (https://github.com/albacore/albacore) with the following parameters (read_fast5_ basecaller.py -i /input_reads_fast5/ -recursive -t 4 -s / output_files -flowcell flo-min107 -kit sqk-lsk108 -o fastq). porechop (https://github.com/rrwick/porechop) was used for adapter-trimming (default setting), barcode-based demultiplexing (default settings), and to trim an additional 21 nucleotides representing the s1 primer sequences (porechop -i input_file.fastq -extra_end_trim 21 -b ./output_ demultiplexed/). after barcode and adapter removal, reads were analyzed with a script-based, 2-step data analysis protocol, which includes centrifuge-kreport as taxonomic read classifier 24 using the sequences in the above-mentioned blast databases. briefly, basecalled reads (fastq) from individual barcoded samples were used as an input. first, the basecalled reads were aligned to the ibv-lineage-typing database using blastn and reads were clustered based on the read sequence alignment to the respective prototype sequence of ibv lineage. these read clusters were used to interpret the presence of ibv genotypes and lineages in the samples. for the identification of ibv types, the lineagebased read clusters were individually aligned to the all-ibv database, which produced subclusters of reads. each of these read subclusters potentially represents a different sequence of ibv and was further used for interpretation. knowing that minion sequencing has a high sequencing error rate in individual reads, further steps were added in the data analysis algorithm to obtain a more accurate consensus sequence. therefore, these read subclusters were mapped, using geneious mapper in geneious software v.11.1.3. (biomatters, auckland, new zealand), to the ibv-lineage-typing database (a reference fasta file) to obtain consensus sequences from each subcluster. a minimum threshold for the number of reads per consensus sequence was not set because in 2 samples (sample 4 in run 1 and sample 12 in run 2), only 1 and 5 ibv reads were obtained, respectively; therefore, consensus sequences were built from all of the available ibv reads per subcluster. the consensus sequences ibv-universal s1 4 ibv-s1-adap fwd 5ʹ-tttctgttggtgctgatattgctgaac ctgaacaaaagac-3ʹ s1 nanopore~1,672 all genotypes and lineages ibv-s1-adap rev 5ʹ-taatgactggcagcgctaag-3ʹ the underlined sequences are oxford nanopore technology adapter sequences for multiplexing samples in a single sequencing run. were compared to genbank using blastn. to select the "top hit" from blastn output, sequence search results were ordered by "sequence identity" and then sequence alignments were evaluated for "minimum mismatches" and "coverage" of query or subject sequence. for each sequence, the blastn output with the highest query or subject coverage and the fewest mismatches was used as "top hit" in the final results. for sanger sequencing amplicon synthesis, cdna from the minion library preparation (as described above) was amplified using the following primer sets (table 2) . first, a primer set based on connecticut sequence ncbi fj904716.1 was used to amplify genotype i-lineage 1 (gi-l1) viruses (e.g., conn and mass serotypes). second, a primer set based on ga98 sequence (ncbi af274439.1) was used to amplify genotype iv-lineage 1 (giv-l1) viruses (e.g., georgia 1998 and delaware 072 serotypes). primer sets were designed using ncbi primer-blast. 51 the pcr reaction mixture (expand high fidelity pcr system; roche diagnostics) is composed of 10 µl of cdna, 0.5 µl of 10 µm forward primer, 0.5 µl of 10 µm reverse primer, 2.5 µl of 10× buffer, 0.5 µl of enzyme mix, and 0.5 µl of 10 mm dntps, then made to the final volume to 25 µl with nuclease-free water. the following thermocycling conditions were used for amplicon synthesis: denaturation at 95°c for 1 min; 35 cycles of 94°c for 30 s, 55°c (conna primer set) or 50°c (ga98 primer set) for 30 s, and 72°c for 45 s; and a final extension of 72°c for 5 min. pcr products were visually inspected after electrophoresis in 1.5% agarose gel, and the correctly sized bands were cut out for dna purification (qiagen pcr purification kit) and quantified (dsdna high sensitivity assay kit; qubit 3.0 fluorimeter; biotium). briefly, purified amplicons were inserted into plasmids, and ligation reactions (10 µl) were set up as per the manufacturer's instructions (pgem-t easy vector system; promega). after ligation, 3 µl of ligation mixture was transformed to jm109 competent cells by heat shock. individual bacterial colonies were checked with pcr, and the positive bacterial colonies were plated on lysogeny broth agar-ampicillin plates at 37°c for 16 h. the plasmids were extracted from these positive bacterial colonies (qiaprep spin miniprep kit; qiagen) and submitted for bidirectional, commercial (genewiz, south plainfield, nj) sanger sequencing. for sanger sequencing data, the chromatogram from each sample was manually checked, and primer sequences were trimmed in mega 6.0. 42 forward and reverse sequences from multiple clones were aligned using mega 6.0 software, and consensus sequences were compared to genbank using blast (as of 2018.03.12) and the top hit from the blastn output was selected, as described above for min-ion sequencing data, to identify the ibv type in samples. for each of the samples in run 1, a pairwise nucleotide identity comparison between the ibv partial s1 gene sequences obtained from sanger sequencing and minion sequencing was performed. briefly, the final ampseq consensus sequences from identical ibv types in each of the samples were aligned using clustalw in mega6, 42 and this alignment was used for pairwise nucleotide identity using mega6. 42 five samples from run 1 had been tested previously with a pan-ibv rt-qpcr assay, 4 and 4 were positive for ibv. samples with pathogenic ibv strains (run 2, n = 5) were tested with the pan-ibv rt-qpcr, and all 5 samples were ibv positive (table 1) . additionally, samples were also tested previously with a ga08-specific rt-rtpcr (in-house validated set of primers used in the poultry diagnostic & research center), which was used on all samples from run 1 (including 5 samples that were tested previously by the pan-ibv rt-qpcr), and 4 of 10 samples were positive. samples 3 and 4, which were positive with the pan-ibv rt-qpcr assay, were negative with the ga08-specific rt-rtpcr assay, indicating that these samples contained ibv serotypes other than ga08 and required further testing. sample 5 tested negative with both assays (table 1) . pcr amplicons obtained directly from 10 clinical swab samples with vaccine ibv serotypes were barcoded, pooled, and sequenced on the minion device (run 1). a total of 38,661 reads were successfully basecalled from the entire sequencing run (42,940 total reads). after demultiplexing, reads per barcode ranged from 831 to 4,114. a total of 14,845 reads were not assigned to any of the used barcodes, and 128 reads were discarded because of middle adapters. the nanopore reads were queried against the ibv-lineage-typing database to determine if the samples contained ibv (fig. 1 ). this ampseq protocol detected ibv reads in 8 of 10 samples. the number of ibv reads per sample (ibv-positive sample) ranged from 56 to 944. the minion-negative samples included sample 5, which was consistently negative with rt-rtpcr and rt-pcr assays (and thus interpreted as ibv negative), and sample 1, which had the highest cycle threshold (ct) values in the pan-ibv rt-qpcr (table 1) . sequencing data were further analyzed to determine if multiple ibv genotypes or lineages could be detected. lineage 1 and lineage 27 from genotype i (gi-l1, gi-l27), and lineage 1 from genotype iv (giv-l1), were the detected lineages in the samples. results from the individual rt-rtpcr assay for ga08 (gi-l27), and rt-pcr assays for giv-l1 and gi-l1, confirmed the presence of multiple ibv genotypes and lineages in 6 of the 9 ibv-positive samples (1-4, 6, 7); ampseq results confirmed multiple genotypes and lineages in 4 (2, 4, 6, 7) of those 6 samples (ampseq failed to detect any ibv in 1, and failed to detect giv-l1 in 3). the presence of a single lineage was confirmed by pcr-based assays and ampseq in 3 samples (8-10; table 3 ). the single-read cluster composed of <5 reads (run 1, sample 4, ga98) yielded a poor-quality consensus sequence, consistent with the known individual read error rate of minion sequencing. high-quality consensus sequences were obtained from the other read clusters (>5 reads per cluster; supplementary table 2 ). each of the obtained consensus sequences was compared to genbank sequences using blast, which revealed >99% sequence identity to respective ibv sequences ( table 4 ). as described in the sanger sequencing section, 2 samples (8 and 9) contained only 1 ibv type per the non-minion assays, and the ampseq results were consistent with these findings. non-minion assays showed that 7 samples contained 2 or more ibvs per sample. six samples (1, 3, 4, 6, 7, 10) contained 2 ibv types per sample (table 4; supplementary tables 1, 2 ). of those 6 samples, ampseq detected both ibv types in 4 samples (4, 6, 7, 10), 1 ibv type in 1 sample (3), and no ibv in 1 sample (1; as mentioned above, sample 1 was the sample with the highest ct value). of note, sample 10 contained 2 ibv types from the same lineage and ampseq was able to identify both types within this sample. finally, the seventh multi-type sample (2) contained 4 ibv types per the non-minion results, and the ampseq detected 2 of those 4 types. these data show that a coinfection of multiple ibv lineages existed in the abovementioned samples, but a single rt-rtpcr, or sanger sequencing of a single clone, may not have detected these coinfecting ibv lineages and multiple assays were required to detect all the ibv types. however, the ampseq protocol accurately detected multiple ibv lineages in 4 of 7 samples, with partial detection in 2 of the remaining 3 samples (table 4 ). in samples that had the same ibv type, blast search of consensus sequences obtained from ampseq and sanger sequencing identified the same (or highly related ibv type for samples 6 [ga98] and 9) in the ncbi nt database as per parameters described in the methods section. to evaluate the utility of this protocol on clinical swab samples with pathogenic ibv variants, the ibv s1 gene was amplified directly from 5 clinical tracheal scrapings, and the pcr amplicons were used to create minion libraries. a total of 146,540 reads were successfully basecalled from the entire sequencing run (156,000 total reads). after demultiplexing, reads per barcode ranged from 4,285 to 41,131. a total of 24,297 reads were not assigned to any of the used barcodes, and 8,109 reads were discarded because of middle adapters in the basecalled reads. real-time analysis of min-ion data, which was obtained within 10 min of the sequencing, was sufficient for the detection of ibv. however, sequencing data obtained from the entire sequencing run was processed with the same protocol as described above. this ampseq protocol detected ibv reads in all of the 5 tested samples using all basecalled reads. the number of ibv reads per sample were 5-4,956. additionally, the sequencing data analysis showed that the ibv reads belonged to gi-l17 and typed the detected ibv variant as dmv1639. after minion sequencing results, these samples were later tested to confirm the presence of ibv variant by mdl_dmv1639 ibv variant-specific rt-rtpcr assay. all 5 samples were positive for the ibv mdl_dmv1639 variant of ibv (table 5, supplementary table 2 ). ten clinical swab samples (run 1) were processed for rt-pcr and sanger sequencing to confirm the presence of ibv lineages. based on the minion results, primers targeting giv-l1 and gi-l1 (ga98 and conn primer sets, respectively) were created (table 3 ). using giv-l1-specific primers showed that 9 of 10 samples were positive, with 1 sample (5) negative for ibv (which is consistent with the pan-ibv rt-qpcr results for this sample). the pcr products were cloned, and multiple (6-24) clones from each sample were submitted for sanger sequencing and type-based analysis ( table 4 ). the consensus sequences from multiple clones obtained by using giv-l1-specific primers showed the rt-rtpcr = reverse-transcription real-time pcr. * g-l = genotype and lineage (e.g., genotype i-lineage 27); neg = negative. †pan-ibv rt-qpcr, ga08 serotype-specific rt-rtpcr, ga98 rt-pcr, and conn rt-pcr. presence of ga98 (top blast hit: "ga/a9dpvaccinated" [eu283069.1]) in 7 of 9 ibv-positive samples. de072 was detected in samples 1 and 9 as the lone giv-l1, but was also detected in 2 samples (2, 10) that also contained ga98 (table 4) ; thus, these samples had 3 and 1 lineages, respectively (table 3) , but 4 and 2 ibv types, respectively (table 4) . by using the gi-l1-specific primer set, which amplified the conn and mass type of ibvs, 3 of 9 ibv-positive samples were positive (2 and 4 for conn; 3 for mass [pdrc_110177]; table 4 ). combining the lineage-based (rt-rtpcr and rt-pcr) and type-based (rt-pcr coupled with sanger sequencing) analyses (table 3 , 4, respectively), only samples 8 and 9 contained a single ibv type; the other 7 samples were positive for 2-4 (e.g., 2 has four, and 4 has two) types of ibv (tables 3, 4 ). in all of the samples (run 1) in which a matching ibv type was identified by both ampseq and sanger sequencing, the ampseq data showed high concordance with sanger sequencing data. in one sample (4), only a single minion read of ga98 was detected by ampseq and had only 96.1% similarity to the respective, shorter sequence from sanger sequencing, consistent with the reported single-read accuracy of minion sequencing. all other samples had 99-100% pairwise identity across the partial s1 fragment generated by sanger sequencing (table 4 ). the accurate detection of ibv as the cause of clinical respiratory disease is contingent on virus typing and differentiating live vaccine viruses from field strains. 11 it has been reported that ibv genotypes are well correlated with the serotypes of ibv 45 ; therefore, accurate genotypic identification of ibv will be useful to identify vaccine and variant viruses in clinical samples. rapid pan-ibv rt-qpcr and serotype-specific rt-rtpcr assays 4,23,36 have been used for serotype identification; however, positive results from rt-rtpcr is insufficient to determine the ibv genotype; thus, sequence analysis of the ibv s1 gene is required. use of partial s1 gene sequences (450 bp) to type ibv is described. 29 however, increasing the length of sequenced s1 gene (~1,620 bp) results in more data to be used for genotyping because more of the hypervariable region is covered. 17, 27, 45 for example, the ampseq protocol that we used was able to differentiate 2 genetically very similar (99.5% at s1 gene of ibv genome) but serotypically different giv-l1 ibvs, de072 and ga98, within a single sample. thus, the detection of highly diverse ibv genotypes, via the s1 subunit, with a single and rapid sequencing protocol is desirable and the ability to detect multiple types of ibv in a single sample could improve ibv detection in clinical samples. currently, pan-ibv rt-qpcr is used to rapidly detect ibv from clinical samples for screening purposes, 4 but this requires additional serotype-specific rt-rtpcr assays to genotype positive samples, including samples containing more than one type of ibv. additionally, if unidentified ibvs are in the sample, then current detection and characterization may also require egg culture followed by various pcr assays. 8 our ampseq method detected ibv in 13 of 14 ibvpositive samples and detected all of the mixed ibvs in 4 of 7 samples containing 2 or more ibvs. although mixed ibvs were not 100% identified (0 of 2 in sample 1; 2 of 4 in sample 2; and 1 of 2 in sample 3) in 3 samples with our ampseq method, detection of those mixed ibvs required several non-ampseq assays. although there is room for improving our new ampseq method, it represents a promising, single-step assay that can be used without egg culture. thus, it is another tool for the detection of ibv, especially in cases in which multiple ibvs may be present or when genotyping is especially important. one area that is problematic for a test such as ampseq is the detection of all genotypes of ibv that may be present. although ampseq did detect all genotypes in 4 samples, in 3 cases not all of the genotypes of ibvs were detected. a potential explanation of partial detection of multiple ibv genotypes could be the relative abundance of ibv genotypes in these clinical samples. it could also be that amplification of these ibv genotypes by serotype-specific rt-rtpcr assays is more efficient given the small targeted fragment size (e.g., 120 bp for pcr and ~1,600 bp for ampseq) and better primer alignment to the target (e.g., degenerate bases are used in the s1 primers used for ampseq). one complicating factor of our current ampseq protocol is that the ibv target sequence 27 was not originally designed for high specificity, especially from clinical samples. as such, a high proportion of total reads were non-ibv reads (e.g., often mapping to the chicken genome, data not shown), consistent with the extra bands visible in the original report for these target sequences. 28 future studies to reduce the proportion of chicken reads may increase the sensitivity of this assay. additionally, it is possible that certain genotypes are better complemented to the s1 primers than others and may outcompete those ibv genotypes for amplification in the ampseq protocol. also, increasing the total number of reads collected by ampseq may improve the ability to detect all of the genotypes in a given sample. it is possible to allow the sequencing to continue longer to obtain more reads per sample. overall, ampseq is a feasible test for ibv characterization, and work is ongoing to improve this new type of assay. cost and time efficiency of a sequencing protocol can be improved through the multiplexing of more samples in a single sequencing run. 3, 47 in our study, samples (run 1 = 10, run 2 = 5) were simultaneously multiplexed (i.e., pooled and then sequenced in one run) while maintaining ibv genotyping from data collected. given that the minion flow cells were not exhausted, and can be washed and re-used, the ampseq method also has the potential for sequential multiplexing. this would decrease the need to hold samples for weeks while waiting for the cost-optimal number of samples for simultaneous multiplexing. the single protocol nature of ampseq, the ability to obtain s1 gene sequence results, realtime data analysis, and flexibility of testing design makes minion-based ampseq a viable sequencing protocol for genotyping and lineage typing of ibv. the advent of real-time, in-house, third-generation sequencing represents a transformative opportunity for diagnostic laboratories by offering the ability to more fully characterize pcr reactions beyond confirming amplicon size (e.g., routine electrophoresis), sanger sequencing rt-pcr products, or by confirming a partial sequence through probe hybridization (e.g., probe-based rt-rtpcr). however, the interpretation of such large data sets represents a challenge to veterinary diagnosticians. read-based classification software such as centrifuge, 24 kraken, 50 qiime, 5 and mothur 37 have been used to identify and profile microbial species; however, the high error rate 20 of nanopore reads translates to poor classification accuracy for many of these tools. alternatively, de novo 3,26 or reference-based 18 assembly methods have been used in minion and other deep-sequencing platforms. using a strategy similar to other unbiased laboratory tests (e.g., standard bacterial cultures), an approach was developed to maximize usage of reads (i.e., reads are not discarded based on pre-set length or abundance requirements, similar to how a single colony may be interpreted as a significant result). this approach uses read-based classification against a database containing an equal number of representative ibv sequences per lineage to detect and classify the ibv reads based on their lineage, before conducting read-based classification of ibv reads by using all available ibv sequences in a separate database. finally, lineage-clustered ibv read assignments are interpreted by a veterinary diagnostician to result in reads available for consensus building. the use of a final consensus alignment helps to overcome the individual error rate of minion sequencing. 3 similarly, the absence of predetermined metrics used in de novo assembly allows for the informed decision as to how many consensus sequences to build, a bioinformatics problem when dealing with clinical cases that can contain more than one type of ibv (e.g., similar to how there is not a predetermined number of significant bacterial colonies). confirmatory follow-up tests (e.g., rt-pcr) may be needed when dealing with low numbers of reads in clinical samples; however, future testing of this new technology will allow for creating standards for such confirmatory testing. our results suggest that the application of minion-based ampseq, specifically for detecting ibv genotypes and lineages from clinical samples within a few days, compared favorably to several days to weeks and multiple detection assays to culture and detect multiple ibv genotypes from a single sample. thus, minion-based ampseq coupled with data analysis workflow for identification, differentiation, and accurate prediction of ibv genotypes from clinical swab samples can be used as an adjunct to other established rapid detection assays 9,12 until extensive testing of this protocol is done to improve and validate ampseq for ibv identification. furthermore, ampseq-based assays can be and are being applied to other viral pathogens, 3,37 demonstrating the power and utility of this method. emergence of novel strains of avian infectious bronchitis virus in sweden phylogenetic analysis of partial s1 and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity and recombination rapid virulence prediction and identification of newcastle disease virus genotypes using third-generation sequencing development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens qiime allows analysis of high-throughput community sequencing data gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication 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natural recombination in the field isolates multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples real-time, portable genome sequencing for ebola surveillance detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-pcr and correlation with virus detection in embryonated eggs introducing mothur: open-source, platformindependent, community-supported software for describing and comparing microbial communities coronaviruses: structure and genome expression minion sequencing to genotype us strains of infectious laryngotracheitis virus differentiation of infectious bronchitis virus vaccine strains ma5 and 4/91 by taqman real-time pcr cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus mega6: molecular evolutionary genetics analysis version 6.0 prevalence of infectious bronchitis and newcastle disease virus among domestic and wild birds in h5n1 outbreaks areas s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus minion nanopore sequencing of an influenza genome rapid multiplex small dna sequencing on the minion nanopore sequencing platform the avian coronavirus spike protein comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses kraken: ultrafast metagenomic sequence classification using exact alignments primer-blast: a tool to design target-specific primers for polymerase chain reaction we thank vanessa gauthiersloan, samantha day, and erich linnemann from the poultry diagnostic & research center, university of georgia for technical help. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. supplementary material for this article is available online. key: cord-001768-8vljd5cv authors: awad, faez; forrester, anne; baylis, matthew; lemiere, stephane; ganapathy, kannan title: protection conferred by live infectious bronchitis vaccine viruses against variant middle east is/885/00-like and is/1494/06-like isolates in commercial broiler chicks date: 2015-09-09 journal: vet rec open doi: 10.1136/vetreco-2014-000111 sha: doc_id: 1768 cord_uid: 8vljd5cv the ability of the infectious bronchitis h120 (a massachusetts strain) and cr88 (a 793b strain) live attenuated vaccine viruses to protect from two middle east infectious bronchitis virus isolates, is/885/00-like (is/885) and is/1494/06-like (is/1494) in broiler chicks was investigated. day-old chicks were separated into three groups, (i) vaccinated with h120 at day-old followed by cr88 at 14 days-old, (ii) vaccinated with h120 and cr88 simultaneously at day-old and again with cr88 at 14 days-old, (iii) control unvaccinated. at 30 days-old, each of the groups was challenged with virulent is/885 or is/1494. protection was evaluated based on the clinical signs, tracheal and kidney gross lesions and tracheal ciliostasis. results showed that administering combined live h120 and cr88 vaccines simultaneously at day-old followed by cr88 vaccine at 14 days-old gave more than 80 per cent tracheal ciliary protection from both of the middle east isolates. in addition, this programme conferred 100 per cent protection from clinical signs and tracheal or kidney lesions. the other vaccination programme, h120 at day-old followed by cr88 at 14 days-old, the tracheal ciliary protection conferred were 60 per cent and 80 per cent from is/885/00-like and is/1494/06-like, respectively. infectious bronchitis virus (ibv) is a ubiquitous virus with high mutation, and a large number of serotypes or genotypes of ibv strains have been reported worldwide (de wit and others 2011). variant strains emerge due to changes in the ibv genome through point mutations, deletions, insertions or rna recombination and these variants are often responsible for ib outbreaks in vaccinated chicken flocks (cavanagh and others 1988 is/885/00 and is/1494/06 or those with high similarities to these strains of ibvs have been reported throughout the middle east and north africa (meir and others 2004) , iraq (mahmood and others 2011) , egypt (abdel-moneim and others 2012), turkey (kahya and others 2013) and libya (awad and others 2014a) . in most cases, severe respiratory distress and renal lesions with high mortality were observed in vaccinated flocks affected by these strains. it appears that the conventional h120 vaccines alone do not provide sufficient protection from these strains (meir and others 2004, kahya and others 2013) . however, development of vaccines against each of these new variants is not generally an option due to the high cost and time required for product registration (jackwood and others 2003, bijlenga and others 2004) . furthermore, it could be a never-ending race as new variant ibvs are constantly emerging in major poultry producing countries. instead, it has been recommended that protection conferred by available live ibv vaccines against new variants should be constantly evaluated (alvarado and others 2003) . vaccination with one serotype does not ensure complete protection from heterologous strains (cook and others 1999) but the use of different combinations of live ibv vaccines has been shown to be able to induce high and broad protection from challenges with several heterologous virulent ibv variants (gelb and others 1991 , cook and others 1999 , cook and others 2001 , alvarado and others 2003 , martin and others 2007 , terregino and others 2008 . the objective of this study is to evaluate the protection conferred by available live ibv vaccines when used in a strategic manner against the two prominent middle east variant ibvs that are related to is/885/00 (meir and others 2004) and is/1494/06 (genbank accesion number: eu780077). in addition to the conventional vaccination programme (mass at day-old followed by variant at 14 days-old), the authors also evaluated another vaccination programme where the mass and variant live vaccines were given simultaneously at day-old followed by a variant vaccine at 14 days-old. following challenge, protection was assessed based on a ciliostasis assay as recommended by european pharmacopoeia (europe 2007 ). in addition, clinical signs and gross lesions were evaluated. day-old commercial broiler chicks with ibv maternally derived antibodies (mdas) were obtained from a commercial hatchery. the parent flock of these chicks had been vaccinated with live ibv h120+ d274 vaccines and with an inactivated ibv m41. chicks were kept in an isolation unit at the university of liverpool. the chicks were reared on deep litter with water and feed provided ad libitum. two different commercial live vaccines were used in this study; (i) h120, vaccine belonging to massachusetts serotype (merial s.a.s, lyon, france) and (ii) cr88, vaccine belong to 793b serotype (merial s.a.s, lyon, france). the vaccines were kept at 4-8°c until used. each vaccine was dissolved in sterile distilled water. ibv challenge strains virulent strains of ibv is/885/00-like (referred here as is/885) and is/1494/06-like (referred here as is/1494) were used as challenge viruses. both viruses had been isolated from a recent outbreak of high mortality and respiratory disease in broiler flocks in egypt. the isolates were submitted to the istituto zooprofilattico sperimentale delle venezie, padova, italy as third passage allantoic fluid. at this laboratory, the allantoic fluid was screened against avian influenza virus, newcastle disease virus and ibv. it was negative for avian influenza virus and newcastle disease virus but positive for ibv. at the university of liverpool, the is/885 strain was identified by reverse transcriptase-pcr and part-s1 sequence analysis showed 90 per cent nucleotide level identity to the israeli strain is/885/00 (meir and others 2004) . based on part-s1 analysis, the is/1494 strain showed 99 per cent nucleotide level identity to the israel strain is/1494/06. a pairwise comparison of the s1 gene sequences of is/885/00-like and is/1494/ 06-like showed low level identity to the mass and 793b types (awad and others 2014a). the titre of the isolates were determined in specific pathogen-free embryonated chickens eggs and expressed as 50 per cent egg infectious doses (eid 50 ) following the method of reed and muench (reed and muench 1938) . ninety one-day old commercial broiler chicks were divided into three groups of 30 chicks and housed in different isolation rooms. at one day of age, group i was inoculated with h120 vaccine. group ii, was inoculated with combined h120 and cr88 vaccines. at 14 days of age, birds in group i and ii were revaccinated with cr88 vaccine. group iii was sham-inoculated at 1-day and 14 days old. each chick was inoculated via oculo (50 µl)-nasal (50 µl) routes. dosages were given as recommended by the manufacturer. following the vaccinations, the birds were observed daily for clinical signs. blood was collected prior to vaccination (at 1 day) and 30 days old from eight chicks in each group for antibody responses. on the same day, 10 chicks from each group (vaccinated and control groups) were transferred to another isolation room and challenged by the oculonasal route with 0.1 ml of virulent is/885 to provide 10 4.66 eid 50 /chick. another 10 chicks from each group were similarly transferred and challenged with 0.1 ml of virulent is/1494 to provide 10 5.00 eid 50 /chick. the remaining 10 chicks (vaccinated or unvaccinated control) in each group were left as unchallenged controls. the birds were observed daily for clinical signs during the postchallenge period. five days after challenge, all ten chicks in each unchallenged and challenged group were humanely euthanased by wing vein injection of 0.5-1 ml of euthenal ( pentobarbitone sodium, rhone merrieux, ireland). vaccine protection was evaluated by a ciliostasis test and examination of trachea and kidneys for gross lesions. sera collected prior to vaccination and 30 days old were tested using commercial elisa (biochek, gouda, the netherlands) following the protocols recommended by the manufacturer. the haemagglutination inhibition (hi) test was carried out as described before (alexander and chettle 1977) . the ibv antigens used for the hi assay were m41 and 793b which were purchased from animal health service, deventer, the netherlands. the haemagglutinin antigens of is/885 and is/1494 were prepared in the authors' laboratory based on the method described before (alexander and chettle 1977, king and hopkins 1983) . assessment of protection from the challenge viruses were carried out as described by others (cook and others 1999) . five days after challenge, birds were humanely killed, a section of trachea (immediately after larynx to thoracic inlet) was carefully dissected out and placed into warm (37°c) tracheal organ culture medium (eagle's serum-free minimum essential medium with glutamine and streptomycin (50 mg/ml) and penicillin (50 iu/ml)). the trachea was cut using a tissue chopper (the mickle laboratory engineering) to give 6 µm thickness of rings, and rings of three from the top and bottom, and four from the middle trachea were examined for ciliary activity using a low-power microscope (×100 magnification). for each of the rings, the ciliary activity was scored; 0, all cilia beating; 1, 75 per cent beating; 2, 50 per cent beating; 3, 25 per cent beating; and 4, none beating (100 per cent ciliostasis). for each bird, out of the 10 rings examined, the maximum possible ciliary score is 40, which indicates a total lack of protection (no cilia beating in all 10 rings). the mean ciliary scores for each bird was calculated and percentage protection for each group using a formula described by others (cook and others 1999) ; [1−(mean ciliostasis score for vaccinated/challenge group)/mean ciliostasis score for corresponding challenge controls)]×100. all chicks that were euthanased at five day post challenge (dpc) were also examined individually for gross tracheal and kidney lesions and scored accordingly using a four-point scale. for the trachea, 0=no lesions, 1=congestion, 2=mucoid exudate in the trachea and bronchi, 3=caseous exudate plug in the trachea and bronchi, air sacculitis, perihepatitis and pericarditis, (ii). for kidneys, 0=no lesions, 1=swollen and pale, 2=swollen with visible urates, 3=large swelling, pale with tubules and ureters distended with urates. total gross lesions scores for each group were calculated based on the mean of scores observed per total number of chicks. the elisa, hi antibody titres and gross lesions between groups were analysed statistically using analysis of variance followed by tukey's test for comparison of means. differences were considered to be significant when p≤0.05. all analyses were conducted using the graphpad prism software, v.6.0.1. there were no clinical signs found in the unvaccinated control chicks. in both vaccinated groups, mild clinical respiratory signs began to appear at six days post vaccination (dpv). the signs included head shaking, tracheal râles and sneezes. these signs subsided by 12 dpv. the mean elisa antibody titre prior to vaccination (at day-old, mda) was 4174±636. the means of ibv hi titres against ibv m41, 793b, is/885 and is/1494 prior to vaccination (at day-old, mda) were log 2 6.3, log 2 5.6, log 2 3.5 and log 2 3.1, respectively. at 30 days of age (on the day of challenge), vaccinated groups showed significantly higher levels of ibv elisa antibody titre than the unvaccinated control group. chicks of group ii (d0:h120+cr88, d14:cr88) exhibited significantly higher antibody titre (p<0.05) than group i (d0:h120, d14:cr88) (fig 1) . the hi response to m41 and 793b were higher than the heterologous antigens (is/885 and is/1494) (fig 2) . using the m41 as antigen, the vaccinated chickens showed high level of hi antibody titre compared with the control group (group iii). the antibody titre against 793b antigen in group ii was significantly higher than group i (fig 2) . the unvaccinated and vaccinated-unchallenged groups remained free of clinical signs. all the 10 chicks of unvaccinated-challenged chicks showed signs of typical ibv infection at 1 dpc which including depression with ruffled feathers, listlessness and huddling, head shaking, tracheal râles, sneezing and coughing. these signs were continued up to 5 dpc. in contrast, no clinical signs were observed in both of the vaccinated-challenged groups. the percentage ciliary protection was calculated for each group. the unvaccinated and vaccinated-unchallenged groups had greater than 98 per cent protection. the unvaccinated (group iii) birds challenged with either is/885 or is/1494 viruses had 0 per cent protection (table 1) . group ii (d0:h120+cr88, d14:cr88) and group i (d0:h120, d14:cr88) showed 83 per cent and 60 per cent ciliary protection from is/885 challenge, respectively (table 1) . following challenge with is/1494, group i and group ii gave 80 per cent and 94 per cent protection, respectively (table 1) . the unvaccinated and vaccinated-unchallenged control groups remained free of gross lesions. five days after the is/885 challenge, congestion of the trachea and pale swollen kidneys were observed in all chicks in group iii (unvaccinated-challenged), which were significantly different from vaccinated groups (p=0.02). these lesions were also found in group i (table 1) . however, no significant difference was observed between group i and group iii regarding kidney lesions (p=0.06). birds in group ii were free of these gross lesions. following the is/1494 challenge, congestion of trachea and pale swollen kidney was observed in one bird in group i, while it appeared normal in the rest of the birds (table 1) . chicks in group ii showed no tracheal or kidney lesions. however, no significant difference was observed between the two vaccinated groups. the evaluation of protection conferred by live vaccines against the virulent is/885 and is/1494 isolates was assessed based on ciliary activity in the tracheal explants prepared from vaccinated-challenged chicks (darbyshire 1980 , andrade and others 1982 , marquardt and others 1982 , snyder and others 1983 , cook and others 1999 and gross lesions in the trachea and kidneys following the challenge with the respective viruses. in this study, similar to other experimental work (martin and others 2007, terregino and others 2008) and field practices, the live vaccines were reconstituted and applied to chicks at dosages recommended by the vaccine manufacturers. the combined vaccination programme where both live h120 and cr88 vaccines were simultaneously given at one day-old followed by cr88 vaccine two weeks later (group ii) provided an excellent protection from both isolates. following the challenges, there were no clinical signs or tracheal/kidney lesions and the ciliary protection was high (83-94 per cent). group 1, which had live h120 vaccine alone at day-old followed by cr88 vaccine two weeks later showed 60 per cent and 80 per cent protection from is/885 and is/ 1494/06-like, respectively. it appears that the protection conferred was much improved when the cr88 was given together (group ii) with the h120 at day-old in contrast to h120 given alone (group 1). it must be noted that the vaccine programme given to group ii also offered 100 per cent kidney protection from both isolates. in a previous study performed in specific pathogen-free chicks, live h120 vaccination afforded protection of the trachea (92 per cent) and the kidney (25 per cent) on the basis of virus isolation when the birds were challenged with is/885/00 (meir and others 2004) . moreover, immunisation with live h120, h52 and d274 given singly was reported to produce little crossprotection from challenge with other nephropathogenic ibv strains (albassam and others 1986 , lambrechts and others 1993 , pensaert and lambrechts 1994 ). the present study shows that the vaccination programme of group ii has further boosted the ciliary and tracheal/ kidney protection from is/885 compared with that of group i. both vaccination programmes used in this experiment provided excellent protection from the virulent is/1494 challenge. a better protection observed in group ii (d0: fig 1: mean elisa antibody titres at 30 days of age. group i=d0:h120, d14:cr88; group ii=d0:h120+cr88, d14:cr88; group; iii=control. bars represent the mean±sd for eight birds in each group. different superscript lowercase letters in the bar indicate significant difference (p<0.05), while data with same letters indicate that there were no significant differences (p>0.05). the cut-off titre=834 fig 2: haemagglutination inhibition (hi) antibody titres log 2 against infectious bronchitis virus (ibv) antigens at 30 days of age. group i=d0:h120, d14:cr88; group ii=d0:h120+cr88, d14:cr88; group iii= control. bars represent the mean±sd for eight birds in each group. different superscript letters in the bar indicate significant difference (p<0.05), while data with same letters indicate that there were no significant differences (p>0.05) (within same antigen). the cut-off titre=3log 2 h120+cr88, d14:cr88) as compared with group i (d0: h120, d14:cr88), could be due to the higher levels of local and cellular immunity at the tracheal site which may have prevented the virulent virus from reaching the kidneys (lambrechts and others 1993) . in an another experiment, cellular and local immunity induced by administration of combined live massachusetts and 793b-type vaccines at day-old showed a significant increase in the expression of cd4+, cd8+ and iga-bearing b cells in the trachea compared with single h120 alone or unvaccinated groups (awad and ganapathy, unpublished) . these results reinforce the importance of optimising local and cell-mediated mucosal immunity at the respiratory lining through strategic heterologous day-old vaccination for enhanced protection from variant viruses. humoral antibody responses following vaccination as measured by elisa and hi are often used for monitoring vaccine-take (raj and jones 1997) . in this study, at 30 days of age (challenge day) the mean elisa titre and the mean 793b hi titre in group ii were significantly higher than the other vaccinated group. this reflects that combined live ibv vaccination at day-old and revaccination at 14 days-old has provided an immunological boost compared with the other vaccination schedule (group 1). although it has been demonstrated before that circulating humoral antibody levels are of minor importance in the protection from ibv infection (raggi and lee 1965 , darbyshire and peters 1985 , endo-munoz and faragher 1989 , pensaert and lambrechts 1994 , in the present study the group with higher levels of humoral antibodies had better protection from both of the middle east isolates. cytotoxic t cell plays a vital anti-viral activity during early stages of ibv infection while serum igg is critical at the late stage of ibv infection (collisson and others 2000) . despite the low level of genetic identity in the s1 protein between the vaccine and challenge strains used in this study, the protection achieved with the vaccination programme employed in the present study was high. the genetic relationship of the hypervariable region of the s1 gene between is/885 and h120 or cr88 was 80 per cent and 79 per cent, respectively, while the genetic relationship between the is/1494 and h120 or cr88 was 81 per cent and 80 per cent, respectively (awad and others 2014a). despite the 19-21 per cent variation in the s1 gene, the successful protection conferred by vaccination programmes employed in this study may be attributable to the shared characteristics of the s1 protein of the vaccine and the challenge ibvs (cavanagh and others 1986) . based on the data presented in this study, it appears that a combination of live h120 and cr88 vaccines given at day-old followed by cr88 vaccine at day 14 of age confer an excellent protection from virulent variant is/885 and is/1494 viruses. more work is needed to establish the underlying immune mechanisms for such higher and broader protection conferred by this vaccination programme. i 60 0 (0/1) a 1 (0/1) b 80 0 (0/1) a 0 (0/1) a ii 83 0 (0/0) a 0 (0/0) a 94 0 (0/0) a 0 (0/0) a iii 0 1 (0/2) b 1 (0/2) b 0 1 (0/1) b 1 (0/1) b group i=d0:h120, d14:cr88; group ii=d0:h120+cr88, d14:cr88; group iii=unvaccinated control *severity of gross lesions induced by virulent ibv infection and data are expressed as the median (minimum/maximum) (number of samples=10 per group). different superscript lowercase letters within same columns indicate significant difference (p<0.05), while data with same letters indicate that there were no significant differences (p>0.05) †protection score=1, the mean score for vaccinated and challenged group/mean score for challenge control group ×100; the higher the score, the better the protection emergence of a novel genotype of avian infectious bronchitis virus in egypt comparison of the nephropathogenicity of four strains of infectious bronchitis virus procedures for the haemagglutination and the haemagglutination inhibition tests for avian infectious bronchitis virus evaluation of the protection conferred by commercial vaccines against the california 99 isolate of infectious bronchitis virus evaluation of ciliary movement in tracheal rings to assess immunity against infectious bronchitis virus detection of variant infectious bronchitis viruses in broiler flocks in libya development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or hemagglutination-inhibiting antibody, or induce chicken tracheal protection amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes assessment of cross-immunity dm chickens to strains of avian infectious bronchitis virus using tracheal organ cultures humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus infectious bronchitis virus variants: a review of the history, current situation and control measures avian infectious bronchitis: cross-protection studies using different australian subtypes avian infectious bronchitis vaccine (live) 01/ 2008:0442. european pharmacopoeia variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens attenuation, safety, and efficacy of an infectious bronchitis virus ga98 serotype vaccine a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains presence of is/1494/06 genotype-related infectious bronchitis virus in breeder and broiler flocks in turkey ankara üniversitesi veteriner fakültesi dergisi evaluation of the hemagglutinationinhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination challenge experiments to evaluate cross-protection induced at the trachea and kidney level by vaccine strains and belgian nephropathogenic isolates of avian infectious bronchitis virus s1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east comparison of ciliary activity and virus recovery from tracheas of chickens and humoral immunity after inoculation with serotypes of avian infectious bronchitis virus evaluation of the effectiveness of two infectious bronchitis virus vaccine programs for preventing disease caused by a california ibv field isolate identification of a novel nephropathogenic infectious bronchitis virus in israel vaccination of chickens against a belgian nephropathogenic strain of infectious bronchitis virus b1648 using attenuated homologous and heterologous strains lack of correlation between infectivity, serologic response and challenge results in immunization with an avian infectious bronchitis vaccine infectious bronchitis virus: immunopathogenesis of infection in the chicken a simple method of estimating fifty per cent endpoints ciliary activity: a criterion for associating resistance to infectious bronchitis virus infection with elisa antibody titer pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes key: cord-284501-5i0w74q4 authors: armesto, maria; cavanagh, dave; britton, paul title: the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity date: 2009-10-09 journal: plos one doi: 10.1371/journal.pone.0007384 sha: doc_id: 284501 cord_uid: 5i0w74q4 we have previously demonstrated that the replacement of the s gene from an avirulent strain (beaudette) of infectious bronchitis virus (ibv) with an s gene from a virulent strain (m41) resulted in a recombinant virus (beaur-m41(s)) with the in vitro cell tropism of the virulent virus but that was still avirulent. in order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the beaudette strain with those from m41. the recombinant ibv was in effect a chimaeric virus with the replicase gene derived from beaudette and the rest of the genome from m41. this demonstrated that it is possible to exchange a large region of the ibv genome, approximately 8.4 kb, using our transient dominant selection method. recovery of a viable recombinant ibv also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the m41 replicase gene with the beaudette derived gene. analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of ibv were able to restore virulence and that therefore, the loss of virulence associated with the beaudette strain resides in the replicase gene. avian infectious bronchitis virus (ibv) is a member of the genus coronavirus, family coronaviridae, order nidovirales [1, 2] . together with the genetically closely related turkey coronavirus [3] [4] [5] [6] , pheasant coronavirus [7] , and recently identified coronaviruses from several species of wild birds [8, 9] , a beluga whale [10] and an asian leopard cat [11] form the group 3 coronaviruses. ibv is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of other organs, including the gut, kidney and oviduct [12] [13] [14] , and is the causative agent of infectious bronchitis, a disease that is responsible for economic losses in the poultry industry throughout the world [15] . coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain a single-stranded, positive-sense rna genome of 28 to 32 kb [16] . ibv has a 27.6 kb rna genome and like all coronaviruses contains the four structural proteins; spike glycoprotein (s), small membrane protein (e), integral membrane protein (m) and nucleocapsid protein (n) which interacts with the genomic rna. all coronaviruses also encode a set of accessory protein genes of unknown function that are not required for replication in vitro [17] [18] [19] [20] [21] [22] [23] , but may play a role in pathogenesis [19, 22] . ibv encodes two accessory genes, genes 3 and 5, which both express two accessory proteins 3a, 3b and 5a, 5b, respectively. in addition to the structural and accessory genes, two-thirds of a coronavirus genome comprises the replicase gene, which expresses two polyproteins, pp1a and pp1ab, in which pp1ab is an extension product of pp1a as a result of a -1 ribosomal shift mechanism. the two polyproteins are cleaved by two types of virus-encoded proteinases usually resulting in 16 non-structural proteins (nsp1-16); ibv lacks nsp1 thereby encoding nsp216. we have previously shown that the ibv accessory genes are not required for replication in vitro [17, 18] ; however, we could not determine any role of the ibv accessory proteins in pathogenicity as our reverse genetics system is based on the avirulent beaudette strain. replacement of the beaudette s gene with the corresponding m41 s gene sequence altered the tropism of the ribv but did not result in a change in virulence [24] [25] [26] . this implied that although the ibv s gene may play a role in virulence, associated with tropism, expression of an s gene from a virulent strain alone was not sufficient to alter the avirulent phenotype associated with beaudette. infectious bronchitis is mainly controlled by the use of live attenuated vaccines derived from virulent viruses by multiple serial passages, usually greater than 50 passages, in 10-11-day-old embryonated chicken eggs [13, [27] [28] [29] . as a consequence of this process the virus becomes more adapted for the embryo, reflected by more efficient replication and higher pathogenicity for the embryo, with concomitant attenuation for chickens and in some cases loss of immunogenicity. however, the mutations associated with attenuation of pathogenicity for the chicken are unknown and variable leading to differing efficacies associated with different vaccines. our previous studies have shown that replacement of the s gene from the avirulent beaudette isolate with that from a virulent virus (m41) did not restore virulence but did alter the tropism of the ribv and restore immunogenicity for subsequent challenge with m41. previously beaudette had been considered to be poorly immunogenic and never used as a vaccine strain [30] . in this study, we describe the generation of recombinant ibvs that consisted of the replicase gene from the avirulent beaudette strain and the structural and accessory genes from the virulent m41 isolate of ibv, to determine whether the replicase or the combination of the structural and accessory genes of ibv play a role in pathogenesis. the growth of ibv in chick kidney (ck) cells was as described previously [31] [32] [33] . the ibv isolates used were: (1) beaudette-ck (beau-ck; [34] ), a virus adapted for growth in ck cells that can grow on but has not been adapted for growth in vero cells, an african green monkey cell line; (2) beau-r, a recombinant ibv (ribv) produced from a full-length cdna of beau-ck using our ibv reverse genetics system [35] ; and (3) m41-ck, an isolate derived from m41 [36] following adaption to growth on ck cells. both the beaudette and m41 strains of ibv belong to the same, massachusetts, serotype. all ibv strains were titrated in ck cells. vaccinia viruses were grown and titrated on vero cells and large stocks for dna isolation were grown in bhk-21 cells [37] . all nucleotide and amino acid residue numbers refer to the positions in ibv beau-r [35] accession n o aj311317. the region of the ibv m41 genome corresponding to the structural, accessory genes and the 39-utr was ligated onto the last 1416 nt of the beau-r replicase gene and inserted into hindiii and sali digested pgpt-neb193 [25] . the resulting plasmid, pgpt-beaur-rep-m41-struct-3utr, consisted of the 39-end of the replicase gene of beau-r, and the region of the m41-ck genome encoding the structural and accessory genes terminated by the m41-ck derived 39utr (fig. 1 ). the ibv cdna within pgpt-beaur-rep-m41-struct-3utr was introduced, by homologous recombination using the transient dominant selection (tds) ( [25, 37] ), into the ibv beaudette cdna within the vaccinia virus genome in rvv-beaur-rep-dstruct containing beau-r-derived sequence corresponding to the replicase gene followed by the first 376 nt of the s gene, part of the n gene and the 39-utr (fig. 1) . briefly, 50% confluent monolayers of vero cells were infected with rvv-beaur-rep-dstruct, containing the beau-r cdna sequence, at a moi of 0.2 and transfected 2 h later with 5 mg of pgpt-beaur-rep-m41-struct-3utr in lipofectin (invitrogen). resultant phenotypically gpt + rvvs were selected by three rounds of plaque purification using vero cells in the presence of 25 mg/ml mycophenolic acid (mpa), 250 mg/ml xanthine and 15 mg/ml hypoxanthine. randomly selected mpa resistant rvvs were grown and plaque purified three times using vero cells in the absence of selection medium. this resulted in a second recombination event (see fig. 1 ) involving the loss of the gpt gene from the rvvs, and either generation of an ibv cdna corresponding to the sequence in rvv-beaur-rep-dstruct or a full-length ibv cdna consisting of a beau-r replicase gene and the rest of the ibv genome derived from ibv m41-ck within the vv genome (fig. 1) . pcr was used to confirm the absence of the gpt gene in resulting rvvs, which were further screened by pcr amplification of the ibv 39-utr, using oligonucleotides bg56 (59-26941 caacagcgcccaaagaag 26958 ) and 93/100 (59-27607 gctctaactctatactagcct 27587 -39), part of the replicase gene, using oligonucleotides bg40 (59-18941 atc-taatttgctcgttca 18958 -39 and bg128 (59-19720 cgccac-tcctttgtcgcttc 19739 -39, and the junction of the replicase and s genes using oligonucleotides bg40 (59-18941 atctaatt-tgctcgttca 18958 -39 and bg134 (59-21398 agcaatt-gaaactgaaagtg 21417 -39. oligonucleotides bg-56 and 93/100 were used to discriminate between the 39-utrs from beau-r and m41 derived sequences; the 39-utr from beau-r results in a 667 bp product but a 483 bp pcr from m41-ck, due to a 184 nt deletion in the m41 39-utr. a rvv, rvv-beaur-rep-m41-struct, that contained a full-length ibv cdna consisting of the replicase gene from beau-r and the rest of the genome derived from ibv m41-ck was identified and used for further work. recombinant vaccinia virus dna from rvv-beaur-rep-m41-struct containing the beaur-rep-m41-struct chimaeric fulllength ibv cdna was purified and used for the rescue of ribvs in ck cells using rfpv/t7 [38] for the generation of infectious ibv rna ( [25, 35, 37] ). resultant chimaeric ribvs were passed three times in ck cells before being used in subsequent experiments. total cellular rna was extracted from ibv-infected ck cells using the rneasy method (qiagen) and rt-pcr (ready-to-go tm rt-pcr beads) for amplification of the 39-utr of the ribv-derived rna, using oligonucleotides bg 56 and 93/100, to confirm the identity of the ribv. confluent monolayers of ck cells in 6-well plates were infected with viruses at a moi of 0.1 pfu in triplicate for each time point. following attachment, for 1 h at 37uc, the cells were washed twice with phosphate-buffered saline (pbs) to remove residual virus and incubated at 37uc. samples of media were collected at 24, 48, 72 and 96 h post-infection and assayed in triplicate for progeny virus by plaque assay using ck cells. chicken tracheal organ cultures (tocs) were prepared from 19-day-old specific pathogen free (spf) rhode island red chicken embryos [39] . groups of five tocs, in triplicate, were inoculated with 0.5 ml of medium containing 5.4610 4 pfu/ml of each virus. after incubation at 37uc for 1 h, the inoculum was removed and the tocs were washed three times with pbs, 1 ml of medium was added and the tocs were incubated at 37uc until the samples were taken. at the selected time points medium from the tocs was removed and analysed for progeny virus by plaque titration on ck cells. all experiments were carried out in accordance with the uk home office guidelines using spf rhode island red chickens obtained from the poultry production unit of the institute for animal health. four groups (n = 12) of 1-day-old spf rhode island red chickens were inoculated via the conjunctival (eye drop) and intranasal routes with 10 6 pfu/ml of each virus in a total of 0.1 ml serum-free bes (n,n-bis(2-hydroxyethyl)-2aminoethanesulphonic acid) medium. the chickens were housed in positive-pressure, hepa-filtered isolation rooms, and each group was housed in a separate room. the birds in the mockinfected group were inoculated with serum-free bes medium. the ibv-associated clinical signs used to determine pathogenicity were snicking, tracheal rales (a sound emanating from the bronchi, also detected by vibrations when holding a chick), wheezing (dyspoena), nasal discharge, watery eyes and ciliary activity of the trachea [26] . chicks were observed daily for clinical signs; snicks (a sound similar to a sneeze) were counted by two persons over 2 minutes. birds were checked individually for the presence of tracheal rales, nasal discharge, watery eyes and wheezing. tracheas were removed from three randomly selected chickens from each group at 4 and 7 days post-inoculation for assessment of ciliary activity. ten 1 mm sections were cut from three different regions of each trachea and the level of ciliostasis of each tracheal section was determined. the remaining regions of the tracheas from the infected birds were cut longitudinally and the epithelial cells scraped from the tracheas and transferred to 1 ml pbs. the samples were analysed for the presence of viable ibv by titration in tocs or used for rna extraction using the rneasy method and analysed by rtthe m41-ck-derived cdna, representing the m41 structural and accessory genes and the m41 39-utr, within pgpt-beaur-rep-m41-struct-3utr was fused to the beau-r replicase gene in the rvv by a homologous recombination event between the beau-r replicase sequence common to both constructs. a potential rvv, rvv-beaur-rep-m41-struct, containing a full-length ibv cdna with the replicase gene from beau-r and the rest of the genome from m41-ck was isolated following the tds process. the complete plasmid dna was fused to the truncated beau-r cdna by a singlestep homologous recombination event; via the beau-r replicase sequence common to both sequences. the initial resultant rvvs had a gpt + phenotype allowing selection in the presence of mycophenolic acid. removal of mycophenolic acid resulted in two types of spontaneous intramolecular recombination events, due to the instability of the ibv cdna with tandem repeats of similar sequences, resulting in either rvv-beaur-rep-d-struct (no modification) or in rvv-beaur-rep-m41-struct; the desired rvv. both recombination events resulted in the loss of the gpt gene. the ibv genes representing the structural and accessory genes are shown along with the 39-utrs and igr sequences, a potential recombination event is indicated between the 1416 nt of the beau-r replicase gene sequence common to both constructs. doi:10.1371/journal.pone.0007384.g001 pcr using oligonucleotides bg56 and 93/100 to determine the identity of the 39-utr. confluent monolayers of ck cells were used for serial passage of rbeaur-rep-m41-struct 25 times. briefly, cells were infected with the ribv and 24 h post-infection medium was collected, diluted 1:10 and used to infect a new monolayer of ck cells. this process was repeated until passage 25 (p 25 ). total rna was extracted from the p 25 infected ck cells and rt-pcr was used to generate a series of overlapping pcr products covering the complete genome of the p 25 ribv. the rt-pcr products were sequenced using a variety of oligonucleotides, derived from the beau-ck sequence [40] . assembly of the sequences was performed using gap4 of the staden sequence software programs [41] . generation of chimaeric ribvs with the replicase gene from beaudette and the rest of the genome m41-ck a full-length ibv-derived cdna, within the vaccinia virus genome, was generated by homologous recombination using the tds method and consisted of the replicase gene from the apathogenic ibv strain beau-r and the structural and accessory genes plus the 39-utr from the pathogenic m41 strain of ibv. this was achieved using a beau-r-based receiver sequence consisting of the complete replicase gene, followed by the first 376 nt of the s gene fused to the n gene and 39-utr (fig. 1) . the donor sequence consisted of the last 1246 nt of the beau-r replicase gene fused to m41-ck-derived cdna from the s gene to the poly(a) tail ( fig. 1 ). following tds, dna was extracted from 20 rvvs, potentially containing the chimaeric ibv full-length cdna. analysis by pcr, using gpt specific primers to confirm the loss of the e. coli gpt gene, on six of the rvv dnas confirmed the loss of the gpt gene following the second tds recombination event (fig. 1 ). the ibv cdnas within these six rvv dnas were analysed (1) for the presence of the m41-ck-derived 39-utr sequence, which is 184 nt shorter than the beau-r 39-utr [42] , (2) the junction between the replicase and s gene using oligonucleotides bg40 and bg134 and (3) for the presence of the m41-ck-derived m gene using oligonucleotides bg52 (59-24945 gaatggtgttctt-tattg 24962 -39) and bg146 (59-25549 tctaacactc-taagttgag 25567 -39); to confirm that the second tds recombination event had not resulted in generation of the starting receiver sequence (fig. 1 ). two rvvs, rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, that did contain the m41-ck-derived 39-utr and m gene were further screened by spot sequence analysis of the ibv cdna to confirm that the region downstream of the beau-r replicase gene had been replaced with the corresponding sequence from m41-ck; the sequences were as expected for the required chimaeric ibv sequence. two infectious ribvs, rbeaur-rep-m41-struct-2 and rbeaur-rep-m41-struct-12, were recovered from dna extracted from rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, respectively, using ck cells, previously infected with rfpv/t7, to provide t7 rna polymerase, and co-transfected with the rvv dna and pci-nuc [35] . the transfected ck cells (p 0 ) were incubated until they showed a cytopathic effect (cpe), the medium was filtered to remove any rfpv/t7 and any potential ribv passaged three times more on ck cells (p 3 ). total rna from the infected p 3 ck cells was extracted and analysed by rt-pcr and spot sequence analysis. sequence analysis showed that rbeaur-rep-m41-struct-12 contained an extra adenosine nucleotide at position 25317 in a six base polyadenosine repeat sequence within the m41 intergenic region (igr) between the end of the m gene and start of gene 5. consequently only rbeaur-rep-m41-struct-2 from the p 3 ck cells was titrated in ck cells and used for further characterisation. sequence analysis of the m41-ck-derived sequence in rbeaur-rep-m41-struct-2 revealed three nucleotide changes when compared to the sequence in rvv-beaur-rep-m41-struct-2 ( table 1 ). the nucleotide changes resulted in one amino acid change in the s protein, the other was a silent mutation and a single amino acid change in the n gene. comparison of the sequence indicated that the changes arose during rescue or during the first three passages of the rescued viruses. twelve one-day-old spf chickens, in four groups, were inoculated with 0.1 ml of 10 6 pfu/ml of rbeaur-rep-m41struct-2, m41-ck or beau-r or using 0.1 ml serum-free medium (mock infection) by eye-drop and intranasally. the birds were observed for clinical signs up to 11 days post-inoculation. at 4 and 7 days post-infection the tracheas of three randomly selected chickens from each group were examined for ciliary activity and presence of ibv. observations for clinical signs, snicking, wheezing and nasal discharge, of an ibv infection were carried out daily on each group from three days post-infection. only chickens inoculated with m41-ck showed any clinical signs of an ibv infection. as can be seen from figure 3, m41-ck induced high rates of snicking, which peaked by day 5 post-inoculation, whereas beau-r and rbeaur-rep-m41-struct-2 induced a much lower rate of snicking, similar to those observed for the mockinoculated chickens (fig. 3a) . similarly, only chickens (11-16%) inoculated with m41-ck showed any nasal discharge three, four and six days post-inoculation (fig. 3b) . again, wheezing was only observed in chickens inoculated with m41-ck, in which 90% of the infected chickens demonstrated wheezing by 7 days postinoculation (fig. 3c) . analysis of the tracheas isolated from the chickens showed that the mock, beau-r and rbeaur-rep-m41-struct-2 infected chickens had .95% ciliary activity whereas the chickens inoculated with m41-ck showed 0% ciliary activity (100% ciliostasis; fig. 3d ). in summary, our observations of the parameters used to assess pathogenicity demonstrated that rbeaur-rep-m41-struct-2 was not pathogenic and that it had the characteristics associated with beau-r rather than m41-ck. in order to determine whether there was any virus in the tracheas of the infected chickens, the epithelial cells were scraped from the tracheas taken from three chickens from each group four days post-infection and any virus present titrated in tocs. the average titre of m41-ck, isolated from the tracheas of the three chickens, was 4.1 log 10 cd 50 /ml, in contrast no detectable virus was isolated from the tracheas taken from the chickens infected with either beau-r or rbeaur-rep-m41-struct-2. this result indicated that if any virus was present in the tracheas of the chickens infected with beau-r or rbeaur-rep-m41-struct-2 the amounts present were at least 4 log 10 cd 50 /ml lower than the m41-ck detected from the tracheas of the chickens infected with m41-ck. analysis of the tracheal epithelial cells isolated from the infected chickens, for the presence of ibv by titration on tocs, had indicated that either there was no beau-r or rbeaur-rep-m41-struct-2 present or that the levels of both viruses were below detection. our previous results had shown that beau-r was not reliably detected in the tracheas of beau-r in infected chickens whereas m41-ck is detectable [26, 43] . to check for the presence of m41-ck-, beau-r-or rbeaur-rep-m41-struct-2-derived rna in the tracheal epithelial cells from the infected chickens, total rna was isolated from the epithelial cells and analysed by rt-pcr using oligonucleotides bg56 and 93/100, corresponding to the 39-utr of the ibv genomes. as can be seen from fig. 4 , the tracheal epithelial cells isolated from chickens infected with m41-ck were positive for the presence of m41-ck-derived rna. in contrast, no ibv-derived rna was detected in the tracheal epithelial cells of chickens infected with either beau-r or rbeaur-rep-m41-struct-2 supporting our virus isolation data. furthermore, sequence analysis of the ibv-derived rt-pcr product amplified from the tracheal epithelial cells of chickens infected with m41-ck confirmed that it was derived from ibv m41-ck. this was consistent with previous results suggesting that beau-r probably does not reach the tracheas of chickens infected by the eye-drop and intranasal routes and that similarly rbeaur-rep-m41-struct-2 did not appear to reach the epithelial cells of the tracheas of infected chickens. a ribv, beaur-m41(s) [24] , which consisted of the beau-r genome but with an m41-derived s gene, had the tropism of m41 in vitro but the characteristics of beau-r in vivo indicating, together with our rbeaur-rep-m41-struct-2 result, that the inability of beau-r-derived viruses to reach the tracheas of infected chickens resides within the replicase gene. infection of tracheal epithelial cells ex vivo is a method of growing ibv strains that have not been adapted for growth either in embryonated eggs or primary cell cultures and results in cessation of ciliary activity (ciliostasis) of the epithelial cells. previous work demonstrated that our ribvs, beau-r and beaur-m41(s), caused ciliostasis of infected tocs with concomitant production of progeny virus [26] . analysis of tracheal epithelial cells from chickens infected with ribv-beaur-rep-m41-struct-2 had shown that there was no virus present. therefore, we decided to investigate the replication of rbeaur-rep-m41-struct-2, for comparison with beau-r and m41-ck, ex vivo in tocs. progeny viruses in toc medium taken from each group of infected tocs at the specific time points were titrated by plaque assay on ck cells to investigate the growth kinetics of the three ibvs in tocs. as can be seen from fig. 5 all three viruses replicated in the tocs. although the viruses reached maximum titres by 24 h postinfection, the titre of rbeaur-rep-m41-struct-2 was between 1-2 log 10 units lower than the titres observed for m41-ck and beau-r, respectively, at this and most later time points. interestingly, although rbeaur-rep-m41-struct-2 replicated and produced following rescue of rbeaur-rep-m41-struct-2 all growth experiments were carried out using virus that had been passaged three times (p 3 ) in ck cells. we routinely use the p 3 -derived ribvs so that there is a lower probability that changes will have occurred within the virus genome, as with other positive strand rna viruses multiple passage will result in changes in both nucleotides and amino acids; for example the acquisition of the extra adenosine residue in rbeaur-rep-m41-struct-12. the ribv, rbeaur-rep-m41-struct-2, showed slightly reduced growth properties when compared with both parental viruses, beau-r and m41-ck. surprisingly, although rbeaur-rep-m41-struct-2 grew in tocs it did not cause ciliostasis, which is induced by both parental viruses. due to the chimaeric nature of the ribv the 59-utr, including the leader sequence, corresponds to beau-r, with the 39-utr derived from m41-ck. there are known nucleotide differences in these regions of the ibv genome between the two viruses ( [33, 42] . this introduces the possibility that if the two utrs interact this could affect the growth properties of the the growth characteristics of rbeaur-rep-m41-struct-2-p 25 were initially examined in ck cells and compared to rbeaur-rep-m41-struct-2-p 3 . as can be seen from fig. 6a the peak titre of rbeaur-rep-m41-struct-2 -p 25 was higher, approximately 0.5 log 10 unit, than for rbeaur-rep-m41-struct-2 -p 3 at 24 h post-infection and remained higher throughout the time course. subsequently, the growth properties of rbeaur-rep-m41-struct-2-p 25 were then studied in tocs and as can be seen from fig. 6b rbeaur-rep-m41-struct-2-p 25 ribv grew to a higher titre than rbeaur-rep-m41-struct-2-p 3 , reaching a 1 log 10 unit difference by 72 h postinfection, although the two viruses had a similar titre by 96 h postinfection. however, analysis of tocs infected with rbeaur-rep-m41-struct-2-p 25 also showed that this virus did not cause ciliostasis. in order to determine whether any nucleotide changes may have been responsible for the observed phenotypic changes of the p 25 ribv we decided to initially sequence the 59-and 39-utrs of rbeaur-rep-m41-struct-2-p 25 for comparison to the corresponding sequences of rbeaur-rep-m41-struct-2-p 3 and the two parental viruses beau-r and m41-ck. no nucleotide changes were found within the 59-and 39-utr sequences of the rbeaur-rep-m41-struct-2-p 25 virus indicating that the change in growth pattern was not due to one of the two utrs changing to the other parental sequence. as a result of this finding we sequenced the complete ribv rbeaur-rep-m41-struct-2-p 25 genomic sequence and identified only three other nucleotide changes (table 1 ) in addition to those identified within rbeaur-rep-m41-struct-2-p 3 . these new nucleotide differences corresponded to a single amino acid change in the s gene and two nucleotide changes within orf 5b of gene 5 when comparing the m41-ck-derived sequences within rbeaur-rep-m41-struct-2-p 3 and ribv rbeaur-rep-m41-struct-2-p 25 sequences; indicating that these changes arose on further passage of rbeaur-rep-m41-struct-2-p 3 . the two nucleotide changes within the orf 5b sequence, nucleotides 25815 and 25816 (uuraa), resulted in the introduction of a premature stop codon in orf 5b and a modification of the n gene transcription regulatory sequence (trs) from uucuuaa-caa to aacuuaacaa, the latter sequence being the more predominant ibv trs. sequence analysis of the rbeaur-rep-m41-struct-2 viruses between passages p 3 and p 25 (fig. 7) indicated that at p 5 , the aa mutation was more prominent than the parental (uu) sequence and that in subsequent viruses, derived from passages p 10 , p 15 , p 20 and p 25 , the original parental sequence gradually decreased and disappeared from detection. our previous work on the swapping of the s gene from the avirulent beaudette (beau-r) strain of ibv with an s gene derived from the pathogenic m41-ck isolate of ibv showed that although the s gene was responsible for tropism it was not responsible for virulence [24] [25] [26] . although our results showed swapping of the s gene did not result in virulence we could not rule out the fact that other ibv structural (e, m and n) and accessory (3a, 3b, 5a and 5b) genes may also play a role and therefore be required for the acquisition of virulence. we therefore decided to exchange the region of the beau-r genome from the end of the replicase gene to the poly(a) tail with the corresponding sequence from m41-ck to investigate whether exchange of the structural and accessory genes was sufficient to confer pathogenicity to a resultant chimaeric ribv, rbeaur-rep-m41-struct. such an approach would also allow us to determine whether or not the replicase gene plays a role in the pathogenicity of ibv. ibv beaudette is a well known apathogenic lab strain of ibv that was attenuated by multiple, several hundred, passages in 11-day-old embryonic chicks [30] , therefore loss of virulence could have resulted in multiple changes throughout the genome. ibv beau-r is a molecular clone of beaudette-ck [35] ) that is also avirulent in chickens [26] . the objective of this study was to determine whether replacing the beau-r structural and accessory genes with those from virulent m41-ck would result in a ribv that was virulent when compared to beau-r. we have used our ibv reverse genetics system [24, 25, 35] to produce chimaeric ibv, rbeaur-rep-m41-struct, consisting of the replicase gene from beaudette (beau-r) and the s, 3a, 3b, e, m, 5a, 5b, n and the 39-utr from m41-ck. the m41-ck-derived structural and accessory gene sequence was fused to the beau-r replicase by homologous recombination using the tds method [25, 37] and ribvs were rescued in ckcs. the m41-ck-derived region, in addition to the structural and accessory genes in common with beau-r, also contains an untranslated region, the intergenic untranslated region (igr), between the m and gene 5, which is 305 nt in beaudette and 350 nt in m41-ck. the m41-ck igr, like some other strains of ibv, contains a potential open reading frame (orf) of 285 nt potentially encoding a 94 amino acid product of 11 kd with the initiation codon immediately downstream of the m gene stop codon. a similar orf has been identified at a similar position in the genome of turkey coronavirus (tcov) [5, 6] , the only trs identified for tcov is 288 nt upstream of the potential orf, within the m gene, but with low identity to the tcov canonical trs. no sg mrna for this potential orf has been identified in tcov or ibv, including m41-ck, infected cells. the lack of a sg mrna, the long distance between the initiation codon and a potential trs and the loss of the potential orf, as a result of several deletions, in some strains of ibv indicates that the orf is probably a pseudogene. two ribvs, rbeaur-rep-m41-struct-2 and rbeaur-rep-m41-struct-12, were rescued from the rvvs, rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, respectively, and analysed for the presence of the m41-ck-derived sequence. analysis of rbeaur-rep-m41-struct-12 identified an extra adenosine nucleotide at position 25317 in a six base polyadenosine repeat sequence within the potential 11.5 kd orf in the m41-ck igr, which had the potential for inactivating this potential gene product. to rule out the possibility that the loss of this potential gene product could affect any pathogenicity of this virus we decided to proceed with rbeaur-rep-m41-struct-2, which had the correct sequence, for subsequent experiments. we have characterised rbeaur-rep-m41-struct-2-p 3 in vitro, ex vivo and tested its pathogenicity in vivo using one-day-old-chickens. the overall growth pattern of rbeaur-rep-m41-struct-2 in ckcs was more similar to that of beau-r (fig. 2) except it grew to a titre of about 1 log 10 less throughout the growth cycle. the main objective of this study was to determine whether swapping of the structural and accessory genes would restore virulence. to this effect we used rbeaur-rep-m41-struct-2 to infect one-day-old chicks, and used clinical signs and measurement of the ciliary activity of the epithelial cells lining the trachea of the infected chickens to assess any pathogenicity associated with the virus. no clinical signs, snicking, wheezing and nasal discharge, associated with an ibv infection were observed in the chickens infected with either rbeaur-rep-m41-struct-2 or beau-r; the latter as in previous experiments did not show any clinical signs [26] . in contrast, chickens infected with m41-ck did show clinical signs of an ibv infection (fig. 3) ; as shown previously [26] . these observations demonstrated that rbeaur-rep-m41-struct-2 was not pathogenic indicating that replacement of the structural and accessory genes did not restore virulence. analysis of epithelial cells, removed from the tracheas of the infected chickens, for the presence of infectious ibv using tocs failed to detect virus from chickens infected with either beau-r or rbeaur-rep-m41-struct-2. in contrast, epithelial cells from chickens infected with m41-ck showed the presence of virus with a titre of 4 log 10 cd 50 in tocs, demonstrating that m41-ck was present in the tracheas of the infected chickens. analysis of the epithelial cells for the presence of any ibv-derived rna by rt-pcr also indicated that no beau-r or rbeaur-rep-m41-struct-2 was present or was below detection in the epithelial cells examined; this was in contrast to the cells examined from chickens infected with m41-ck, in which virus-derived rna was detected by rt-pcr (fig. 4) . subsequent analysis of rbeaur-rep-m41-struct-2 ex vivo using tocs surprisingly showed that the ribv did not cause ciliostasis when directly used to infect tocs, in contrast to beau-r and m41-ck which caused ciliostasis. this observation raised the possibility that the lack of ciliostasis and detection of rbeaur-rep-m41-struct-2 in the tracheal epithelial cells of chickens infected by the virus using tocs for our pathogenicity experiments may have been interpreted incorrectly as the read out for these tests was ciliostasis. however, it should be noted no clinical signs were observed nor was any rbeaur-rep-m41-struct-2-derived rna detected. growth analysis of rbeaur-rep-m41-struct-2 in tocs showed that the lack of ciliostasis observed with rbeaur-rep-m41-struct-2 was not due to the inability of the virus to replicate in tocs (fig. 5) . the ribv rbeaur-rep-m41-struct-2 was able to replicate in tocs, though the amount of virus produced was about 1 log 10 less than for the growth of beau-r and m41-ck, somewhat analogous to the growth pattern observed for the virus on ckcs. ibv rbeaur-rep-m41-struct-2 consisted of the beau-r replicase and m41 structural and accessory genes and as a consequence had a 59-utr derived from beau-r and a 39-utr from m41. there are known differences between ibv 39 utrs, in fact the m41-ck 39-utr is 184 nt smaller than the beau-r 39-utr, the remaining part of the m41-ck 39-utr representing the conserved region of the ibv 39-utr and contains the predicted rna secondary structures believed to be involved in replication [44] . there are 7 and 3 nucleotide differences between the 59-and the conserved region of the ibv 39-utrs, respectively, of beau-r and m41-ck ( [33, 42] , raising the possibility that if the two utrs interact during replication their heterologous nature could be responsible for the observed decrease in growth for rbeaur-rep-m41-struct-2 in both ckcs and tocs. impairment in growth on tocs may also have been responsible for the loss of ciliostasis. we hypothesised that an increase in growth would be a selective advantage to the virus and therefore decided to serially passage rbeaur-rep-m41-struct-2 25 times on ckcs to see whether any adaption could result in a higher growth rate and a virus that could cause ciliostasis in tocs. for example, due to the very few differences in the 59-and 39-utrs, between the two parental viruses, a nucleotide substitution in either utr that may result in a more homogenous interaction, as seen with either parental virus, may result in an increase growth rate. analysis of the passaged virus, rbeaur-rep-m41-struct-2-p 25 , on ckcs and tocs showed an altered growth rate in comparison to rbeaur-rep-m41-struct-2-p 3 (fig. 6) . however, rbeaur-rep-m41-struct-2-p 25 still did not cause ciliostasis in tocs. sequence analysis of the 59-and 39-utrs of rbeaur-rep-m41-struct-2-p 25 showed that there were no nucleotide substitutions within these regions when compared to rbeaur-rep-m41-struct-2-p 3 , indicating that the change in the growth characteristics was not associated with changes in the utrs. we therefore sequenced the entire genome of rbeaur-rep-m41-struct-2-p 25 for comparison with the sequence in rvv-beaur-rep-m41-struct, used for generating rbeaur-rep-m41-struct-2-p 3 , in order to identify any potential changes that may have been responsible for the phenotypic change in growth. we identified three nucleotide changes between the p 3 and p 25 viruses, one resulted in an amino acid change, pherleu, in the s1 region of the s gene and two adjacent nucleotides in the orf-5b sequence that had two potential effects, (1) the introduction of a premature stop codon in orf-5b and (2) modification the n gene trs. these observations indicate that the increase in growth associated with rbeaur-rep-m41-struct-2-p 25 could have arisen from the amino acid change in the s protein, the loss of orf-5b or a potential change in the expression levels of sg mrna 6, which is responsible for the expression of the n protein. overall, the main conclusion from our results is the fact that rbeaur-rep-m41-struct-2 was nonpathogenic; indicating that the loss of virulence associated with beaudette is not determined by the structural and the accessory genes of ibv but resides within the replicase gene. our results demonstrating that the ibv replicase gene is a determinant of pathogenicity differs from the conclusion by navas-martin et al. [45] who reported that the differences in pathogenicity between two strains of the murine coronavirus mouse hepatitis virus (mhv), mhv-jhm and mhv-a59, mapped to the mhv genome encoding the structural and accessory genes downstream of the haemagglutinin esterase (he) gene. the replicase gene was interchangeable between the two virus genomes but the differences in pathogenicities associated with two viruses did not appear to correlate with the replicase genes of the rmhvs generated. however, a major difference between the two mhv isolates and the two ibv viruses we used was that the two mhvs were pathogenic but demonstrated different pathogenicities, neurovirulence and hepatitis, respectively, whereas the difference between the two ibv isolates used in this work was that one causes disease and the other does not. it is clear from the mhv work that both replicase sequences are of a ''virulent'' phenotype and that the pathogenicity phenotype of the recombinant viruses is associated with the structural and accessory genes. in contrast our results demonstrated that loss of virulence per se can be determined by some attenuating modification to one or more of the coronavirus replicase components. recent work involving the attenuation of a nephropathogenic strain of ibv by serial passage in embryonated chicken eggs, reported that the virus became fully attenuated by passage p 110 [46] . sequence analysis of the 39-7 kb (s gene to poly(a) tail) region of the genome from the p 110 virus identified several amino acid substitutions and a 109 nt deletion within the 39 utr when compared to p 0 virus. the authors reported that the changes identified were potentially responsible for attenuation; they did not report any changes within the replicase gene of the p 110 virus and indicated that other changes, apart from those identified in the 39-7 kb region, within the genome may also be involved in attenuation. other workers attenuated the virulent ark dpi 11 strain of ibv following 101 passages in embryonated eggs (ark dpi 101) and compared the complete genomes of both viruses; identifying 21 nucleotide changes corresponding to 17 amino acid changes [47] . the nucleotide changes resulted in eight amino acid changes in nsp2 (1), nsp3 (3), nsp6 (2), nsp10 (1) and nsp13 (1) of the replicase protein, eight amino acid changes in the s protein and one amino acid in orf 5a and the n protein. the authors were unable to confirm which amino acid changes were responsible for loss of pathogenicity. taking into consideration our results that loss of virulence associated with the beaudette strain resides in the replicase and the results of ammayappan et al. [47] , it is possible that very few amino acid substitutions within the replicase gene can result in attenuation following serial passage in embryonated eggs. the replicase gene of ibv encodes 15 nsps, some of them with known enzymatic functions [48] . how these proteins function in the context of pathogenesis is still not well understood, however, some of the nsps for other coronaviruses have been linked to loss of virulence. for example, the loss of nsp1 from mhv did not affect replication in tissue culture but severely attenuated the rmhv in vivo [49] ; inactivation of the mhv adp-ribose-10phosphatase activity in nsp3 caused a reduction in virus replication in the livers of infected mice but did not induce liver disease [50] and a single amino acid change in the mhv nsp14 did not alter the replication of the rmhv in tissue culture but resulted in attenuation in mice [51] . our previous spike swapping results demonstrated that introduction of an s protein from a virulent isolate of ibv did not confer virulence on beau-r indicating that loss of virulence was not receptor-mediated. results from work described here has shown that replacing all the beaudette structural and accessory proteins with those from a virulent isolate of ibv did not restore virulence. generation of the ribv produced in this work can either be viewed as replacing the structural and accessory genes of an avirulent virus with those of a virulent virus or replacing the replicase gene of a virulent isolate (m41-ck) with one from an avirulent virus (beau-r). in either scenario our results indicate that loss of virulence associated with ibv beaudette resides within one or more of the 15 ibv replicase proteins comprising the ibv replicase gene. virus taxonomy classification and nomenclature of viruses a comparative sequence analysis to revise the current taxonomy of the family coronaviridae turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian 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sequence of the genome of the coronavirus avian infectious bronchitis virus a new dna sequence assembly program sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus open reading frames 3a and 3b of infectious bronchitis virus cis-acting sequences required for coronavirus infectious bronchitis virus defective-rna replication and packaging replicase genes of murine coronavirus strains a59 and jhm are interchangeable: differences in pathogenesis map to the 39 one-third of the genome altered pathogenicity, immunogenicity, tissue tropism and 39-7 kb region sequence of an avian infectious coronavirus strain after serial passage in embryos. vaccine identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi coronavirus replicative proteins coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines mouse hepatitis virus liver pathology is dependent on adp-ribose-10phosphatase, a viral function conserved in the alpha-like supergroup singleamino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice we thank drs. francesca culver and abu-bakr abu-median for help with removal, processing and extraction of rna from the tracheas control chickens or chickens infected with ibv. key: cord-004810-g0y7ied0 authors: lee, s. k.; sung, h. w.; kwon, h. m. title: s1 glycoprotein gene analysis of infectious bronchitis viruses isolated in korea date: 2003-11-13 journal: arch virol doi: 10.1007/s00705-003-0225-3 sha: doc_id: 4810 cord_uid: g0y7ied0 fifteen isolates of infectious bronchitis virus (ibv) were obtained from the kidney, trachea, and cecal tonsil of ib suspected chickens between 2001 and 2002 years in korea. the s1 glycoprotein gene of ibv isolates were amplified by reverse transcriptase – polymerase chain reaction (rt-pcr) and analyzed by restriction fragment length polymorphism (rflp) analysis. fifteen korean ibv isolates were classified into 4 groups by their rflp patterns using restriction enzymes, haeiii, bstyi, and xcmi. the rflp patterns for 3, 1, and 1 of 15 isolates corresponded to the patterns of ibv arkansas, connecticut, and massachusetts strains, respectively. ten of 15 isolates generated unique km91 rflp pattern that was observed in the ibv km 91 strain previously isolated in korea. to confirm genetic diversity in the s1 genes of ibv isolates, viral rnas of representative 9 of 15 ibv isolates were amplified, cloned, sequenced and compared with published sequences for non-korean ibv strains. korean ibv isolates showed amino acid sequence similarity between 61.8% (k446-01 and k161-02) and 96.1% (k281-01 and k210-02) with each other and they showed amino acid sequence similarity between 42.9% (k161-02 and ga980470) and 96.5% (k203-02 and kb8523) compared to non-korean ibv strains. by phylogenetic tree analysis, korean ibv field isolates were branched into five clusters in which 3 clusters were differentiated from non-korean ibv strains. especially, korean ibv isolates k069-01, k507-01, k774-01 and k142-02 formed a separate cluster. it seems that ibvs continue to evolve and ibvs showing various genetic differences may cocirculate in korea. infectious bronchitis virus (ibv) is the etiological agent of infectious bronchitis (ib), which is an acute and highly contagious disease of the respiratory and sometimes the urogenital tracts of chickens causing tracheal rales, sneezing, coughing, a poor weight gain and reduced feed efficiency in broilers and a decline in egg production and egg shell quality in layers [8] . ibv belongs to the family coronaviridae [24] . it is a pleomorphic enveloped virus with club-shaped surface projections (spikes) on the surface of the virion and its genome consists of the single stranded positive-sense rna genome of approximately 27 kilobases [1] . the virion contains four major structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the envelope (e) glycoprotein, and the nucleocapsid (n) protein [24, 30] . the s glycoprotein of ibv is posttranslationally cleaved into n-terminal s1 and c-terminal s2 subunits [7, 30] . the s1 glycoprotein forms the distal, bulbous part of the spike, and the s2 glycoprotein anchors the s1 glycoprotein to the viral membrane [3] . the s1 subunit is known to contain regions that induce neutralizing, serotype-specific, and hemagglutination-inhibiting antibodies [5, 18, 27] . a number of ibv serotypes and variants have been isolated and identified worldwide [10, 17] . these antigenic ibv variants do not completely cross-protect [13] . therefore, ib continues to be an economically important disease to the poultry industry although ibv vaccines have been used to prevent ib outbreaks worldwide. different serotypes and variants of ibv are thought to be generated by amino acid changes resulting from nucleotide insertions, deletions, or point mutations in the s1 subunit made by the viral polymerase [4, 20, 32] . since ibv was first reported in 1986 in korea and nephropathogenic ibv was recognized in 1990, various serotypes of ibv have been reported in korea. and these ibv isolates showed different patterns from each other and non-korean ibv isolates in reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism (rt-pcr-rflp) analysis [29] . however, only massachusetts (mass) type live attenuated vaccines as well as inactivated oil-emulsion vaccines were used to control ib [16, 17, 28, 29] . the purpose of the present study was to genetically characterize ibv strains isolated recently in korea. the s1 glycoprotein genes of the korean ibv strains were amplified by rt-pcr. amplified s1 genes were first classified by rflp analysis and then the representative strains were cloned, sequenced and compared to other non-korean published ibv sequences. by the rt-pcr-rflp and phylogenetic analysis, recent korean ibv isolates were classified into at least 5 different groups at the genetic level in which one group included only korean ibv strains. field ibvs were isolated from kidney, trachea and cecal tonsil of ib suspected chickens using spf embryonated eggs between 2001 and 2002 according to the standard procedure [11] . the history of these isolates is listed in table 1 . ibv isolates were propagated in 10-dayold specific pathogen free embryonated eggs and identified as ibv by ibv-specific rt-pcr as described below [22] . the harvested allantoic fluids were used to prepare viral rna. s1 gene of korean ibv isolates 483 viral rna extraction and rt-pcr of s1 gene the viral rna was extracted from allantoic fluid as described previously [22] . briefly, sodium dodecyl sulfate (final concentration, 2% wt/vol) and proteinase k (final concentration, 250 µg/µl) were added to the allantoic fluid and the mixture was incubated for 5 min at 55 • c. viral rna was extracted with acid phenol : chloroform (5:1, ph 4.7, ambion, woodward, u.s.a.) and chloroform : isoamylalcohol (iaa) (49:1), and further purified using the rnaid kit (bio101, carlsbard, u.s.a.). finally, the rna was resuspended in diethyl-pyrocarbonate (depc) treated water and stored at −70 • c until used in the reverse transcriptase (rt) reaction. amplification of the s1 gene by rt-pcr was performed using the forward s1 oligo5 (5 tgaaaactgaacaaaaga 3 ) and reverse s1 oligo3 (5 ctaaactaacataagg gc 3 ) primer pair [22] . the rt reaction to synthesize cdna contained purified rna, 25 pmol s1 oligo3 primer and rt premix accupower rt premix (rtase, stabilizer and tracking dye) (bioneer, korea). the mixture was incubated at 42 • c for 60 min, and then heated for 5 min at 94 • c to stop the reaction. for the pcr reaction, 20 pmol each primer (s1oligo5 and s1oligo3 ) and cdna were added to accupower pcr premix (taq dna polymerase, each dntp, tris-hcl, kcl, mgcl 2 , stabilizer and tracking dye) (bioneer, korea). the pcr was performed by 35 cycles of denaturation at 94 • c for 90 sec, annealing at 45 • c for 30 sec, and polymerization at 72 • c for 90 sec. the final polymerization step was performed at 72 • c for 10 min. the pcr products were analyzed on a 1.0% agarose gel. pcr products with the expected size (about 1720 bp) were excised from an agarose gel and purified using geneclean kit (bio 101). the restriction enzymes haeiii, bstyi and xcmi were used to digest the pcr products of s1 gene as described by kwon et al [15, 23] . the rflp patterns were observed after electrophoresis on a 2% agarose gel. nine representative ibv isolates (k069-01, k281-01, k446-01, k507-01, k774-01, k142-02, k161-02, k203-02, k210-02) of 15 isolates after rflp analysis were sequenced. pcr products were cut from 1% agarose gels and purified using geneclean (bio 101). purified pcr products were cloned into the ta cloning vector (invitrogen, carlsbed, ca) and transformed into competent cells (top10) (invitrogen). cells carrying recombinant plasmid were selected on lb agar plates containing ampicillin and x-gal. plasmid dna for sequencing was prepared by plasmid maxi kit (qiagen, santa clarita, ca, u.s.a.). sequencing was performed with the m13 forward and m13 reverse primers. dna sequencing was performed using an abi prism 3700 dna analyzer (applied biosystems, foster city, ca, u.s.a.). nucleotide sequence data were compiled and analyzed using the clustal v method in megalign software (dnastar, inc. madison, wi). phylogenetic trees for s1 glycoprotein were generated using the maximum parsimony method with 100 bootstrap replicates in a heuristic search with the paup 4.0 software program (sinauer associates inc., sunderland, ma, u.s.a.). the sequence data of s1 gene reported have been deposited in the genbank database (table 1) . sequences used for comparison or phylogenetic analysis in this study were obtained from the following genbank database accession numbers: arkansas 99 (m85244), fifteen ibvs were isolated from trachea, kidney and cecal tonsil of broiler chickens (table 1) . six, 5 and 4 of 15 ibvs were isolated from trachea, kidney, and cecal tonsil respectively. the age of chickens with ib outbreaks was from 15 to 70 days. rt-pcr-rflp analysis was initially performed to classify ibv isolates. fifteen korean ibv isolates were classified into four groups by rflp analysis (fig. 1) . the rflp pattern for isolate k446-01 corresponded to a pattern for ibv connecticut strain. isolate k203-02 showed both massachusetts and km91 rflp patterns. three isolates (k281-01, k434-01, and k210-02) were identical to the ibv arkansas strain and 10 isolates (k069-01, k507-01, k748-01, k774-01, k044-02, k058-02, k117-02, k142-02, k161-02 and k234-02) had same rflp pattern as the ibv km91 strain which was isolated in 1991 in korea [29] . the whole s 1 gene of representative 9 of korean ibv isolates was sequenced to further characterize the isolates. the nucleotide and deduced amino acid sequences of those ibv isolates were determined and compared with the sequences of published non-korean ibv strains ( table 2 , fig. 2 ). korean ibv isolates had nucleotide sequence similarity between 62.4% (k281-01 and k161-02) and 96.6% (k069-01 and k142-02) with each other and they had nucleotide sequence similarity between 47.1% (k281-01 and de072) and 98.9% (k203-02 and h120) with non-korean ibv strains. korean ibv isolates had amino acid sequence similarity between 61.8% (k446-01 and k161-02) and 96.1% (k281-01 and k210-02) with each other and they had amino acid sequence similarity between 42.9% (k161-02 and ga980470) and 96.5% (k203-02 and kb8523) compared to non-korean ibv strains. the korean ibv k446-01 isolate had an s1 amino acid sequence most similar to the mass41 strain (95.5%) and 93.8% similar to the h120 strain. korean ibv isolate k203-02 had an s1 amino acid sequence most similar to h120 (98.0%) and 92.6% similar to mass41. isolates k281-01 and k210-02 had amino acid sequences similar (79.2%-79.6%) to the ark dpi strain. isolates k069-01, k507-01, k774-01 and k142-02 were 91.4% to 97.2% similar to one another, and they were 72.8% to 79.7% similar to eight published non-korean strains (ark dpi, beaudette, connecticut, d41, gray, h120, kb8523, and mass41). isolates k161-02 had an s1 amino acid sequence 61.8% to 64.3% similar to other korean isolates and 61.6% to 64.2% similar to 8 published non-korean strain (ark dpi, beaudette, connecticut, d41, gray, h120, kb8523, and mass41). the deduced amino acid sequences of korean ibv isolates were aligned with the sequences of published non-korean ibv strains (fig. 2) . the most variations were observed between residues 53-96, 115-152, and 376-392 (numbering is in reference to mass41 strain). phylogenetic trees were constructed from the nucleotide and deduced amino acid sequences of the s1 glycoprotein genes of korean ibv isolates and non-korean ibv strains (fig. 3) . nine korean ibv isolates were grouped into five distinct branches. the k446-01 isolate formed the first branch in which mass41 was included. the k203-02 isolate formed the second branch that related to non-s1 gene of korean ibv isolates 489 fig. 3 . phylogenetic relationship based on the deduced amino acid sequences of the s1 glycoprotein of the korean ibv field isolates (k069-01, k281-01, k446-01, k507-01, k774-01, k142-02, k161-02, k203-02, and k210-02) and non-korean ibv strains generated by the maximum parsimony method with heuristic search and 100 bootstrap replicates. the tree was rooted to a sequence of the ibv beaudette strain. the length of each branch represents the number of amino acid changes between sequences korean ibv isolates, kb8523, h120, and d41. the k281-01 and k210-02 isolates formed the third branch and the k161-02 isolate formed the fourth branch. k069-01, k507-01, k774-01 and k142-02 isolates formed the fifth independent group. the spike glycoprotein of ibv is translated as a precursor protein (s 0 ), and then cleaved into two subunits s1 and s2 [7, 22] . the ibv spike glycoprotein cleavage recognition site for korean ibv isolates k069-01, k446-01, k507-01, k774-01, k142-02, k161-02 and k203-02 isolates had the sequence arg-arg-phe-arg-arg, which was found in the published ibv beaudette, d41, h120, kb8523, and mass41 strains [7, 14] . whereas korean ibv isolates k281-01 and k210-02 isolates had the sequence arg-arg-ser-arg-arg which was found in the published ibv ark dpi and gray strains [14, 22] . ib has been a continual problem in korea although both mass type live attenuated vaccine and inactivated vaccine were widely used to control the disease. fifteen korean ibv isolates were initially analyzed by rt-pcr-rflp and followed by nucleotide sequencing of the s1 glycoprotein gene in this study. korean ibv field isolates between 1986 and 1997 were characterized using rt-pcr-rflp analysis and pathogenicity testing but the sequences of those viruses were not reported [29] . according to rt-pcr-rflp analysis, 40 ibv field isolates were classified into five genotypes (i, ii, iii, iv, and v). six genotype i ibv isolates showed similar rflp patterns to mass type of ibv and only 2 genotype ii ibv isolates were only isolated in 1986. twenty-nine genotype iii ibv (km91 type) isolates showed distinct rflp patterns in pcr-rflp analysis using restriction enzymes haeiii, ecori, and bamhi [29] . the km91 isolate is the representative genotype iii isolate. genotypes iv andv were newly isolated in 1995. in this study, 10 of 15 field ibv isolates were classified as the km91 type and 3 isolates were classified as arkansas and 1 as connecticut types. in pathogenicity tests, isolate km91 caused 50% mortality, severe nephritis and renal urate deposition in the kidneys of infected chicks, but genotypes i, ii, iv and v only induced respiratory distress at 1 to 2 days after inoculation [29] . the h120 vaccine could not protect chicks against challenge with the km91 isolate, genotype iii. therefore, the km91 type has seemed to be a major ibv circulating in korea. the korean ibv k203-02 isolate showed both massachusetts and km91 type rflp patterns in pcr-rflp analysis. the detection and differentiation of two different kinds of viruses in a single sample is a significant advantage of rt-pcr-rflp analysis which other researchers have also reported [2] . korean ibv isolates (k069-01, k507-01, k774-01, k142-02 and k161-02) sequenced among ibv isolates classified as km91 type by rflp analysis showed 63.4% to 97.2% nucleotide sequence similarity and 66.7% to 98.6% amino acid sequence similarity among themselves. it seemed that ibvs with differences in genetic composition existed in field condition although they showed identical rflp patterns. s1 gene of korean ibv isolates 491 by alignment of the s1 glycoprotein of korean ibv isolates with published non-korean ibv strains, the most sequence variations were observed between residues 53-96, 115-152 and 376-392. regions between 53-96 and 115-152 showing high amino acid variations herein were similar to hypervariable region 1 (hvr 1, residues 56-69) and hypervariable region 2 (hvr 2, residues 117-131) of ibv [6, 20] . the hvrs are associated with two separate viral neuralizing and conformationally dependent epitopes [6, 20] . amino acid variation region 376-392 was similar to region iii (274-387) associated with a neutralizing epitope [19] . virus neutralization tests will be needed to definitively classify korean ibv isolates into a distinct serotype although pcr-rflp pattern correlates with serotype [23] . in the phylogenetic tree, korean ibv isolates formed five distinct clusters. korean ibv k446-01 isolate was clustered into mass41 group although it was classified into the connecticut group by pcr-rflp analysis. korean ibv k203-02 isolate was clustered into the same genotypic clade as non-korean ibv strains kb8523, h120, and d41 that were an attenuated vaccine strains [21, 26, 31] . therefore, k203-02 isolate appears to be originated from a vaccine strain. korean ibv k281-01 and k210-02 isolates formed distinct clusters that were related to non-korean ibv ark99, ark dpi, gray and jmk strains although k281-01 and k210-02 isolates were classified into the arkansas type by pcr-rflp analysis. two korean ibv isolates k281-01 and k210-02 had the same arg-arg-ser-arg-arg on spike glycoprotein cleavage recognition sites as the non-korean ibv strains ark dpi, gray, and jmk [14] . spike glycoprotein cleavage recognition site does not appear to correlate with serotype or pathogenicity [14] . korean ibv k161-02 isolate formed a distinct cluster although it was classified into the km 91 type with other several ibv isolates by pcr-rflp analysis. it showed 61.8% to 64.3% amino acid sequence similarity compared to other korean ibv isolates, which was lower similarity than other ibv isolates. it was noteworthy that k161-02 isolate was related to non-korean ibv isolate de072 and ga980470 in the phylogenetic tree although it showed 42.9% (k161-02 and ga980470) to 43.4% (k161-02 and de072) amino acid sequence similarity. the ibv de072 strain was involved in ib outbreaks of broilers on the delmarva peninsula, u.s.a. in 1992, and it was classified into the delaware variant (de var) type by virus-neutralization tests, cross-challenge tests and s-1 gene analysis [12] . the ibv ga980479 strain was isolated from broilers in georgia, u.s.a. and is similar to the ibv de072 strain [25] . but it was genetically distinct from ibv de072 and shared very low antigenic relatedness with ibv de072 and other ibv isolates and thus was designated into a new serotype, georgia 98 [25] . it seems that the korean ibv k161-02 isolate may be one of the korean ibv variants. further characterization by virus-neutralization tests and cross-challenge tests using several ibv strains will be needed. the korean ibv k069-01, k507-01, k774-01, and k142-02 formed a distinct cluster that consists of only korean ibv isolates. they were classified into the ibv km91 type by pcr-rflp analysis, which has been isolated mainly in korea [29] . the evolution of ibv is seemed to be influenced by a number of factors such as the use of multiple strains for vaccination, population density and host immune status [9] . in addition, ibv has a high error rate during the genomic transcription and then, produces a quasispecies phenomenon where many different viral genotypes will cocirculate in the host, with each virus potentially having different levels of fitness for the host environment [9, 24] . widespread uses of various vaccines made from heterologous ibvs in the field may play an important role in increasing the number of new genetic variants in korea. in conclusion, 15 korean ibv field isolates were different from published non-korean ibv strains in nucleotide and amino acid sequences and were clustered into different groups from non-korean ibv isolates by phylogenetic tree analysis. and 15 korean ibv isolates were clustered into at least 5 groups in which 3 groups were differentiated from non-korean ibv isolates. virus-neutralization test and cross-challenge tests using several ibv strains will be needed to further characterize the ibv isolates. completion of the sequence of the coronavirus avian infectious bronchitis virus molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing of haemagglutination-inhibiting antibody, or induce chicken tracheal protection amino acids within hypervariable region i of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptice of ibv strains beaudette and m41 infectious bronchitis the quasispecies (extremely s1 gene of korean ibv isolates heterogeneous) nature of viral rna genome population; biological relevance-a review variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens a laboratory manual for the isolation and identification of avian pathogens antigenic and s-1 genomic characterization of the delaware variant serotypes of infectious bronchitis virus cross-immunity in chickens using seven isolates of avian infectious bronchitis virus spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus further development and use of molecular serotype identification test for infectious bronchitis virus an outbreak of nephropathogenic infectious bronchitis in commercial pullets identification of recent infectious bronchitis virus isolates that are serologically different from current vaccine strains antigenic domains on the peplomer protein of avian infectious bronchitis virus; correlation with biological functions epitopes of neutralizing antibodies are localized within three regions of the s1 spike protein of infectious bronchitis virus. world veterinary poultry association sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus phylogeny of antigenic variants of avian coronavirus ibv molecular cloning and sequence comparison of the s1 glycoprotein of the gray and jmk strains of avian infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis the molecular biology of coronaviruses identification and analysis of the georgia 98 serotype, a new serotype of infectious bronchitis virus sequencing and analysis of s1 gene of avian infectious bronchitis virus strain d41 epitopes on the peplomer protein of infectious bronchitis virus strain m41 as defined by monoclonal antibodies outbreaks of infectious bronchitis in korea epidemiology classification of infectious bronchitis virus isolated in korean between coronavirus proteins; biogenesis of avian infectious bronchitis virus virion proteins cloning and sequencing of genes encoding structural proteins of avian infectious virus evidence of natural recombination within the s1 gene of infectious bronchitis virus author's address: dr. hyuk moo kwon the authors thank dr. chang-won lee for his excellent assistance to make phylogenetic tree and dr. mark w. jackwood for editorial comments. this work was supported by korea research foundation grant (krf-2001-002-g00074), korea. key: cord-279495-zxerb7de authors: liu, xiaoli; shao, yuhao; ma, huijie; sun, chuyang; zhang, xiaonan; li, chengren; han, zongxi; yan, baolong; kong, xiangang; liu, shengwang title: comparative analysis of four massachusetts type infectious bronchitis coronavirus genomes reveals a novel massachusetts type strain and evidence of natural recombination in the genome date: 2012-11-21 journal: infect genet evol doi: 10.1016/j.meegid.2012.09.016 sha: doc_id: 279495 cord_uid: zxerb7de four massachusetts-type (mass-type) strains of infectious bronchitis coronavirus (ibv) were compared genetically with the pathogenic m41 and h120 vaccine strains using the complete genomic sequences. the results revealed that strains ck/ch/lnm/091017 and ck/ch/ldl/101212 were closely related to the h120 vaccine, which suggests that they might represent re-isolations of vaccine strains or variants of vaccine strains that have resulted from the accumulated point mutations after several passages in chickens. in contrast, strains ck/ch/lhlj/07vii and ck/ch/lhlj/100902 had a close genetic relationship with the pathogenic m41 strain. in addition, molecular markers have been identified that distinguish between field and vaccine (or vaccine-like) mass-type viruses, which may be able to differentiate between field and vaccine strains for diagnostic purposes. phylogenetic analysis, and pairwise comparison of full-length genomes and the nine genes, identified the occurrence of recombination events in the genome of strain ck/vh/lhlj/07vii, which suggests that this virus originated from recombination events between m41and h120-like strains at the switch site located at the 3′ end of the nucleocapsid (n) genes. to our knowledge, this is the first time that evidence for the evolution and natural recombination under field conditions between mass-type pathogenic and vaccinal ibv strains has been documented. these findings provide insights into the emergence and evolution of the mass-type ib coronaviruses and may help to explain the emergence of mass-type ibv in chicken flocks all over the world. in 1931, schalk and hawn described ''an apparently new respiratory disease of chicks'' in north dakota in the united states, which was considered to be infectious bronchitis (ib) by later researchers of avian respiratory diseases (schalk and hawn, 1931) . currently, ib still occurs in nearly all poultry-producing countries; it is a highly contagious, acute, and economically important viral disease of chickens. the etiology of ib, which was first demonstrated by beach and schalm (1936) , is infectious bronchitis virus (ibv). ibv is grouped in the genus gammacoronavirus of the family coronaviridae in the order nidovirales (de groot et al., 2011) . the coronavirus genomes are the largest among the known rna viruses and are polycistronic, generating a nested set of subgenomic rnas with common 5 0 and 3 0 sequences (masters, 2006) . like those of all other coronaviruses, the 5 0 two-thirds of the ibv genome consists of two large replicase open reading frames (orfs), orf1a and orf1b. the orf1a polyprotein (pp1a) can be extended with orf1b-encoded sequences via a à1 ribosomal frameshift at a conserved slippery site (brierley et al., 1987) , which generates the polyprotein pp1ab, comprising more than 7000 amino acids, which includes the putative rna-dependent rna polymerase (rdrp) and rna helicase (hel) activity (ziebuhr et al., 2001) . the pp1a and pp1ab of ibv are processed autocatalytically by two different viral proteases, encoded by a papain-like protease (plp) and a 3c-loke protease (3cl pro ) (lee et al., 1991; ziebuhr et al., 2000 ziebuhr et al., , 2001 . other putative domains, presumably associated with a 3 0 -to-5 0 exonuclease (exon) activity, a poly(u)-specific endo-rnase (xendou) activity, and a 2 0 -o-methyltransferase (2 0 -o-mt) activity, have been predicted in pp1ab (ivanov et al., 2004; snijder et al., 2003) . the 3 0 end of a coronavirus genome includes the viral structural and accessory protein genes: a spike (s) glycoprotein gene; an envelope (e) protein gene; a membrane (m) glycoprotein gene; a nucleocapsid (n) phosphoprotein gene; and several orfs that encode putative non-structural accessory proteins (masters, 2006) . of the virus-encoded proteins, the s1 subunit of the s protein carries virus-neutralizing activity, determines the serotype of ibv and is responsible for viral attachment to cells. it is also a major determinant of cell tropism in culture (casais et al., 2003) . the accumulation of point mutations, deletions, insertions and recombination events that have been observed in multiple structural genes, especially the s1 gene, of ibv recovered from naturally occurring infections have been considered to contribute to the genetic diversity and evolution of ibv, and consequently, to a number of ibv serotypes (cavanagh, 2007) . the occurrence and emergence of multiple serotypes of the virus have complicated control by vaccination because many serotypes and variants do not confer complete cross-protection against each other (cavanagh and gelb, 2008) . the originally discovered massachusetts (mass) type of ibv was identified in the united states, beginning in the 1950s (fabricant, 2000; johnson and marquardt, 1975; mondal et al., 2001) . mass-type strains have been isolated in europe and asia since the 1950s and up to the present day (cavanagh and gelb, 2008) , together with dozens of other serotypes that have been isolated in africa, asia, india, australia, europe, and south america (cavanagh, 2001 (cavanagh, , 2003 (cavanagh, , 2005 . the first mass-type ''h'' vaccines were developed in about 1960. they include h120 and h52 (bijlenga et al., 2004) , and are used very commonly and widely around the world. however, virus of this type is occasionally isolated from massachusetts-vaccinated and -unvaccinated flocks with respiratory clinical signs. some of the viruses have shown close genetic relationships with pathogenic mass-type, rather than vaccine, strains by s1 gene analysis. however, conclusions based on the genetic analysis of a single gene sequence, and sometimes even a partial gene sequence, require caution because the true phylogeny can only be demonstrated by analyzing complete genomic sequences. herein, we sequenced the complete genome of four ibv mass-type strains that showed s1 gene diversity (liu et al., 2009; ma et al., 2012; sun et al., 2011) , and we present evidence for in-field recombination between pathogenic and vaccinal strains. furthermore, we characterized the molecular variability of the four mass-type strains to gain insight into the emergence and evolution of these viruses. four mass-type ibv strains were used for complete genomic sequence comparison and analysis in this study. strain ck/ch/lhlj/ 07vii was isolated in 2007 from the kidney of a layer hen vaccinated with h120 and 4/91 in heilongjiang province, china (liu et al., 2009) . strain ck/ch/lnm/091017 was isolated in 2009 from the swollen proventricular tissues of a broiler vaccinated with h120 in neimenggu province, china . strains ck/ ch/ldl/101212 and ck/ch/lhlj/100902, both of which were isolated in 2010, were obtained from laying hens in dalian and heilongjiang provinces, respectively, in china; the birds were suffering from nephropathogenic lesions and respiratory signs, respectively. in addition, the diseased birds in both flocks were suffering from proventriculitis (ma et al., 2012) . all of the ibv strains have been associated with various ib outbreaks in recent years in china and were assigned to the mass-type strains by s1 sequence analysis. to avoid the possible mutation in the viral genome after serial passages in specific-pathogen-free (spf) embryonated chicken eggs, the first passage of each original virus stock was used and purified once by propagating in 9-to 11-day-old spf chicken eggs with a dose of l0 4 -fold dilutions per egg, and the presence of viral particles in the allantoic fluids of inoculated eggs was confirmed with a negative contrast electronic microscope (jem-1200, ex) and reverse transcriptase-polymerase chain reaction (rt-pcr) as described previously . in addition, since these viruses were isolated from chickens vaccinated with h120, it is possible that mixed ibv infections are present in one chicken flock. to exclude this, nine clones of s1 gene of each virus obtained from three independent pcr reactions were sequenced and compared. sequences of each virus identical to the previously results were obtained (liu et al., 2009; ma et al., 2012; sun et al., 2011) . fertile white leghorn spf chicken eggs were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. to determine the full-length genomic sequences of the four viruses, 15 pairs of overlapping primers encompassing the entire genome were used. the primers were designed in regions that are conserved among most of the ibv strains available in the gen-bank database. the sequences and locations of the primers used in rt and pcr in this study are presented in table 1 . viral rna was extracted from 200 ll of infectious allantoic fluid using trizol reagents (invitrogen, grand island, usa), following the manufacturer's protocol. complementary dna (cdna) was synthesized using 80 ll of the first strand mixture (invitrogen) containing 20 lm of primers n (à), 0.5 mm each of dntp (takara, dalian, china) and 40 ll of total rna. the mixture was incubated at 70°c for 5 min and then quick-chilled on ice for 2 min. the rt master mix was composed of 16 ll 5â rt buffer (invitrogen), 8 ll 10 mm dtt, 200 u of m-mlv reverse transcriptase (invitrogen), and 20 u rnase inhibitors (invitrogen). this rt master mix was incubated at 37°c for 3 h. the reaction was terminated by heating at 70°c for 10 min then chilling on ice for 5 min. the pcr was performed in a 25 ll reaction containing 2 ll first strand cdna; 15 nmol each of downstream and upstream primers; 5 ll of 10â pcr buffer (mg 2+ plus, takara); 4 ll of 2.5 mmol dntps; 2 u taq polymerase (takara); and 18 ll of water. the reaction was conducted at 95°c for 5 min, and 30 cycles of 94°c for 1 min; 50°c for 1 min; 72°c for 2 min, and a final extension step of 72°c for 10 min. a product, detectable by ethidium bromide staining, of the expected size was generated. 2.4. the 5 0 -and 3 0 -ends of the genome a cdna clone representing the 5 0 and 3 0 ends of the genome of the four ibv strains was synthesized according to the 5 0 race and 3 0 race system for rapid amplification of cdna ends (takara). pcr was performed according to the instructions accompanying the kits. the sense and antisense primers used to amplifying the 5 0and 3 0 -ends of the genome had been designed on the basis of the sequences obtained above that were constant in the four ibv strains, respectively. the outer and inner primers used to amplify the 5 0 -end of the four ibv strains were 5 0 -cagctatggcaatgcg cag-3 0 and 5 0 -catctttggtgtctca/tcc-3 0 , respectively. the primer used to amplify the 3 0 -end was 5 0 -gaggagaggaacaatgc aca-3 0 . the dna generated by pcr amplification was cloned using a ttailed vector, pmd18-t (takara), and transformed using jm109 competent cells (takara) according to the manufacturer's instructions. at least five clones of each fragment in each strain were sequenced and the consensus sequence was determined. the sequences were analyzed using the sequencher 4.5 sequence analysis program, and a single contiguous sequence comprising the entire ibv genome of each of the four ibv strains was constructed. the nucleotide and amino acid sequences of the entire genome of the four ibv strains were assembled, aligned, and compared with those of other reference ibv and turkey coronavirus (tcov) strains using the megalign program in dnastar (version 7, lasergene corp, madison, wi). the orfs were determined using the gene runner program version 3.00 (http://www.generunner.com) by comparison with those of other reference ibv and tcov strains. a total of 39 ibv and 7 tcov reference strains, for which entire genomic sequences were available in genbank database, were selected for phylogenetic analysis of full-length genomes. the selected avian coronavirus reference strains and their accession numbers are provided in table 2 . phylogenetic analysis, accurate estimation and comparison of the 5 0 -utr, gene 1, s1, s2, gene 3, m, gene 5, n and 3 0 -utr of the four ibv strains was conducted with those of the mass-type strains selected in this study using the clustal v method of dnastar software and mega4 (liu et al., 2009) , and the alignments were edited manually and adjusted to remove mistakes. deletion, insertion and gene recombination were determined according to the results of the phylogenetic analysis and pairwise comparisons. the full genomic sequences of the four mass-type ibv strains described in this report have been deposited in the genbank database with accession numbers ck/ch/lnm/091017 jf330899, ck/ ch/lhlj/07vii jf274479, ck/ch/ldl/101212 jf828981 and ck/ ch/lhlj/100902 jf828980. four mass-type ibv strains were subjected to genome sequencing and phylogenetic analysis in this study. the sequences of each the four strains were assembled into one contiguous sequence to represent the entire viral genomes. sequences of 27630, 27678, 27630 and 27473 nucleotides were obtained from strains ck/ch/ lnm/091017, ck/ch/lhlj/07vii, ck/ch/ldl/101212 and ck/ch/ lhlj/100902, respectively, excluding the polyadenylation tail at the 3 0 end. the genomes of the viruses were similar overall in their coding capacity and genomic organization to those of other ibvs. the genome of each of the viruses contained two large slightly overlapping orfs in the 5 0 two-thirds of the genome and multiple additional orfs in the 3 0 one-third of the genome. both termini were flanked with untranslated regions (utrs). ten orfs were identified within the genome. gene 1 contained motifs common to all coronaviruses, including ribosomal frameshifting and slippery sequences, because orf1b is translated in the à1 frame. the typical coronavirus structural genes encoding the s, e, m and n proteins were identified following gene 1 (fig. 1) . the genome organization was determined to be as follows: 5 0 -utr-gene 1 (orf1a, 1b)-s-gene 3 (orfs 3a, 3b, e)-m-gene 5 (orfs 5a, 5b)-n-utr-3 0 . the analysis of the complete genome showed that strains ck/ch/ lnm/091017 and ck/ch/ldl/101212 possessed 99.9% and 99.8% nucleotide identity with h120, respectively. however, they shared 91.3% and 91.4% identity with m41, respectively. phylogenetic analysis using the full-length genome and the 5 0 -utr, gene 1, s1, s2, gene 3, m, gene 5, n and 3 0 -utr showed that the ibv strains ck/ ch/lnm/091017 and ck/ch/ldl/101212 consistently formed the same clade with vaccine-related strains of mass-type (figs 2 and 3 ). the analysis of the s1 gene showed that strains ck/ch/lnm/ 091017 and ck/ch/ldl/101212 had high nucleotide identities (99.8% and 99.8%, respectively) with h120, while they had 97.7% and 97.7% identity with m41. multiple alignments revealed that there were 2 and 1 nucleotide mutations within the s1 gene between strains ck/ch/lnm/091017 and ck/ch/ldl/101212 and h120; however, there were 36 and 39 mutations between strains ck/ch/lnm/091017 and ck/ch/ldl/101212 and m41. all these results suggest that strains ck/ch/lnm/091017 and ck/ch/ldl/ 101212 are closely related to the h120 vaccine strain. the percent nucleotide similarity between strain ck/ch/lhlj/ 100902 and h120 for the full-length genomes was 91.3%; however, the percent similarity was up to 99.7% between strain ck/ch/lhlj/ 100902 and m41. in addition, in all of the nine trees constructed for the 5 0 -utr, gene 1, s1, s2, gene 3, m, orf5, n and 3 0 -utr, the strain ck/ch/lhlj/100902 constantly fell into the same clusters as the pathogenic m41 strain, and both belonged to the mass-type. pairwise comparison of the s1 protein gene revealed that strain ck/ ch/lhlj/100902 had 3 and 41 nucleotide mutations with respect to m41 and h120, respectively. taken together, these results demonstrate that strain ck/ch/lhlj/100902 exhibits a close genetic relationship to pathogenic m41. pathogenic and non-pathogenic mass-type strains were clustered into different clades by phylogenetic analysis of full-length genomic sequences and the 5 0 -utr, gene 1, s1, s2, gene 3, m, gene 5, n and 3 0 -utr. in addition, insertions and deletions were also observed that distinguished between the genomes of pathogenic and non-pathogenic mass-type strains, as illustrated in fig. 4 and supplementary material 1. in non-pathogenic strains, five deletions: of 31 nucleotides, 5 nucleotides, 9 nucleotides, 10 nucleotides, and 9 nucleotides, respectively, were observed to be located in the nsp3 of gene 1. they were found to occur between genomic positions 2897-2929, 3219-3225, 3241-3251, 3257-3268, and 3398-3407, respectively , by comparing the sequences with the homologous regions of pathogenic strains. in contrast, a 3-nucleotide and a 9nucleotide insertion were found in nsp6 and between the m gene and gene 5, respectively. additionally, a cluster of insertions was found at the 3 0 -utr region in the non-pathogenic strains. these changes might not only account, at least partly, for viral fitness when the pathogenic virus has become adapted to egg embryos hewson et al., 2012) , but may act also as molecular markers, able to differentiate between vaccine and field strains, for diagnostic purposes. comparative sequence analysis based on full-length genomic sequences and the sequences of the 5 0 -utr, gene 1, s1, s2, gene 3, m, gene 5 and n showed that strain ck/ch/lhlj/07vii clustered with pathogenic mass-type strains. the exceptions were the trees constructed using the 3 0 -utr and s1 gene, in which ck/ch/lhlj/07vii was grouped with non-pathogenic strains; this suggests that a possible recombination event may have occurred. thus the n and 3 0 -utr of ck/ch/lhlj/07vii were carefully compared pairwise with those of strains ck/ch/lnm/091017, ck/ch/ldl/101212, ck/ch/ lnm/091017, h120 and m41. parallel to the result of the phylogenetic analysis, ck/ch/lhlj/07vii showed high similarity with m41 at the 5 0 -end of the n gene; however, it showed high similarity with vaccine strain h120 at the 3 0 -end of the n gene (supplementary material 2). the data strongly suggest that ck/ch/lhlj/07vii arose from a homologous rna recombinant event that involved a template switch between massachusetts pathogenic m41-like and non-pathogenic h120-like strains. we located the switch site at the 3 0 -end of the n gene (supplementary material 2), which implies that the template switch occurred within the n gene. the percent nucleotide similarity between strain ck/ch/lhlj/ 07vii and h120, and ck/ch/lhlj/07vii and m41, for the full-length genomes was 92.5% and 99.4%, respectively. alignment revealed that a 3-nucleotide insertion was located in nsp6 of the ck/ch/ lhlj/07vii strain between genomic positions 24009 and 24013 (supplementary material 3). in addition, the s1 gene of strain ck/ ch/lhlj/07vii showed extensive mutations by pairwise comparison (supplementary material 4) though it was grouped with h120 by s1 gene phylogenic analysis. these and our previous results (liu et al., 2009) showed that, with the exception of the occurrence of recombination events, ck/ch/lhlj/07vii has experienced multiple mutations and deletions in the genome over time. understanding the evolution of mass-type ibv is important because not only is this virus circulating worldwide but information on virus genomics will aid our understanding of the evolution and emergence of ibv with infectious potential in vaccinated chicken flocks. in this study, we focused on the full-length genomic sequences of four ibv isolates which had been shown to be of the mass-type by s1 gene analysis (liu et al., 2009; ma et al., 2012; sun et al., 2011) . based on the high degree of similarity in the full genomic sequence, it could be concluded that two ibv strains, ck/ ch/lnm/091017 and ck/ch/ldl/101212, were very similar to the vaccine strain h120. they might therefore represent re-isolations of vaccine strains, although they were isolated from vaccinated chickens with respiratory disease. similarly, ibv strains that showed a very close relationship to the h120 vaccine strain have been isolated from unvaccinated broiler flocks in slovenia with respiratory problems (krapež et al., 2010) . alternatively, these strains might be variants of vaccine strains that have resulted from accumulated point mutations after several passages in chickens. a few key mutations in the s1 subunit of the spike protein might result in a change to a new serotype, which is defined as a lack of cross-neutralization with specific sera against different ibv serotypes (cavanagh et al., 1992) . the point mutations found in the genome that distinguish between the two isolates and vaccine strain h120 might be the result of adaptive evolution driven by the host immune response when the vaccine strain was transmitted among chickens. adaptive evolution is the process by which genetic changes in the viral genome leading to a more fit virus population become fixed over time, and it has been reported to occur in many coronaviruses (hasoksuz et al., 2007; lee and jackwood, 2001; shi et al., 2006; tang et al., 2009; zhang et al., 2006) . as shown in this study, and as also occurs in other countries (dolz et al., 2008; rimondi et al., 2009; roussan et al., 2009) , the isolation of mass-type ibv is expected, because attenuated vaccine strains are used extensively in chicken flocks in china. however, vaccination is not likely to be the only explanation for the circulation of mass-type virus, because ck/ch/lhlj/100902 and ck/ch/ lhlj/07vii were most closely related to a massachusetts pathogenic type strain, m41. the isolation of a massachusetts pathogenic strain from h120-vaccinated chicken flocks may be due to vaccination failure in these flocks . alternatively, molecular studies have shown that only a few changes in the amino acid composition of the s1 spike protein can result in immune failure, even when the majority of the virus genome remains unchanged (cavanagh et al., 1992) . our findings showed that mutations had occurred in the genomes of both ck/ch/lhlj/100902 and ck/ch/ lhlj/07vii, especially in the s1 genes of ck/ch/lhlj/07vii though it was in the same group with h120 strain in the phylogenetic analysis, implicating that strain ck/ch/lhlj/07vii has experienced evolution over time. it has been reported that amino acid changes may result from immunological pressure caused by the widespread use of vaccines (cavanagh et al., 1988 . the occurrence of recombination events is another process that allows new strains to emerge, and this has been well documented in ibv (hughes, 2011; jia et al., 1995; kottier et al., 1995; kusters et al., 1990; wang et al., 1993) and other coronaviruses baric, 1992, 1994; makino et al., 1986) . it is believed that the conditions for recombination amongst ibv strains in the field are as follows: an extremely large number of chickens, most maintained at high density; the ease of spread of the virus; and serotype cocirculation, including proof of co-infection with more than one serotype in a given flock (cavanagh, 2007) . in china, intensive chicken farms are concentrated in many provinces, including heilongjiang, where ck/ch/lhlj/07vii was isolated (liu et al., 2009) . almost all the chickens in china receive mass-type vaccines at a very young age and subsequently receive this vaccine a couple more times during the rest of their life span. therefore the vaccine virus exists constantly in chickens; it may persist in various internal organs for 163 days or longer (cavanagh and gelb, 2008) . generally, vaccination using the h120 vaccine provides full protection against pathogenic mass-type pathogenic ibvs and prevents the same type of pathogenic strain from being replicated and spreading in the flocks. however, a single amino acid substitution at position 63 of the s1 subunit of the spike has resulted in escape mutants of mass 41 (cavanagh et al., 1988) . this may have occurred in the case of the ck/ch/lhlj/07vii s1 gene (liu et al., 2009) , which might have made it possible for both pathogenic and vaccine strains to co-exist in a given flock, leading to the occurrence of recombination. similarly, an escape mutant could be a result of adaptive evolution driven by the host immune response. consequently, it is likely that genetic changes due to adaptive evolution and recombination both contributed to the origin and evolution of strain ck/ch/lhlj/07vii: it is possible that adaptive evolution created a mutant, followed by recombination between mass 41-and h120-like strains to create a novel virus. the recombination events from which the ck/ch/lhlj/07vii virus resulted can be explained by a scenario in which the recombination may have involved two parental viral strains, with initiation of rna replication in a m41-like template of either negative or positive polarity (liao and lai, 1992) . this would be followed by switching of the polymerase-nascent crna complex to an h120like virus template. the switch may have occurred at the 3 0 -end of the n gene. in general, for a recombinant virus to emerge and establish itself in the field, it must be viable and have selective advantages. it has been reported that uptake of canine coronavirus (ccv) sequences by type ii feline coronavirus (fcov) may have led to increased viral fitness when compared with type i fcov (herrewegh et al., 1998) . recombination can also result in the emergence of new strains with distinct characteristics, such as pathogenicity and tissue tropism (worobey and holmes, 1999) . in addition, in the cov genome, as with most rna viruses, the 3 0 -utrs usually harbor important structural elements that are involved in replication and/or translation (chang et al., 1994; raman et al., 2003; raman and brian, 2005; goebel et al., 2006; züst et al., 2008) . in ibv, the 3 0 -utr-binds to the n protein, which is essential for the synthesis of negative-strain viral rna. perhaps the acquisition of the 3 0 end of the n gene and the 3 0 -utr from h120-like virus by an m41-like virus (e.g. ck/ch/lhlj/07vii) can alter the efficiency of viral replication. this alteration may in turn affect pathogenicity. however, it remains unknown whether this is the true origin of ck/ ch/lhlj/07vii, and therefore this strain is of particular importance to the surveillance of ibv in china. it will be of equal importance to examine future outbreaks of ibv in chickens by full-length genomic sequence analysis in the context of novel recombination events among ibv strains. furthermore, investigations using 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online version, at http://dx.doi.org/10.1016/j.meegid.2012. 09.016. key: cord-278324-eqqvwwh6 authors: wang, huanan; cong, feng; guan, jianchi; xiao, li; zhu, yujun; lian, yuexiao; huang, ren; chen, meili; guo, pengju title: development of a sensitive and specific xmap assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses date: 2018-09-21 journal: virol j doi: 10.1186/s12985-018-1048-x sha: doc_id: 278324 cord_uid: eqqvwwh6 background: a serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (iltv) and infectious bronchitis virus (ibv) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. method: the microspheres coated with purified recombinant glycoprotein d (gd) of iltv or nucleocapsid (n) protein of ibv were incubated with serum samples. the simultaneous quantification of iltv and ibv antibodies were achieved through the interrogation of microspheres by luminex 200 detection system. . results: this xmap detection demonstrated no nonspecific reactions with avian influenza virus (aiv), avian leukosis virus (alv), newcastle disease virus (ndv), and marek’s disease virus (mdv). the results also demonstrated that the xmap assay was four times more sensitive than the enzyme-linked immunosorbent assay (elisa) for iltv detection and two times more sensitive for ibv detection. a total of 90 chicken serum samples from a chicken farm were tested by xmap and elisa assays. the results showed that the coincidence rates were 84.44 and 100% for iltv and ibv detection, respectively. conclusion: this study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination. infectious laryngotracheitis (ilt) and infectious bronchitis (ib) are common respiratory diseases in poultry and are currently present at epidemic levels around the world, including in china, the united states, and india. because ilt and ib have high incidence and infectivity in chickens at different ages (in days), they have caused huge economic losses to the poultry industry. both diseases have similar clinical symptoms and pathological changes, leading to significant difficulties in clinical differential diagnosis, especially in cases of mixed infection [1, 2] . in addition, the use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases, such as infectious laryngotracheitis and infectious bronchitis [3] . therefore, it is important to establish a method to simultaneously detect iltv and ibv antibodies for the differential diagnosis and immune response evaluation after vaccination. infectious laryngotracheitis virus (iltv), an alphaherpes virus, possesses at least 10 envelope glycoprotein, two main proteins of which are the glycoprotein b (gb) and gd, respectively, which are highly conserved herpesvirus structural glycoproteins [4] . the gd of herpes virus has an important role by binding to the host receptors [1, 5] . the gd protein has been demonstrated to be a candidate antigen for recombinant vaccines [6, 7] . the infectious bronchitis virus (ibv) genome encodes four major structural proteins: the spike glycoprotein (s), the membrane glycoprotein (m), the nucleocapsid (n) protein, and the envelope or small membrane protein (e) [8] . the n protein is thought to be an appropriate diagnostic reagent for antibody detection [9] . in this study, gd and n proteins were selected as antigen molecules for the diagnosis of ilt and ib. at present, the methods used for the diagnosis and effect evaluation of vaccine immunity of avian respiratory diseases primarily include virology detection, serological detection, and molecular biological detection. traditional methods, such as virus isolation, animal inoculation experiments, elisa, hemagglutination (ha), and hemagglutination inhibition (hi) assays are characterized by complex procedures and long periods of time required for diagnosis. despite its unique, sensitive, simple features and rapidity, polymerase chain reaction (pcr) is unable to meet the requirements for high-flux quarantine and is not favorable for the urgent screening of bulk samples. therefore, it is imperative to establish a new technology for the effect evaluation of vaccine immunity and the rapid differentiation or correct identification of important avian respiratory diseases. a new high-flux detection technology, flexible xmap (x = unknown, map = multi-analyte profiling) assay, uses variously colored polystyrene beads that is carboxylated to allow covalent coupling of protein. conjugated beads can be used to capture specific antibodies in serum, and a fluorescent secondary antibody is incubated to bind to the captured serum antibodies. a red laser is used to determine the color of the bead and a green laser to detect fluorescence intensity of bound secondary antibodies through the luminex 200 detection system [10] . this method is applicable for the simultaneous and rapid detection of multiplex pathogen , s antibodies, with simple operation and high accuracy that are superior to conventional methods. in this study, a method employing luminex xmap technology to simultaneously detect iltv and ibv antibodies in serum was established, optimized and used for the differential diagnosis of ibv and iltv. this assay can also be used to simultaneous monitoring of ibv and iltv antibody levels for the evaluation of immunity after vaccination. a singleplex xmap for iltv was established by testing six concentrations of the iltv gd envelope protein (2.5 μg, 5 μg, 10 μg, 20 μg, 40 μg, and 80 μg per 5 × 10 5 magbeads), seen in table 1 . the p/n value is the ratio of mfi (median fluorescence intensity) value between the positive sample and negative sample. the optimal conjugation protein concentration was 2.5 μg per 5 × 10 5 magbeads for singleplex xmap detection of iltv. the mean p/n value under the condition was 10.18. a singleplex xmap for ibv was established by testing six concentrations of the ibv n envelope protein (3 μg, 6 μg, 12 μg, 24 μg, 48 μg, and 96 μg per 5 × 10 5 magbeads), seen in table 2 . the p/n value is the ratio of mfi value between the positive sample and negative sample. the optimal conjugation protein concentration was 12 μg per 5 × 10 5 magbeads for singleplex xmap detection of ibv. the mean p/n value under the condition was 17.16. based on the results in tables 1 and 2, the optimum serum dilution ratio for iltv and ibv were 1:25 and 1:100, respectively. when singleplex xmap assay was performed. to determine the optimal serum dilition ration for simultaneous detection of iltv and ibv, a serial serum dilutions from the range of 1:25 to 1:200 were prepared and were incubated with iltv and ibv antigen conjugated microspheres. from the p/n values showed in fig. 1 , it is obvious that the optimal serum dilution ratio for duplex xmap assay was 1:100. the threshold is considered as the sum of the average plus three times of standard deviation, which is also named cut-off value. to determine the threshold of xmap assay for iltv and ibv, 32 special pathogen free chicken serum samples at 1:100 dilution were used to obtain the average mfi value and standard deviation. from the table 3 , it showed that the threshold of iltv and ibv detection were 154.95 and 266.75, respectively. the results in figs. 2 and 3 demonstrated that xmap duplex assay for iltv and ibv has a high specificity since there were no cross-reactions with serum positive for other avian diseases, such as avian influenza virus intra-assay repeatability experiments showed that the coefficient of variation (cv) was 5.09% for iltv and 8.05% for ibv. inter-assay repeatability experiments showed that the cv was 8.11% for iltv and 9.48% for ibv. all cvs were less than 10%, indicating that the method has high repeatability. the results were shown in tables 4 and 5 . comparison of xmap assay with elisa using clinical samples the sensitivities of xmap and elisa were compared using 1:50 to 1:3200 iltv-positive sera and 1:100 to 1:6400 ibv-positive sera. the results showed that xmap detected iltv-positive sera at 1:1600 and ibv-positive sera at 1:3200, while elisa detected iltv-positive sera at 1:400 and ibv-positive sera at 1:1600 (see table 6 ). to compare duplex xmap assay with elisa for iltv/ibv detection, 90 chicken serum samples from a chicken farm in huizhou, china were used and the results are shown in table 7 . for iltv, xmap detected 76 positive cases whereas elisa detected 69 positive cases; among them, 67 samples were detected positive by xmap assay and elisa. all 90 samples were detected as positive for ibv by both methods. ib, an acute infectious disease characterized by acute watery diarrhea, kidney necrosis, and high mortality, is caused by ibv. thus far, ib has resulted in economic losses to the poultry industry in various countries [8, 11] . ibv infects chickens at different ages. chicks infected with ibv are characterized by noisy breathing, panting, sneezing, nasal fluid, and other symptoms; furthermore, laying hens exhibit significantly reduced egg production, broilers exhibits low growth and bad meat quality, and breeding hens exhibit significantly reduced breeding efficiency [12] . ilt is an acute, contact respiratory infectious disease in chickens. chickens infected with ilt are characterized by nasal fluid, conjunctivitis, panting, coughing, and the production of bloody mucus, as well as swelling, erosion, necrosis, and extensive bleeding in the throat after analysis; laying hens exhibit reduced egg production or even no production, as well as poor egg quality [13] . the disease spreads rapidly and has a wide distribution. the high mortality rate (> 70%) results in huge economic losses to the poultry industry [4] . given the similar clinical symptoms between ilt and ib, the development of diagnoses for both diseases is significant. this is the first case report of differential diagnosis of iltv and ibv with xmap assay. luminex xmap assay is an emerging technology that uses small carboxylated polystyrene microspheres that are internally dyed with both a red and an infrared fluorophore [14] . by changing the ratio of the two fluorophores, it is possible to distinguish up to 100 different color-coded microsphere sets, and each microsphere set may be coupled with a different biological probe. the microspheres are detected and characterized by a dedicated flow cytometer [15] . the luminex xmap technology is useful for many different applications. one review describes the use of this technology for the multiplex detection of viral, bacterial, parasitic, and fungal agents using the microsphere-based multiplex nucleic acid assay (mbmna) and the microsphere-based multiplex immunoassay (mbmi) [16] . mbmis are typically biochemical tests that allow the detection or measurement of the concentration of a protein in a solution via an antibody or immunoglobulin [17] . mbmis are often used in the diagnosis of various pathogens, such as human papillomavirus and human cytomegalovirus, to detect antibodies in serum samples [18] [19] [20] . the advantages of this technology are its simple operation, high accuracy, and reduction in the number this study describes the establishment and validation of single xmap and duplex xmap assays. first, to achieve effective and rapid clinical diagnosis of ilt and ibv, single xmaps were established for the separate diagnosis of these two diseases after screening to identify the best concentrations of microsphere antigens for iltv gd and ibv n (2.5 μg/5 × 10 5 magbeads and and 12 μg/5 × 10 5 magbeads, respectively), with an optimal serum dilution of 1:100. however, it is not ideal to diagnose only one type of pathogen antibody because more than one disease often affects the respiratory tract in poultry. given the similar clinical symptoms of ilt and ibv, the differential diagnosis of these two pathogens with two single xmap assays using one sample wastes time and energy. to achieve the rapid differential diagnosis of clinical samples, it is necessary to establish a duplex xmap. in addition to iltv and ibv, aiv and ndv are also common respiratory viruses for poultry. these four diseases have high incidences, and it is difficult to make a differential diagnosis based on clinical symptoms, which are all similar. figures 2 and 3 showed that our xmap methods was able to simultaneously detect the antibodies to iltv or ibv in the sera and had the high specificity. a duplex xmap can quickly identify iltv and ibv infection, but it does not indicate possible infection by aiv and ndv. therefore, based on this study, we propose that quadruplex xmap should be established in the future for the detection of iltv, ibv, aiv and ndv as a fast and effective differentiation tool for these four viruses. since elisa was established to detect iltv antibodies in 1982 [21] , it has demonstrated strong advantages, as well as higher sensitivity than virus neutralization tests (vnt), making it widely applicable. elisa antibody detection of ibv is also feasible at earlier time points [22, 23] and is currently broadly applied in the poultry industry. single-analyte elisa does not support the detection of multiple specific antibody responses simultaneously for a single serum sample [24] and possesses other disadvantages, such as the requirement for a relatively large amount of sample, negligible nonspecific binding, and increased background. however, with the development of the poultry industry, requirements dictate that many samples must be detected simultaneously; thus, a new detection method for diagnosis with iltv and ibv is significant. luminex xmap assay represents an alternative for commonly employed indirect tests such as elisa. the conversion of an elisa to the xmap format is uncomplicated, efficient and cost-saving, producing an assay with superior dynamic range and sensitivity [25] . mbmi represents increased sensitivity and the potential to quantify antibodies, antigens, and other substances (e.g., hormones, cytokines, and tumor markers), unlike conventional elisa tests [26] . luminex xmap has also been compared with a western blot assay for antibody detection [27] . the results obtained from both methods showed that the sensitivity of xmap was 82.7%, while the western blot assay sensitivity was 74.7%. therefore, xmap may be more efficient and precise than western blots for the diagnosis of diseases. although elisa provides relatively accurate results and has been used for many years, this study demonstrates that xmap is much more sensitive than elisa for the diagnosis of ilt, as indicated by the results presented in table 6 . in addition, xmap may be used for the rapid diagnosis of many samples, saving time and effort. the results presented in table 7 , demonstrate that xmap is highly accurate, with similar detection results to those obtained by elisa in positive clinical samples; thus, xmap is able to be widely applicable. this study describes how to determine the optimal conditions for establishing duplex xmap assay based on the singleplex xmap result, thus providing a reference for establishing multiplex xmap assay for simultaneous detection of animal pathogen , s.antibodies. the antigens used for iltv were a recombinant gd [28] , expressed, purified, preserved in our laboratory. for the detection of antibodies against ibv, the recombinant n protein was expressed as gst fusion protein in escherichia coli bl21 (de3) cells [29] . prior to coupling, the recombinant proteins was desalted by gel filtration using microbio-spin 6 columns (bio-rad, california, usa) based on the manufacture's protocol to exchange the buffer from sodium azide or imidazole to pbs. all the antigens were quantified using pierce bca protein quantification kit (thermo scientific, usa). coupling of proteins to fluorescent microspheres was performed as described by karanikola et al. [30] . map mc10007 beads (luminex, usa) were coupled by gd protein of iltv and map mc10015 beads (luminex, usa) were coupled by n protein of ibv. a certain number of beads was transferred to one coupling reaction tube, followed by 100 μl bead wash buffer and suspended in 80 μl bead activation buffer. then 10 μl of 50 mg/ml edac (sigma-aldrich) and 10 μl of 50 mg/ml s-nhs (sigma-aldrich, germany) were added in the reaction buffer. the reaction tube was gently vortexed for 20 min at room temperature (rt). pbs (ph 7.4, 150 μl) was added twice and the recombinant protein was added. incubation was carried the xmap assays were carried out in 96-well polystyrene microplates using luminex 200 detection system (luminex, usa). relevant positive and negative sera have been prepared for the establishment of the singleplex xmap assay to detect iltv antibody or ibv antibody in the serum. iltv gd protein at the following concentrations (2.5 μg, 5 μg, 10 μg, 20 μg, 40 μg, and 80 μg) and ibv n protein at the following concentrations (3 μg, 6 μg, 12 μg, 24 μg, 48 μg, and 96 μg) were coupled with 5 × 10 5 magbeads to determine the optimum antigen concentration for singleplex xmap assay. a serious dilution of sera at the range from 1:25 to 1:200 have been made to determine the optimal sample dilution. cross-reactivity or specificity was evaluated by making the assay with sera from chicken infected with aiv, alv, ndv, and mdv. then, a duplex xmap assay was established based on the result of two separated singleplex xmap assay. in briefly, a 50 μl aliquot of the simplex beads (50 beads/μl) or the duplex beads (100 beads/μl, 50 beads/μl each kind) was transferred into the wells, added in 50 μl of diluted sera. incubation was conducted on a plate shaker (800 rpm) at rt for 60 min. the centrifugation was conducted in the magnetic separator for 60 s. the supernatant was removed from each well. beads were washed 5 times in 100 μl pbs. each well was added with 100 μl of a biotinylated secondary antibody (goat anti-chicken igy, beijing solarbio science & technology, china) at 2 μg/ml. the plate was shaken (800 rpm) for 30 min at rt and washed as described above. finally, 2 μg/ml of streptavidin-phycoerythrin (sape, life technologies gmbh) was added to each well at 100 μl/well. the plate was shaken and the supernatant was removed. finally, 100 μl of assay buffer was added to each well. the plate was shaken for approximately 15 s and analyzed according to the manufacturer's protocol. all samples were conducted in triplicates in each assay. commercial iltv and ibv elisa kit (biochek, netherlands) were used to detect presence of iltv or ibv antibodies in poultry sera. the assay was performed according to the manufacturer's protocols. herpes virus fusion and entry: a story with many characters avian infectious bronchitis virus viral respiratory diseases (ilt, ampv infections, ib): are they ever under control? molecular biology of avian infectious laryngotracheitis virus structure of herpes simplex virus glycoprotein d bound to the human receptor nectin-1 antibody-induced conformational changes in herpes simplex virus glycoprotein gd reveal new targets for virus neutralization newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges coronavirus avian infectious bronchitis virus an elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications response of young rabbits to infectious bronchitis virus (wachtel strain) review of infectious bronchitis virus around the world diseases of poultry quantitative, multiplexed detection of bacterial pathogens: dna and protein applications of the luminex labmap (tm) system report from a workshop on multianalyte microsphere assays xmap technology: applications in detection of pathogens a collection of methods and protocols for developing multiplex assays with xmap technology simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay 2 optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18 development of a multiplex bead-based assay for detection of hepatitis c virus enzyme-linked immunosorbent assay (elisa) for detecting infectious laryngotracheitis viral antibodies in chicken serum enzyme-linked-immunosorbent-assay for the detection of infectious-bronchitis virus (ibv) antigen with monoclonal-antibody a standardized enzyme-linked immunosorbent-assay for infectious-bronchitis viruscomparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody-levels in chickens a novel bead-based assay to detect specific antibody responses against toxoplasma gondii and trichinella spiralis simultaneously in sera of experimentally infected swine conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method validation and comparison of luminex multiplex cytokine analysis kits with elisa: determinations of a panel of nine cytokines in clinical sample culture supernatants luminex xmap combined with western blot improves hiv diagnostic sensitivity preparation of polyclonal antibodies against gd protein of iltv and application cloning and prokaryotic expression of nucleoprotein gene of infectious bronchitis virus (ibv) zz2004 strain development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against cooperia oncophora, dictyocaulus viviparus and fasciola hepatica in cattle availability of data and materials all data generated or analyzed during this study are included in this published article. contact corresponding author for requests. all authors contributed to the interpretation of the data, critically revised the manuscript for important intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work.ethics approval and consent to participate not applicable. our manuscript does not contain any individual person's data in any form. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-003208-lwirkob3 authors: yan, liping; hu, jianhua; lei, jing; shi, zhiyu; xiao, qian; bi, zhenwei; yao, lu; li, yuan; chen, yuqing; fang, an; li, hui; song, suquan; liao, min; zhou, jiyong title: novel protein chip for the detection of antibodies against infectious bronchitis virus date: 2018-09-17 journal: bmc vet res doi: 10.1186/s12917-018-1586-x sha: doc_id: 3208 cord_uid: lwirkob3 background: infectious bronchitis (ib) caused by the ib virus (ibv) can cause acute damage to chickens around the world. therefore, rapid diagnosis and immune status determination are critical for controlling ibv outbreaks. enzyme-linked immunosorbent assays (elisas) have been widely used in the detection of ibv antibodies in the early infection and continuous infection of ib because they are more sensitive and quicker than other diagnostic methods. results: we have developed two indirect microarray methods to detect antibodies against ibv: a chemiluminescent immunoassay test (cit) and a rapid diagnostic test (rdt). ibv nonstructural protein 5 (nsp5) was expressed, purified from escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near “zero” background for serological assays. compared with the idexx ibv ab test kit, cit and rdt have a sensitivity and specificity of at least 98.88% and 91.67%, respectively. no cross-reaction was detected with antibodies against avian influenza virus subtypes (h5, h7, and h9), newcastle disease virus, marek’s disease virus, infectious bursal disease virus, and chicken anemia virus. the coefficients of variation of the reproducibility of the intraand inter-assays for cit ranged from 0.8 to 18.63%. the reproducibility of rdt was consistent with the original results. the application of the ibv nsp5 protein microarray showed that the positive rate of the cit was 96.77%, that of the nsp5 elisa was 91.40%, and that of the rdt was 90.32%. furthermore, the rdt, which was visible to the naked eye, could be completed within 15 min. our results indicated that compared with nsp5 elisa, the cit was more sensitive, and the rdt had similar positive rates but was faster. furthermore, the two proposed methods were specific and stable. conclusions: two microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against ibv. these methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination. infectious bronchitis (ib) is an acute, highly contagious, and economically important respiratory disease in chickens; it is caused by the ib virus (ibv), which is a significant respiratory pathogen that causes considerable economic losses in the commercial poultry industry worldwide [1] . the ibv genome is a single-stranded, positive-sense rna that is 27.6 kb in size [2] . it encodes four major structural proteins, namely, glycosylated spike protein (s), membrane protein (m), phosphorylated nucleoprotein (n), and envelope protein (e) [3] , and 15 nonstructural proteins (nsp2-nsp16). generally, nonstructural proteins are present in infected cells but not in the virus, and they only play a role in the process of virus infection and replication [4] . chickens immunized with an inactivated vaccine will produce no antibodies or low levels of antibodies against viral nonstructural proteins. thus, nonstructural proteins have the potential application in differentiating natural infection from inactivated vaccine immunity [5] . ib diagnosis is complicated due to the continual emergence of new serotypes [6] and the difficulty in differentiating ib from other upper respiratory diseases [7] . virus isolation is regarded as the gold standard for the diagnosis of ibv infection, but it is time-consuming and costly [8] . the agar gel precipitation test is used in ibv antibody detection; however, this method has low sensitivity. hemagglutination inhibition (hi) assays are suitable for the rapid diagnosis of ib, which requires a series of methods to treat the antigen; however, the hi titer is not related to protection. the virus neutralization test correlates with protection and has the highest specificity among ib diagnostic methods, but it is tedious and laborious [9] . compared with these methods, enzyme-linked immunosorbent assay (elisa) has been widely used for testing ibv early infection and continuous infection, and this technique can be used for both antigenic and antibody detection. the immunogenicity of the coating antigen is one of the crucial factors when performing an elisa test for antibody detection. an inactivated whole virus is the most commonly used coating antigen in commercial diagnosis kits for ibv diagnosis. recombinant antigenic protein expressed using prokaryotic, yeast, or baculovirus systems has been widely used in preparing specific coating antigens for elisa kits [10] [11] [12] [13] . elisas based on purified recombinant protein may have higher specificity and sensitivity as the target antigen is immune-dominant and devoid of any nonspecific immune responses [14] . elisas based on whole virus particles as well as recombinant s1 (spike protein 1 subunit) and n proteins (nucleoproteins) can provide a rapid and large-scale detection method for ibv infection. however, few ibv detection methods have been developed based on nonstructural proteins (nsps). our laboratory has established an nsp5 elisa to detect ibv infection [4] . the nsp5 antibodies detected are likely to be non-neutralizing and exist in lower numbers than the ones generated by other proteins. based on previous studies, we developed a rapid, highly sensitive protein microarray and a visible detection method to detect ibv nsp5 antibodies for epidemiological investigation and antibody level monitoring. initiator integrated poly(dimethylsiloxane) (ipdms) membrane 26 ( in this study, 328 clinical serum samples were collected from a chicken farm. forty-two negative sera were obtained from different ages of specific-pathogen-free (spf) chickens raised in spf isolators in zhejiang university. three-month-old spf chickens, which were purchased from shennong company (zhejiang, china) and reared in spf isolators, were used to prepare negative serum and positive serum. we prepared standard positive serum samples from chickens infected with h5, h7, and h9 avian influenza virus (aiv); newcastle disease virus (ndv); ibv; infectious bursal disease virus (ibdv); and chicken anemia virus (cav). a microarray was prepared in a 100,000-grade clean room. proteins were first dissolved with 30% acetonitrile solution (v/v, in milli-q water) to 1 mg/ml stock solution and then diluted into the optimized concentration (200 μg/ml) with printing buffer (0.3 m phosphate buffer, 0.2% glycerin, 0.01% triton x-100, and 1.5% mannitol) for further printing. ipdms membranes were first activated with 0.1 m edc and 0.1 m nhs mixtures for 30 min, rinsed with milli-q water, and immediately used for printing. to determine the optimal antigen concentration, the protein was diluted with 0.3 m phosphate buffer to different concentrations. each dilution of protein was printed on ipdms using a protein microarray (scienion, germany). once the antigen concentration was determined, the optimized concentration of nsp5 was achieved by dilution with printing buffer and printed on ipdms for subsequent experiments in triplicate. the protein microarray was prepared using the smartarrayer 48 contact printer (capitalbio, china) with approximately 0.6 nl of printing solution for each sample. each subarray had a positive control with chicken-igy at a concentration of 0.1 mg/ml and negative control with printing buffer. the procedure for the cit is shown in fig. 1 . serum samples were first diluted with serum-dilution buffer (1% bovine serum albumin, 1% casein, 0.5% sucrose, 0.2% polyvinylpyrrolidone, 0.5% tween 20 in 0.01 m phosphate-buffered saline, ph = 7.4). in total, 100 μl of the diluted serum samples was then added into each protein microarray and incubated for 30 min on a shaker (thermo fischer, usa) at 500 rpm and 37°c. microarrays incubated with serum-dilution buffer were used as negative controls. each microarray was then rinsed thrice with washing buffer and incubated with 100 μl of 1 mg/ml hrp-igg diluted 1:20,000 in peroxidase conjugate stabilizer/diluent for another 30 min on the shaker (500 rpm, 37°c), followed by the same washing steps described above. a total of 15 μl of the chemiluminescent substrate was added to the microarray, and images were taken at a wavelength of 645 nm with the amersham imager 600 (ge, usa). chemiluminescent signals were acquired using genepix pro 6.0 software, and the signal-to-noise ratio (snr) was calculated. the purified recombinant nsp5 was printed on the ipdms membrane to form a microarray with a concentration of 0.05, 0.1, 0.2, and 0.4 mg/ml. subsequently, the serum samples were added to microplates at the following dilutions: 1:100, 1:200, 1:400, 1:600, 1:800, 1:1600, 1:3200, and 1:6400. to identify the optimal time of exposure, the images were taken at an exposure time of 30 s, 1 min, 2 min, 3 min, and 4 min. to determine the cit threshold, a total of 184 serum samples, including 142 positive samples and 42 negative samples, identified by the idexx ibv ab test kit were tested according to the optimal working conditions. results were then compared with those obtained using the idexx ibv ab test kit. finally, receiver operating characteristic (roc) curve analysis was conducted to determine the accuracy of the ibv protein microarray test. the specificity of the cit was evaluated by detecting the positive sera against aiv (h5, h7, and h9), ndv, mdv, ibdv, and cav. the evaluation of the cit reproducibility within and between runs was carried out as described by jacobson [15] . thirteen field serum samples (nine idexx positive samples and four idexx negative samples) were selected for the reproducibility experiments. for intra-assay reproducibility, three replicates of each serum sample were analyzed within the same plate. for inter-assay reproducibility, three replicates of each sample were run in different plates. the mean snr, standard deviation (sd), and coefficient of variation (cv) were then calculated. the procedure of the rdt is also shown in fig. 1 . serum was first diluted 1:100 with serum-dilution buffer, and 100 μl of the diluted serum sample was added into each protein microarray and incubated for 5 min on a shaker (500 rpm, 37°c). the microarray incubated with serum-dilution buffer was used as a negative control. the microarray was then rinsed thrice with washing buffer and incubated with 100 μl of 1 mg/ml hrp-igg diluted 1:2000 in peroxidase conjugate stabilizer/diluent for another 5 min on a shaker (500 rpm, 37°c), followed by the same washing steps described above. a total of 60 μl of tmb was added to the microarray and incubated for 5 min in the dark; then, the results were observed. to confirm the concentration of the nsp5 protein in the rdt, the purified recombinant nsp5 was printed on an ipdms membrane to form a microarray with concentrations of 0.05, 0.1, 0.2, and 0.4 mg/ml. the specificity of the rdt was evaluated by detecting the positive sera against aiv (h5, h7, and h9), ndv, mdv, ibdv, and cav. the sensitivity experiments of the rdt were conducted by detecting the ibv positive serum with different titers. then, the results were observed, and the detection limit was determined. to further evaluate the cit and rdt, 186 clinical serum samples were detected by the cit, rdt, and nsp5 elisa antibody test kit [4] . subsequently, the positive rate of each method was determined. step 1, the prepared chip was rinsed thrice with pbst; step 2, 100 μl of diluted serum was added and incubated on a constant temperature oscillator and then washed with pbst thrice; step 3, 100 μl of goat anti-chicken igy conjugated to hrp was added, and the plate was incubated on a constant temperature oscillator and washed with pbst thrice; step 4, for chemiluminescence, 15 μl of chemiluminescent substrate was added to each well, and images were taken at a wavelength of 645 nm with amersham imager 600; step 5, for rdt, 60 μl of tmb was added to each well and incubated for 5 min in a dark place; then, the results were observed chemiluminescent signals were acquired using gene-pix, and the snr was calculated as follows: snr = (signal intensity − background)/background. graph-pad prism 6 and microsoft excel were used for the statistical analysis of all data, including the determination of the threshold and the calculation of the snr value, means, sds, and cvs. the roc curve was obtained using graphpad prism 6. sensitivity and specificity were calculated according to the following formulas: sensitivity = true positive/(true positive + false negative) × 100%; specificity = true negatives/(false positives + true negatives) × 100%. the area under the curve (auc) was used to validate the diagnostic application of the cit. the area under the roc curve quantifies the overall ability of the test to discriminate between those individuals with the disease and those without the disease. a truly useless test (one no better at identifying true positives than flipping a coin) has an auc of 0.5, whereas a perfect test (one that has zero false positives and zero false negatives) has an auc of 1. for the cit, the optimal antigen concentration was 0.2 mg/ml (fig. 2a, b) , and the dilution for the serum samples was 1:600 (fig. 2c, d) , on the assumption that the snr between the positive and the negative sera was the highest. the dilution of the hrp-conjugated goat anti-chicken antibody was defined as 1:20,000. when the exposure time was more than 2 min, the snr of the negative serum rose rapidly; thus, we set the exposure time to 2 min (fig. 3) . roc analysis showed that the ibv nsp5 microarray had high selectivity (p < 0.0001) between the positive and the negative samples, and the auc was 0.9993 (fig. 4a) . based on the roc analysis of the ibv nsp5 microarray, the snr value of the idexx-negative serum samples varied from a minimum of 0.01 to a maximum of 1.964, whereas the snr value of the idexx-positive serum samples was from a minimum of 1.82 to a maximum of 23.59 (fig. 4b) . a threshold snr value of 2 for ibv nsp5 microarray was found to provide optimal results, with a (table 1) . thus, the samples with snr < 2 were considered negative, whereas those with snr ≥ 2 were considered positive. the specificity of the cit was evaluated by detecting the cross-reactivity of the antibodies against aiv (h5, h7, and h9), ndv, mdv, ibdv, and cav. the snrs of all sera from the previously mentioned viruses were all below the threshold of 2. these data revealed that no cross-reactivity occurred between the ibv gst-fused nsp5 antigen and antibodies against other avian viruses. this result demonstrated that the antigen has a high specificity. the reproducibility of the cit detection was determined by comparing the snr value of each clinical serum sample from the below tests. the within-plate cvs of nine positive and four negative serum samples tested ranged from 0.8 to 18.63% (table 2) , whereas the between-run cvs of these serum samples ranged from 1.89 to 18.01% (table 3 ). these results showed that the cit detection results were reproducible and had low and acceptable variation. one hundred and forty-four clinical serum samples (130 samples were positive for antibodies against ibv, and 14 samples were negative as confirmed by the idexx ibv ab test kit) were subjected to visual rapid detection following the procedure described above. the data showed that 130 serum samples were positive for antibodies against ibv, and 14 samples were negative, similar to the results of the idexx ibv ab test kit with the nsp5 concentration of 0.2 mg/ml (table 4 ). if ibv antibodies exist in the serum, the spot with the ibv antigen turns blue, thereby allowing us to determine the concentration of nsp5 as 0.2 mg/ml. the specificity of the rdt was evaluated by detecting the cross-reactivity of antibodies against aiv (h5, h7, and h9), ndv, mdv, ibdv, and cav. the specific experiments of the rdt showed that no cross-reaction fig. 4 a distribution of the snrs of the idexx-positive (n = 142) and idexx-negative (n = 42) serum samples of the clinical sera obtained from the ibv protein microarray. the threshold was defined as 2. the diagnostic sensitivity and specificity of the assay were greater than 90%. b roc curve obtained with graphpad prism 6 software with positive (n = 142) and negative (n = 42) samples. the auc was 0.9993, indicating that the ibv protein microarray is a reliable test occurred between the ibv gst-fused nsp5 antigen and the antibodies against other avian viruses. the sensitivity experiments demonstrated that when the positive serum was diluted 1:1000, the spot still turned blue (fig. 5) . (table 5 ). most serological assays, including the idexx elisa kit, use viral particles of ibv as an antigen for the detection of antibodies against ibv. however, the preparation of purified virions for use as an antigen is time-consuming and expensive. in the present study, recombinant nonstructural proteins expressed in escherichia coli antigen-based protein microarray was evaluated for the first time in the serological diagnosis of ib [4] . protein microarrays have high sensitivity and good reproducibility in quantitative and qualitative assays, and they are a valuable asset when analyzing complex biological samples [16] . in clinical sample testing, many factors, including time, cost, accuracy, sensitivity, and throughput, determine the performance and usefulness of an immunoassay. in this study, a new solidly supported material, ipdms membrane, which has a near "zero" background for identification, was used. it achieved high sensitivity in detecting antibodies in serum [17] . these unique features of ipdms not only simplify data analysis but also reduce nonspecific interactions [18] . elisa detection has been widely used in the detection of ibv antibodies in early infection and continuous infection of ib and vaccine-immune, and no diagnosis method is more sensitive and quicker than elisa. in this study, two microarray methods (cit and rdt) were established. except for the method of the observation, the reaction processes of the two methods are akin to the detection process of elisa. however, unlike elisa, the established methods only require 2 ng of antigen coating on each spot, and the amount of hrp-igg required for each reaction well is only 5 ng. the antigen and hrp-igg used in both methods were less than those used in elisa, thereby reducing the cost of detection. in addition, the cit can detect antibodies against ibv nsp5 quantitatively and is more sensitive than the ibv nsp5 elisa kit. the rdt was developed to detect antibodies against ibv visually, and the results can be obtained within 15 min with great sensitivity and specificity. compared with elisa, rdt has a shorter detection time and better detection efficiency. in this study, we only used one antigen of ibv for testing and verification. in the future, we will apply antigens of different diseases to ipdms to achieve high-throughput test results. for the establishment of the ibv nsp5 protein chip, we first optimized the procedure and determined the cit threshold as 2 with the idexx ibv antibody detection kit. with the threshold of 2, the cit showed high sensitivity (98.59%), specificity (100%), and accuracy (98.91%) in the antibody detection of the samples compared with those of other thresholds ( table 1 ). the rdt demonstrated a high success rate compared with the commercial idexx ibv ab test kit, suggesting that the rdt is a reliable assay for the detection of ibv infection. clinical serum samples were also subjected to rapid detection. furthermore, the rdt has higher sensitivity than the commercial idexx ibv ab test kit. it is also simpler and faster than elisa methods. to further evaluate ibv nsp5 protein chip, 186 clinical serum samples were detected by the ibv nsp5 protein chip and the nsp5 elisa antibody test kit. the positive rates of the cit, nsp5 elisa, and rdt were 96.77%, 91.40%, and 90.32%, respectively. compared with nsp5 elisa, the cit was more sensitive, and the rdt had similar positive rates but was faster. protein chips are a high-throughput monitoring system that monitors the interaction among protein molecules through the interaction between a target molecule and a capture molecule. although protein chips have been produced in the context of proteomics research, its application is not limited to proteomics alone. with the development of protein chip technology, researchers have gradually applied this technology to other fields, such as food inspection, disease diagnosis, drug screening, agriculture, forestry, animal husbandry, and forensic science. at present, this technology is rarely studied and applied in veterinary medicine. high throughput is an [19, 20] and enable their use for determining antibody responses to infectious diseases [21] . in the future, we will print recombinant antigenic proteins of different avian viruses to achieve high-throughput detection results with the same serum. the nsp5 protein chips were developed for the detection of antibodies against ibv. these assays are comparable to the commercial idexx ibv ab test kit in terms of sensitivity and specificity. the rdt can generate results within 15 min and may be a suitable alternative to screen for the presence of ibv in chickens. raw data is available from the corresponding author on reasonable request. authors' contributions lpy, jhh, and jl performed the experiments, interpreted the results, and drafted the manuscript. lpy, zys, and qx analyzed the data. zwb, ly, yl, yqc, af, and hl collected the clinical samples. sqs and ml analyzed the data and revised the manuscript. lpy and jyz designed the study, analyzed the data, and revised the manuscript. all authors reviewed the results and approved the final version of the manuscript. this study was performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the ministry of health, china. the protocol of the current study was reviewed and approved by the institutional animal care and use committee of nanjing agricultural university (approval no. syxk 2017-0007). written informed consent to use 328 clinical serum samples, which were collected from a chicken farm, were obtained from the owner of the animals. all efforts were made to minimize animal suffering during sample collection. not applicable vaccination against infectious bronchitis virus: a continuous challenge completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus the molecular biology of coronaviruses development and 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immunosorbent assay for the detection of antibodies against infectious bronchitis virus identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection recombinant lipl32 antigen-based single serum dilution elisa for detection of canine leptospirosis validation of serological assays for diagnosis of infectious diseases overview of protein microarrays integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays initiator integrated poly(dimethysiloxane)-based microarray as a tool for revealing the relationship between nonspecific interactions and irreproducibility the analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays protein microarrays: potentials and limitations antigen microarrays for serodiagnosis of infectious diseases the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-259505-7hiss0j3 authors: kong, qingming; xue, chunyi; ren, xiangpeng; zhang, chengwen; li, linlin; shu, dingming; bi, yingzuo; cao, yongchang title: proteomic analysis of purified coronavirus infectious bronchitis virus particles date: 2010-06-09 journal: proteome sci doi: 10.1186/1477-5956-8-29 sha: doc_id: 259505 cord_uid: 7hiss0j3 background: infectious bronchitis virus (ibv) is the coronavirus of domestic chickens causing major economic losses to the poultry industry. because of the complexity of the ibv life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. results: apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%), molecular chaperone (18%), macromolcular biosynthesis proteins (17%), cytoskeletal proteins (15%), signal transport proteins (15%), protein degradation (8%), chromosome associated proteins (2%), ribosomal proteins (2%), and other function proteins (3%). interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, tenp protein, ovalbumin, and scavenger receptor protein. following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified ibv preparation was verified by western blotting and immunogold labeling detection. conclusions: the results present the first standard proteomic profile of ibv and may facilitate the understanding of the pathogenic mechanisms. infectious bronchitis virus (ibv), the coronavirus of domestic chickens that causes acute, highly contagious respiratory disease, is one of the most important causes of economic loss in the poultry industry. ibv is an enveloped virus with continuous, positive and single-stranded rna genome, which is the largest of any rna virus characterized [1] and encodes four types of structural proteins. the spike (s) glycoprotein, together with small envelope (e) protein and matrix (m) glycoprotein, consists of the viral envelope, whereas the nucleocapsid (n) protein interacts with genomic rna of the virus to form the viral nucleocapsid, in the invariable order 5'-s-e-m-n-3'. proteins s, e, and m have been studied for their important roles in receptor binding and virus budding. s mediates attachment to cellular receptors and entry by fusion with cell membranes, whereas m interacting with s and n proteins is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation [2, 3] . in addition, s protein can inhibit host cell translation by interacting with eif3f [4] and the interaction between m and actin facilitates virion assembly and budding [5] . e is a poorly characterized small envelope protein present in low levels in the virions. the significance of the e protein appears to be critical for viral budding. another role for protein e is that it can promote apoptosis [6, 7] . viruses constantly adapt to and modulate the host environment during replication and propagation. to govern egress from the host cell and initiation of replication in the target cell, viruses will carry some of the host proteins when released from infected cells. enveloped viruses particularly encoding only small proteins have the capability of incorporating numerous host proteins into or onto the newly formed viruses. it is an important prerequisite for the functional studies to know the protein composition of the purified viral particles, as it allows the analysis of specific proteins and their roles during the virus life cycle, resulting in better understanding of the infection process and the pathogenesis of viruses. as a large number of virus complete genomes have been sequenced since 1980s, more and more host proteins in different enveloped viruses have been studied using viral proteomic approaches. herpesviruses have been the most extensively studied in this respect, such as kaposi's sarcoma-associated herpesvirus (kshv) [8, 9] , marek's disease virus (mdv) [10] , epstein-barr virus (ebv) [11] , human cyotomegalovirus (hcmv) [12] and murine cyotomegalovirus (mcmv) [13] . other doublestranded dna (dsdna) viruses including vacciniavirus have also contributed to a better understanding of this intriguing phenomenon [14] [15] [16] . furthermore, recent studies on identification of the incorporated host proteins in rna viruses have also been undertaken. for retrovirus, various studies in this research area have been performed on human immunodeficiency virus type 1 (hiv-1) [17] [18] [19] [20] and moloney murine leukemia virus (mmlv) [21] . for paramyxovirus, numerous host proteins have been found incorporated into avian influenza virus (aiv) particles and respiratory syncytial virus (rsv) particles [22] [23] [24] . to date, no study of the host proteins in the virions of coronavirus has been performed yet. in this study, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified ibv particles. our analysis resulted in the identification of 2 virus-encoded structural proteins and 60 incorporated host proteins. in addition, we also discussed the functional implications of some host proteins in ibv infection and pathogenesis. viral proteomic analysis requires a highly purified preparation of virions. there was no available permissive cell line capable of supporting productive replication of ibv. although primary chick embryo kidney cell (cek) and chick kidney cell (ck) are capable of supporting productive replication of ibv, their poor yields prohibit them from being used for producing large quantity of ibv. in order to obtain large quantity of ibv virions, this study selected 10-day-old spf embryonated chicken eggs for the growth of ibv strain h52. the af with enrichment of h52 was clarified by differential centrifugation in order to remove the contamination of nuclei, mitochondria, lysosomes, peroxisomes and so on from the chicken embryo. the virus was concentrated through a 20% (wt/vol) sucrose cushion before purified over a non-linear 20%-50% sucrose-tne (tris-buffered saline including 50 mm tris, 100 mm nacl, 1 mm edta, ph 7.4) gradient. two distinct types of ibv particles were isolated by sucrose density gradients. the higher density particles banded at 30%-40% sucrose-tne gradients while the less density particles banded at 20%-30% sucrose-tne gradients. the purity of ibv was confirmed by electron microscopy analysis following negative staining to ensure that the virions have normal viral morphology and to exclude the possible inclusions of vesicles, other cellular organelles and debris (fig. 1a ). an abundance of intact virions were observed without obvious contamination from host cellular materia. proteins in purified virions were separated on 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) and stained withc coomassie brilliant blue (fig. 1b ). there were also some lighter bands visible that may represent cellular proteins besides the conjectured major virus-encoded structural proteins. furthermore, the four virus-encoded structural proteins were confirmed by immunoblotting test (fig. 1c) . taken together, the best purification of the ibv was obtained after differential centrifugation to remove the cellular contamination and condensation through a 20% (wt/vol) sucrose cushion with a non-linear sucrose gradient. to obtain a detailed protein composition profile associated with the ibv particles, the proteins in purified ibv particles were extracted for 2-de experiments. to authenticate the results and to compensate the variability figure 1 analysis of purified avian infectious bronchitis virus preparations. a: specific pathogen free (spf) chick embryo-grown major particles h52 from 30%-40% sucrose density gradients, negatively stained with 2% potassium phosphotungstate, ph 6.5. b: sds-page separation of proteins in a purified h52 preparation. 8 μg of proteins were separated on an 5-17.5% polyacrylamide gel and stained with coomassie blue. c: western blotting of the purified h52 virions. viral proteins were separated on 12% polyacrylamide gel and analyzed by western blot with chicken polyclonal antibody against infectious bronchitis virus (massachusetts). the identified viral proteins are indicated. s: spike, n: nuclecapsid, m: mebrane, e: envelope. of gel electrophoresis, three independent experiments were performed with three replicate gels for each experiment. the viral protein profiles were analyzed by 2-d with 250 μg of protein. after the electrophoresis separation, gels were stained with silver and processed for image analysis. for ibv particle-associated proteins separated in the ph3-10 range, 88 protein spots were detected (fig. 2) . to identify the proteins associated with ibv particles, all protein spots detected in the gels were excised and in-gel digested with trypsin followed by maldi-tof/tof (matrix-assisted laser desorption/ionizationtime of flight mass spectrometry) analysis. database search analysis revealed that 2 virus-encoded structural proteins and 60 host proteins were successfully identified. detailed information of the full set of the identified proteins is listed in table 1 ; additional file 1. to better understand the host proteins incorporated with ibv virion and their roles played in ibv infection, these proteins were categorized with biological processes according to uniprot knowledge database (swiss-prof/ trembl) and gene ontology database. the identified 60 host proteins were comprised of cytoskeleton proteins, molecular chaperone, macromolecular biosynthesis proteins, signal transport proteins and glycolytic enzymes (table s1 ; additional file 1). these host proteins were located mainly in the cytoplasm, including cytoskeleton, cytosol and mitochondrion (fig. 3) . to confirm the presence of host proteins in the purified ibv particles after the identification of them by pro-teomic method, we performed immunoblotting experiments. ibv preparation purified from af was analyzed for the presence of n protein, actin, hsp90, annexin a2 and tubulin (fig. 4) . extracts from 10-day-old specific pathogen free (spf) embryonated chicken eggs were included as a positive control. when analyzing the results of virion proteomic studies, the challenge is to prove that the host proteins are really an part of the virion and that they are not just attached non-specifically to the outside of the virus. to address this question, the af from uninfected 10-day-old spf embryonated chicken eggs were parallelly subjected to our standard density centrifugation procedure and the protein extracts from 30%-40% sucrose gradient was used as negative control. gradientpurified virions and the control were separated on 12% sds-page gels, transferred to pvdf membranes, and probed with the appropriate antibodies. as shown in fig. 4 , actin, tubulin and annexin a2 were both found in the purified virions and positive control but not in the af extracts from uninfected 10-day-old spf embryonated chicken eggs. hsp90 is a member of the heat shock protein family which is upregulated in response to stress and has low abundance in unstressed cells. in present study, we detected it only in the purified virions but not in normal cells. it is an expectable result that we also detected actin and tubulin in the af extracts from uninfected 10day-old spf embryonated chicken eggs which resulted from their high concentrations in all eukaryotic cells and subcellular fractions. to provide additional evidence that the host proteins are not just derived from a microvesicle or exosome that co-purified with the virus, we used the bromelain protease protection assay which has been shown to efficiently remove microvesicles from ibv virion preparations [25] . protease treatment of the purified virus preparation strips proteins off any contaminating microvesicles and off the outside of virus particles, such as s protein. in doing so, the microvesicles become lighter than the virions and therefore the virions can be isolated by density centrifugation. proteins that are inside the virion are protected by the lipid envelope and therefore will remain after the protease treatment. and then immunogold labeling of the purified virions was performed ( fig. 5 ). virions were either mock treated or subjected to digestion with bromelain and then were incubated with antibodies against actin, annexin a2, hsp90, ibv massachusetts strain and secondary gold antibodies followed by negative staining. one or two gold particles located on the surface of a virion could be seen for hsp90. this was significantly less compared with the degree of other labelings which is consistent with the fact that there is most likely far more actin, annexin a2 present on the virions than hsp90. in addition, the abundance of actin detected in the 2-de gels is much higher than that of hsp90. virus exploits multiple host proteins during infection for successful entry, replication, egress, and evasion. this is especially true for rna viruses because they encode only little proteins. learning the protein composition profile of the infectious viral particle is prerequisite for studying the role of host proteins during infection. to our knowledge, incorporation of host proteins in the envelopedvirus family coronaviridae has not been investigated so far. in this study, we revealed the presence of virusencoded proteins in infectious bronchitis particles and for the first time confirmed the incorporation of host proteins. a total of 2 viral and 60 host proteins associated with purified ibv particles were identified. in the present study, we failed to obtain m and e protein while other two structural proteins n and s were identified successfully. n protein is easy to identify because it is the most abundant virus-derived protein produced throughout the process of the virus infection, whereas s protein as a major structural protein of ibv located on the surface of viral particles is also easy for identification because it is large (about 175 kda) and has many tryptic cleavage peak in the ms analysis. the identification of m and e protein of coronavirus by ms has been thought to be a difficult task due to their properties, especially in the case of e protein for the following reasons [26] . first, e protein is a very hydrophobic protein. second, it is low-abundant in the virions. third, e is a low molecular weight protein with the mass about 12 kda. fourth, e protein contains two cysteines, which may form disulfide bonds within itself or with other proteins and make e protein difficult to be reduced and subsequently digested. of the total host proteins, 39 have also been described to be present in virions of quite diverse virus families, such as herpesviruses, poxviruses, paramyxovirus and retroviruses [27, 28] . there are some explanations that the incorporated host proteins are common to other virus families. first, they are all enveloped viruses. enveloped viruses contain the viral genome and core proteins wrapped within one or more membranes which are acquired from the host cell during virus assembly and budding. these viruses all share some fundamental feature in the particular stage of their life-cycle and these host proteins are involved in the common process. second, these host proteins would be either highly abundant cytosolic proteins or enriched at the virus budding sites. several of the highly abundant cytosolic proteins found within both ibv and other viral particles are beta actin, tubulin, annexins and enolase. proteins enriched at the virus budding sites including hsp70, hsp90 and gapdh are also identified in ibv and other viruses [9, 11, 12, 14, 19, 24] . some host proteins may be specially incorporated into the virions. in this study, 21 of the total host proteins are reported for the first time. the identified host protein functions in diverse biological processes and some functional groups are analyzed. these proteins participate in a broad array of cellular functions and are involved in many processes in the viral life cycle. the potential roles of some of these proteins are discussed below in relation with ibv infection, pathogenesis and early host antiviral response. numerous viral proteins interact with cytoskeletal elements. many viruses, such as retroviruses, herpesviruses and picornaviruses, even contain the main cytoskeletal element actins in their infectious particles. the transport machinery of actins are proven to be critical at almost every step along the infectious cycle [29] . actin has been found in preparations from several types of retroviruses and paramyxoviruses. for coronavirus, an association of m with cytoskeletal elements has been reported [5] , which indicates an essential function of actin in the replication cycle of coronavirus ibv. in our studies, actin and tubulin were all present in the interior of infectious bronchitis particles and this observation most likely reflects their active participation in moving the viral components to assembly sites. actin and tubulin have been characterized as the major folding substrates for cct (chaperonin containing tcp-1, also termed tric). both cytoskeletal proteins require in vivo and in vitro the interaction with cct to fold to their native states [30] . cct is the most different and complicated protein of all group ii chaperonins in eukaryotic cytosolic chaperonins, which might be involved in the assistance of the folding of a small set of proteins. in addition to the already mentioned actin and tubulin, cct has been found to interact either in vitro or in vivo with other cytoskeletal proteins, cell division control protein 20, protein phosphatase type 2a, and guanine nucleotide-binding protein (g protein) beta subunit [31] , which are all found to be associated with infectious bronchitis particles in present study. it's pleasantly surprising to find that certain viral proteins such as the epstein barr virus-encoded nuclear protein (ebna-3), the hepatitis b virus capsid and the type d retrovirus gag polyprotein are also folded by cct [32] [33] [34] . thus, cct may have an important role in infectious bronchitis viral proteins assembly. other cytoskeletal proteins found to be associated with infectious bronchitis particles are actin-related proteins, wd repeat containing protein, destrin and annexin. several annexin family members (a2, a5 and a11) were identified in purified infectious bronchitis particles. annexins are a well-known multigene family of ca 2+ regulated phospholipid-binding and membrane binding proteins with diverse functions. the presence of annexin a2 is thought to support viral binding, fusion and replication [35] [36] [37] [38] [39] . annexin a5, which interacts with annexin a2, has the opposite effect by preventing fusion, which possibly indicates a potential regulatory role [38] . annexin a2 tightly binds to a member of the s100 family of calciumbinding proteins, s100a10 (p11). upon binding, annexin a2 and p11 form a heterotetramer which is capable of binding two membrane surfaces simultaneously, which potentially promotes fusion events and also plays a role in exocytosis [40] . the p11 protein was also detected by our analysis, suggesting that ibv is also incorporated this complex. other s100 family members such as s100a6 and s100a11 were also detected in viral samples and could play various roles in fusion and membrane organization [41, 42] . heat-shock proteins (hsps) have been known as multifunctional proteins. they facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. most, but not all, hsps are molecular chaperones. several viruses require host molecular chaperones for entry, replication, and assembly, as well as other steps in viral production [43, 44] . hsp70 and hsp90 have been found incorporated into ibv. hsp70 interacts with various viral proteins and may be involved in the assembly of adenovirus [45] , enterovirus [46] , vaccinia virus [47] and hantaan virus [48] . alternatively, upon entry into susceptible target cells, virion-associated hsp70 might participate in early events of infection. for example, hsp70 might actively uncoat the viral capsid in a manner similar to its role in the uncoating of clathrin cages [49] . hsp70 and hsp90 have been shown to interact with hepatitis b virus reverse transcriptase and to facilitate the initiation of viral dna synthesis from hepatitis b virus pregenomic rna [50, 51] . for sendai virus (sv), the viral proteins synethsis will be inhibited as long as hsp70 synthesis occurs [52] . thus, hsp70 in ibv virions might serve a similar function in the virus life cycle. the chaperone hsp90 has been identified as an essential factor in the folding and maturation of picornavirus capsid proteins [53] . the involvement of hsp90 in viral replication has also been reported for many viruses and it has been demonstrated that hsp90 inhibition blocks viral replication [54] . recently, a role for hsp90 in the control of hepatitis c, flock house and influenza virus polymerase function has been shown [55] [56] [57] [58] [59] [60] [61] and it has been proposed that hsp90 is a major host factor that is of central importance for viral replication for a wide spectrum of rna viruses [56] , which implies the crucial roles of hsp90 in ibv replication. the importance of hsp90 for the replication of multiple viruses opens up an interesting possibility for developing new antiviral therapies which have not yielded drug-resistant viruses [62] . some proteins involved in the glycolytic pathway were identified, such as aldehyde dehydrogenase 9 family, member a1 (aldh9a1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), alpha-enolase, which were identified in other viral particles, like hiv-1, mmlv, hcmv, kshv and aiv [8, 12, 19, 21, 24] . some studies have suggested that several glycolytic enzymes interact with microtubules and tubulin [63] [64] [65] and may also contribute to transcription of rna virus genomes. in higher eukaryotes, enolase is found as a dimer of subunits, α, β, or γ. all enolase isoforms from mammalian have been reported that are capable of stimulating transcription of svgenome [66] . gapdh is a well-characterized key enzyme in glycolysis, but recent evidence suggests it also has rna binding properties and binds to the untranslated rna sequences of several different viruses, including human parainfluenza virus type 3(hpiv3), japanese encephalitis virus (jev), hepatitis a virus (hav) and hepatitis b virus (hbv) as well as hepatitis c virus (hcv) [67] [68] [69] [70] [71] . in the case of hpiv3, gapdh has been reported to inhibit actin-dependent in vitro transcription and is also present in purified virions [67, 72] . in vitro data indicates that gapdh serves a negative regulatory role in hpiv3 transcription and in a phosphorylation-dependent on manner [72] . in addition to these host proteins associated with enveloped viruses, the roles of which in the virus life cycles have been studied well, we also identified 21 host proteins in purified infectious bronchitis particles, which have not been described to be present in other virions of quite diverse virus families, such as apolipoprotein a-i (apoa-i), fatty acid-bingding protein 3, ovalbumin, tenp protein, tumor protein translationally controlled-1, transthyretin and so on. apoa-i, a major constituent of highdensity lipoproteins, alters plasma membrane morphol-ogy by participating in the reverse transport of cholesterol binding with atp-binding cassette transporter a1 [73] , and activates the small gtp-binding protein cdc42associated signaling including apoa-i induced cholesterol efflux, protein kinases, and actin polymerization [74] . what important is that apo a-i can inhibit herpes simplex virus (hsv)-induced cell fusion at physiological concentrations. this function may be related to the structure of apoa-i and before long its amphipathic peptide analogue was also found to inhibit cell fusion, both in hiv-1infected t cells and in recombinant vaccinia-virusinfected cd4+ hela cells expressing hiv envelope protein on their surfaces [75] . the results indicate that amphipathic helices may be useful in designing novel antiviral agents that inhibit penetration and spreading of enveloped viruses. ovalbumin is the main protein found in egg white, making up 60-65% of the total protein. the chicken ovalbumin upstream promoter transcription factors (coup-tfs), members of the steroid/thyroid hormone receptor superfamily, binds to a negative regulatory region in the human immunodeficiency virus type 1 long terminal repeat (ltr). ltr contains a negative regulatory element which downregulates the rate of ltr-directed transcription and hiv-1 replication [76] . the interaction between ovalbumin and np from influenza a virus as well as glycoprotein c from the herpes simplex type i virus was reported long time ago [76] . the tenp protein from g. gallus, however, was isolated as a transiently expressed gene in neural precursor cells in retina and brain, and has been proposed to function in the transition to cell differentiation in neurogenesis. after expressed in chicken embryonic fibroblast cells, tenp was immunodetected in membrane fractions, implying that tenp might be a membrane protein as predicted by a computer analysis of its primary sequence [77] . to date, there have been no reports about tenp associated with virus, but it's an enriched and abundant protein identified in purified infectious bronchitis particles which suggests to us that it may be a requisite host protein in ibv life cycles. the present study 1) provides the first proteomic analysis of infectious bronchitis particles, 2) establishes the most comprehensive proteomic index of ibv and 3) shows that most of the virion incorporated host proteins have central roles in virus life cycle. although some proteins may be associated with virus biology, further investigation of the function of these host proteins may facilitate the understanding of the pathogenic mechanisms. the ibv strain h52 was obtained from qianyuanhao biological corporation limited (beijing, china). virus was propagated in 10-day-old specific pathogen free (spf) embryonated chicken eggs (beijing merial vital laboratory animal technology co, ltd, beijing, china) for 48 h at 37°c. the allantoic fluid (af) with enrichment of ibv h52 was clarified by differential centrifugation. af was first centrifugated at 3,000 × g for 30 min and then the supernatant was centrifugated at 12,000 × g for 30 min. clarification and all subsequent centrifugations were performed at 4°c. the virus was sedimented through 5.5 ml of 20% (wt/vol) sucrose in tne buffer (50 mm tris, 100 mm nacl, 1 mm edta, ph 7.4) by centrifugation in a 70ti rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge) at 75,000 × g for 1.5 h. condensed virions were then diluted with 1.0 ml tne buffer and centrifuged to equilibrium in 11.5 ml non-linear 20%-50% sucrose-tne gradients at 75,000 × g for 2.5 h in a sw41 rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at 75,000 × g for 1.5 h in a sw41 rotor to remove the sucrose. the purified ibv pellets were stored at -80°c until use. the purified ibv particles were dissolved in about 300 μl lysis buffer (7 m urea, 2 m thiourea, 2% triton x-100, 65 mm dtt, 2% biolyte ph 3-10) and incubated for 60 min at 4°c. then the lysis solution was sonicated for 4 min (pulse durations of 2 s on and 3 s off ) in an ice bath sonicator. the viral protein samples were prepared when the indiscerptible sediments were wiped off by centrifugation at 12,000 × g for 30 min. the supernatant was collected and the concentration of the prepared protein samples was determined by the bio-rad protein assay kit ii according to the manufacturer's instructions. the samples were then aliquoted and stored at -80°c until used for further analysis. purified virus particles treated with bromelain (bb0243, bbi) at 0.2 mg/ml in 50 mm dtt (ph 7.2) in dulbecco's phosphate buffered saline (pbs) at 37°c for 15 min. after incubation, the treated virus was directly centrifuged to equilibrium in 11.5 ml non-linear 20%-50% sucrose-tne gradients at 75,000 × g for 2.5 h in a sw41 rotor (beckman coulter, optima™ l-100xp preparative ultracentrifuge). purified virions were diluted with tne buffer and pelleted by sedimentation at 75,000 × g for 1.5 h in a sw41 rotor to remove the sucrose and then subjected to immunogold labeling and electron microscopy analysis. two-dimentional gel electrophoresis analysis was performed using 18 cm immobile drystrip (ipg strips, ph 3-10 non-linear, ge healthcare). first, 100 μl samples containing 250 μg protein were added into 400 μl sample rehydration buffer (7 m urea, 2 m thiourea, 2% (w/v) chaps, 65 mm dtt, 0.2% bio-lyte ph 3-10) and incubated for 30 min at 37°c prior to their separation by isoelectric focusing (ief) in the first dimension. the ipg strips were rehydrated at 20°c for 12 h by a passive rehydration method. ief was carried out for a total of 45 kvh at 20°c on an ettan ipgphor iii electrophoresis unit (ge healthcare). second, ipg strips were further transferred onto the second dimension of gel electrophoresis. before this step the ipg strips were reduced and alkylated in a equilibration buffer containing 50 mm tris-hcl, ph 8.6, 6 m urea, 2% sds and 30% glycerol supplemented with 1% (w/v) dl-dithiothreitol (dtt) or 2.5% iodoacetamide (iaa) instead of dtt for 15 min. subsequently, the viral protein samples were separated at 140 v on linear 5%-17.5% sodium dodecyl sulfate gradient polyacrylamide gel (sds-page) in tris: glycine buffer (192 mm glycine, 25 mm tris, 0.1% sds, ph 8.3) for about 10 h. third, proteins in the gel were stained by the modified silver staining method compatible with ms [78] and the gels were scanned at a resolution of 600 dpi using imagescanner™ iii (ge healthcare). gel pieces (1.0 mm 3 ) containing the whole protein spots from the 2d gel were cut and washed three times with 50 mm carbonic acid, monoammonium salt (nh 4 hco 3 , amresco). these gel pieces were destained with 15 mm potassium ferricyanide (k 3 fe(cn) 6 , amresco) and 50 mm sodium thiosulfate (nas 2 o 3 , amresco) in 50 mm nh 4 hco 3 and dehydrated in 100% acetonitrile (acn, wako) until gel pieces turn to white. after dring in speedvac concentrator (thermo savant, usa) for about 100 min, gel pieces were incubated with 12.5 ng/μl trypsin (sequenceing grade, promega) to cover dry gel pieces completely at 37°c overnight. the gel pieces were then extracted three times in 50% acn water solution containing 5% trifluoroacetic acid (tfa, wako). the supernatant was pooled and dried thoroughly in speed-vac. protein digestion extracts were resuspended with 5 μl of 0.1% tfa and then the peptide samples were mixed (1:1) with a matrix consisting of a saturated solution of αcyano-4-hydroxycinnamic acid (α-cca, sigma) in 50% acn containing 0.1% tfa. 0.8 μl aliquot was spotted onto stainless steel target plates. peptide mass spectra were obtained on an applied biosystem/mds sciex 4800 maldi tof/tof plus mass spectrometer. data were acquired in positive ms reflector using a calmix5 standard to calibrate the instrument (abi4800 calibration mixture). mass spectra were obtained from each sample spot by accumulation of 900 laser shots in an 800-3500 mass range. for ms/ms spectra, the 5-10 most abundant precursor ions per sample were selected for subsequent fragmentation and 1200 laser shots were accumulated per precursor ion. both the ms and ms/ms data were interpreted and processed by gps explorer software (v3.6, applied biosystems), then those obtained ms and ms/ms spectra per spot were combined and submitted to mascot search engine (v2.1, matrix science, london, u.k.) by gps explorer software and searched with the following parameters: trypsin as the digestion enzyme, one missed cleavage site, partial modification of cysteine carboamidomethylated and methionine oxidized, none fixed modifications, ms tolerance of 60 ppm, ms/ms tolerance of 0.25 da. mascot protein score in ipi_chicken (v3.49) database (based on combined ms and ms/ms spectra) of greater than 57 (p ≤ 0.05) or in ncbinr database of greater than 67 (p ≤ 0.05) was accepted. mouse monoclonal antibodies against actin (mab1501) and hsp90 (05-594) were purchased from millipore. rabbit polyclonal antibodies against annexin a2 (ab40943) and tubulin alpha-1 (ab4074), and chicken polyclonal antibody against ibv (massachusetts) (ab31671) were purchased from abcam. mouse monoclonal antibody against nucleoprotein of ibv (3bn1) was purchased from hytest ltd. for control, the af from 10-day-old spf embryonated chicken egg performed with the same protocol as the purification of ibv particles and the protein extracted from the normal 10-day-old spf embryonated chicken eggs included for western blot analysis. samples were separated at 120 v on linear 5%-17.5% sds-page with 5% stacking gels in tris: glycine buffer for about 3 h. for purified virus, 10 μg of total proteins were used per lane. for the control, a total of 15 μg proteins were loaded. after separated by sds-page, the proteins were transferred to a polyvinylidene fluoride membrane (pvdf, p/n 66485, biotrace, pall corporation). the membrane was blocked in freshly prepared 5% bovine serum albumin (bsa) with 0.05% tween-20 for 2 h at room temperature with constant agitation. the pvdf membrane was washed three times with tris buffered saline plus 0.2% tween 20 (tbst) and then incubated with properly diluted primary antibodies for 2 h at room temperature or overnight with agitation at 4°c. anti-rabbit or anti-mouse immunoglobulin g antibody conjugated to horseradish peroxidase (hrp) (00001-14, proteintech group, inc) was used as the secondary antibody and the pvdf membrane was incubated in it for 1 h at room temperature. the chemiluminescence system (ar1022, boster bio-technology co. ltd) was used for detection of antibody-antigen complexes. rabbit polyclonal antibody against chicken igg (15 nm gold) (ab41500), goat polyclonal against rabbit igg (5 nm gold) (ab27235) and goat polyclonal against mouse igg (10 nm gold) (ab27241) were purchased from abcam. purified ibv particles were suspended in pbs (ph 7.4) and then were collected onto 230-mesh formwar-coated nickel grids and adsorbed on the grids for 5 min. the viruses were fixed in 2% paraformaldehyde for 5 min at rt and treated with triton x-100 (0.2%) in pbs (ph 7.4) for 5 min and then blocked with 5% bsa in pbs-tween 20 (ph 7.4) for 30 min at rt. all grids were then blocked with blocking buffer (5% bsa, 5% normal serum, 0.1% cold water skin gelatin, 10 mm phosphate buffer, 150 mm nacl, ph 7.4) for 30 min. after washing with pbs, immobilized virions were incubated for 1.5 h with 50 μg/ml primary antibody (in 1% bsa), and washed three times for 5 min in pbs/1% bsa. anti-rabbit or anti-mouse immunoglobulin g coupled to 10 nm colloidal gold particles was used as the secondary antibody and virions were incubated in it for 40 min at room temperature. the grids were then washed extensively with pbs, washed twice more with distilled water to remove excess salt and negatively stained with 2% sodium phosphotungstate for 1 min. negatively stained virions were examined on a scan and transmission electron microscope. abbreviations 2d: two-dimensional; 2-de: two-dimensional electrophoresis; sds-page: sodium dodecylsulfate polyacrylamide gel electrophoresis; ms: mass spectrometry; maldi-tof: matrix-assisted laser desorption/ionization time of flight mass spectrometry; spf: specific pathogen free; af: allantoic fluid; bsa: bovine serum albumin; dtt: dithiothreitol; iaa: iodoacetamide; acn: acetonitrile; tfa: trifluoroacetic acid; α-cca: α-cyano-4-hydroxycinnamic acid; tne: tris-buffered saline including 50 mm tris; 100 mm nacl; 1 mm edta: ph 7.4; pbs: phosphatebuffered saline; tbs: tris-buffered saline; tbst: tris buffered saline plus 0.2% tween 20; hrp: horseradish peroxidase; pi: isoelectric point; mw: molecular weight. coronavirus avian infectious bronchitis virus envelope glycoprotein interactions in coronavirus assembly characterization of the coronavirus m protein and nucleocapsid interaction in infected cells coronavirus spike protein inhibits host cell translation by interaction with eif3f interaction of the coronavirus infectious bronchitis virus membrane protein with betaactin and its implication in virion assembly and budding induction of apoptosis in murine coronavirusinfected 17cl-1 cells induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer host and viral proteins in the virion of kaposi's sarcoma-associated herpesvirus virion proteins of kaposi's sarcomaassociated herpesvirus a mass spectrometry-based proteomic approach to study marek's disease virus gene expression proteins of purified epstein-barr virus identification of proteins in human 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intracellular and extracellular roles of s100 proteins s100-annexin complexes: some insights from structural studies recruitment of hsp70 chaperones: a crucial part of viral survival strategies synthesis and quality control of viral membrane proteins association of hsp70 with the adenovirus type 5 fiber protein in infected hep-2 cells association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells vaccinia virus infection induces a stress response that leads to association of hsp70 with viral proteins increased expression of hsp70 and co-localization with nuclear protein in cells infected with the hantaan virus uncoating atpase is a member of the 70 kilodalton family of stress proteins hsp90 is required for the activity of a hepatitis b virus reverse transcriptase two-dimensional blue native/sds-page analysis reveals heat shock protein chaperone machinery involved in hepatitis b virus production in hepg2.2.15 cells selective inhibition of virus protein synthesis by prostaglandin a1: a translational block associated with hsp70 synthesis evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is important for vaccinia virus growth in cells hsp90 inhibitors suppress hcv replication in replicon cells and humanized liver mice hepatitis c virus rna replication is regulated by fkbp8 and hsp90 the cellular chaperone heat shock protein 90 facilitates flock house virus rna replication in drosophila cells identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus development and application of hsp90 inhibitors enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules a glycolytic enzyme binding domain on tubulin glycolytic enzyme-tubulin interactions: role of tubulin carboxy terminals enolase, a cellular glycolytic enzyme, is required for efficient transcription of sendai virus genome specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and la protein with cis-acting rnas of human parainfluenza virus type 3 human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(u) tract of the 3' noncoding region of hepatitis c virus genomic rna identification of glyceraldehyde-3-phosphate dehydrogenase as a cellular protein that binds to the hepatitis b virus posttranscriptional regulatory element glyceraldehyde-3-phosphate dehydrogenase (gapdh) interaction with 3' ends of japanese encephalitis virus rna and colocalization with the viral ns5 protein functional significance of the interaction of hepatitis a virus rna with glyceraldehyde 3-phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro specific binding of apoa-i, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing abc1 apolipoprotein a-i activates cdc42 signaling through the abca1 transporter apolipoprotein a-i and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation immunoprecipitation, with an antiserum to ovalbumin, of protein np from influenza a virus and of glycoprotein c from the herpes simplex type i virus identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis a modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry proteomic analysis of purified coronavirus infectious bronchitis virus particles proteome science the authors declare that they have no competing interests. qk performed the main proteomic experiments and data analysis and drafted the manuscript. cx created the detailed experimental design. xr and cz contributed to the initial phase of the proteomic experiments. ll and ds assisted in the propagation and purification of ibv. yb and yc helped conceive the research. all authors read and approved the final manuscript. key: cord-272437-gvzfl8c3 authors: zhao, jing; zhang, keran; cheng, jinlong; jia, wenfeng; zhao, ye; zhang, guozhong title: replicase 1a gene plays a critical role in pathogenesis of avian coronavirus infectious bronchitis virus date: 2020-08-20 journal: virology doi: 10.1016/j.virol.2020.08.009 sha: doc_id: 272437 cord_uid: gvzfl8c3 avian coronavirus infectious bronchitis virus (ibv) is an important pathogen threatening poultry production worldwide. here, two recombinant ibvs (ryn-1a-ayn and ryn-1b-ayn) were generated in which orf1a or orf1b of the virulent yn genome were replaced by the corresponding regions from the attenuated strain ayn. the pathogenicity and virulence of ribvs were evaluated in ovo and in vivo. the results revealed that mutations in the orf1a gene during passage in embryonated eggs caused the decreased pathogenicity of virulent ibv yn strain, proven by determination of virus replication in eces and cek cells, the observation of clinical signs, gross lesions, microscopic lesions, tracheal ciliary activity and virus distribution in chickens following exposure to ribvs. however, mutations in orf1b had no obvious effect on virus replication in both eces and cek cells, or pathogenicity in chickens. our findings demonstrate that the replicase 1a gene of avian coronavirus ibv is a determinant of pathogenicity. . those of the ryn group at 5 and 7 dpi (p < 0.01 or 0.001) (fig. 4b ). in the lungs, the 264 virus copy numbers in the ryn-1a-ayn infected group were also significantly lower 265 than those of the ryn group at 7 and 10 dpi (p < 0.01 or 0.001) (fig. 4c) . the numbers of viral rna copies in the kidneys of the ryn-1a-ayn infected group were 267 lower than in the ryn group at 10 dpi (p < 0.001) (fig. 4d ). there were no orf1a limited viral replication in chicken tissues. ibv is a highly infectious pathogen in domestic fowl that replicates mainly in the this may indicate that avirulent strain beaudette was not the ideal background in 296 which to study the role of ibv-specific mutations in pathogenicity. we suspect that 297 mutations in the replicase gene may play an important role in attenuation of ibv. in this study, we used an ibv reverse genetics system based on virulent strain fig. 2. growth characteristics of ibv ryn, ryn-1a-ayn or ryn-1b-ayn, and 466 clinical scores and survival rates of chickens inoculated with these ibv strains. ibv ryn, ryn-1a-ayn and ryn-1b-ayn strains. statistical significance was considered as follows: highly significant at p < 0.01 (**) 482 and extremely significant at p < 0.001 (***). is critical for viral virulence infectious bronchitis virus variants: a 425 review of the history, current situation and control measures chapter three -the nonstructural proteins directing 427 coronavirus rna synthesis and processing recombinant live attenuated avian coronavirus 431 vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious 432 bronchitis in chickens coronavirus pathogenesis attenuation, safety, and efficacy of a qx-like infectious bronchitis virus serotype 445 vaccine porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, 447 prevalence and genetic evolution attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine successful establishment of a reverse 452 genetic system for qx-type infectious bronchitis virus and technical improvement of the 453 rescue procedure s gene and 5a accessory gene 455 are responsible for the attenuation of virulent infectious bronchitis coronavirus coronavirus replicative proteins virus-encoded proteinases and proteolytic 460 processing in the nidovirales key: cord-276550-1in7m56w authors: abdel-moneim, ahmed s; el-kady, magdy f; ladman, brian s; gelb, jack title: s1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt date: 2006-09-20 journal: virol j doi: 10.1186/1743-422x-3-78 sha: doc_id: 276550 cord_uid: 1in7m56w background: infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. ibv is endemic in probably all countries that raise chickens. it exists as dozens of serotypes/genotypes. only a few amino acid differences in the s1 protein of vaccine and challenge strains of ibv may result in poor protection. tropism of ibv includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. results: infectious bronchitis virus (ibv) strain closely related to massachusetts (mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. the isolate was serologically identified by dot-elisa and further characterized by rt-pcr then genotyped using s1 gene sequence analysis. alignment of the s1 sequence of the isolate with 16 ibv strains revealed high homology to isolates related to mass serotype. inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. a protection study using the h120 live attenuated vaccine showed low protection rate in spite of high s1 sequence homology (97%). protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as ibv antigens detection by immunofluorescent antibody technique in kidney sections. conclusion: periodical evaluation of cross-protective capabilities of ibv vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of ibv. avian infectious bronchitis virus (ibv) is a highly contagious pathogen of chickens that replicates primarily in the respiratory tract and also in some epithelial cells of the gut, kidney and oviduct [1] . ibv is a virus member of genus coronavirus, family coronaviridae, order nidovirales [2] . the virus possesses a positive stranded rna genome that encodes phosphorylated nucleocapsid pro-tein (n), membrane glycoprotein (m), spike glycoprotein (s) and small membrane protein (e). the spike glycoprotein is post-translationally cleaved into two subunits, s1 and s2 [1, 3] . the s1 protein forms the n-terminal portion of the peplomer and contains antigenic epitopes mainly within three hvrs [4] [5] [6] . neutralizing and serotype specific epitopes are associated within the defined hvrs [4, 7, 8] . variation in s1 sequences [9] [10] [11] , has been recently used for distinguishing between different ibv serotypes. diversity in s1 probably results from mutation, recombination and strong positive selection in vivo [12] . antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. the generation of genetic variants is thought to be resulted from few amino acid changes in the spike (s) glycoprotein of ibv [13, 14] . in egypt, isolates related to massachusetts, d3128, d274, d-08880, 4/91 and the novel genotype; egypt/beni-suef/ 01 were isolated from different poultry farms [15] [16] [17] [18] . the commonly used ibv attenuated vaccine is h120 while the mass 41 (m41) strain is commonly used in inactivated vaccines. in the present study, egypt/f/03 was isolated from 25day-old broiler chickens in fayoum governorate, identified by dot-elisa, rt-pcr and sequenced to determine its serotype. pathogenicity test to 1-day-old chickens and protection afforded by the commonly used h120 live attenuated vaccine were also performed. the allantoic fluid of the first chicken embryo passage of egypt/f/03 was harvested at 48 h pi. four additional egg passages were performed. five eggs of the 4 th passage were incubated till being 18-day-old and all of them (100%) showed typical lesions of the ibv (stunting and dwarfing). the virus identity was ascertained by performing dot-elisa on the cam homogenate (fig. 1) . polymerase chain reaction and s1 gene cycle sequencing rt-pcr of egypt/f/03 resulted in a product of 1600 base pairs using s1 primers oligo 5' and oligo 3'. egypt/f/ 03 is closely related to the beaudette us reference strain; 98% nucleotide identity and 96% amino acid identity (table 1 and fig. 2 ). it showed 97% similarities both in nucleotides and amino acids to h120 and 98% nucleotide and 96% amino acid homology to m41 (table 1) . egypt/ f/03 showed 34 point mutations from h120; 20 silent and 14 non silent mutations. on the other hand, it showed 30 point mutations; 10 silent and 20 non silent mutations from m41 (fig. 3, 4) . sixteen potential glycosylation sites were found in egypt/f/03 while 17 were found in h120 and m41 (fig. 4) . all potential glycosylation sites found in egypt/f/03 were shared with those found in h120 and m41 (fig. 4) . chickens inoculated with egypt/f/03 exhibited snicking and rales in approximately 50% of infected birds at 3 rd day of inoculation. conjunctivitis was observed in 20/30 at 3 rd day pi that was elevated to 22/30 by the 5 th day pi of infected birds whereas no birds exhibited watery eyes. six birds were dead after egypt/f/03 experimental infection; 4 birds in the 7 th day pi and 2 birds in the 8 th day pi. postmortem examination of dead birds, revealed petechial haemorrhages in larynx and thymus, severe congestion of liver, spleen and lungs as well as renal haemorrhages. these changes appeared but in milder form in birds sacrificed at 5 days pi. histopathological examination of sacrificed birds at 5 days pi and freshly dead birds at 7 days pi revealed mucus, marked loss of cilia, desquamation, mononuclear infiltration, epithelial hyperplasia and vascular congestion of the trachea (fig. 5 ). kidneys showed severe changes including haemorrhages, degenerative changes in renal tubules and hypercellularity of the renal glomeruli as well as focal lymphocytic infiltration (fig. 5) . in general the tracheal and renal histopathological lesions were more severe in dead birds (fig. 5b , d) than birds sacrificed at 5 days pi (fig. 5a, c) . chickens vaccinated with h120 (group a) showed 58.3% protection (7/12) by virus reisolation procedure and 66.6% (8/12) protection by histopathology after challenge with egypt\f/03 while all control unvaccinated birds (group b) were not protected (5/5) ( table 2) . on day four pi, the kidneys of (3/12) birds in group a (vaccinated and challenged with ibv) showed focal lymphocytic infiltration, urates deposition and degenerative changes in renal tubules while birds of group b (unvaccinated and challenged with ibv) showed multifocal lymphocytic infiltration, urates deposition and degenerative changes in renal tubules in 4/5 birds, while only focal lymphocytic infiltration, urates deposition and degenera-dot-elisa shows positive reaction in tested (chorioallantoic membrane homogenate) and control positive sample control +v e tested sample control -ve tive changes in renal tubules were observed in 1/5 birds. birds in group c (vaccinated unchallenged group) did not show any abnormalities (0/12) either in tracheae or kidneys. immunofluorescenct assay on kidneys of challenged and unchallenged groups showed that all challenged unvaccinated and 3/12 of challenged vaccinated groups possessed kidney immunofluorescence. none of control unchallenged groups possessed kidney immunofluorescence (table 2 ). in this study, an egyptian ibv strain; egypt\f/03 was isolated from a tissue pool of kidney and trachea from unvaccinated broiler flock with a history of respiratory and renal disease. the strain produced typical lesions of ibv in inoculated embryos and identified as ibv by dot-elisa and rt-pcr. the isolate was found to be devoid of major concomitant viruses; avian influenza virus, newcastle disease virus, infectious laryngotracheitis virus, reovirus and adenovirus (data not shown). s1 sequence analysis of egypt/f/03 revealed its close relatedness to mass serotype. it showed high nucleotide similarities to gx1-98.china (99% nucleotide and amino acid identities), beaudette-us (98% nucleotide and 96% amino acid identities), is/385/97 (98% nucleotide and 97% amino acid identities), h120 (97% nucleotide and amino acid identities) and m41 (98% nucleotide and 96% amino acid identities). ibv s1 gene sequence relationships expressed as a phyloge-netic tree of egypt/f/03 isolate and selected ibv reference strains figure 2 ibv s1 gene sequence relationships expressed as a phylogenetic tree of egypt/f/03 isolate and selected ibv reference strains. it is known that the most severe clinical response of ibv appears in very young chickens and severity is alleviated in older chickens [19, 20] . this fact explains the high mortality rate observed in 1-day-old chickens that experimentally inoculated with egypt/f/03 compared to mortality pattern in the original flock (25-day-old chickens). the presence of acute interstitial nephritis on days 5 and 7 post infection indicated that egypt\f� is a nephrogenic ibv. the microscopic findings of the renal tubules matched the general findings recorded with nephrogenic ibv strains [21, 22] . the microscopic findings in tracheal sections appeared similar to those recorded by [19, 21] including: loss of cilia, degenerative changes of the tracheal mucosa, irregular loss of epithelium, desquamation of the sloughed epithelium in the tracheal lumen and lymphocytic infiltration that ranged from focal aggregation to diffuse massive infiltration. severe renal haemorrhages observed grossly and in hisopathological sections of birds dead after experimental infection with egypt/f/03 denote that deaths resulted from acute renal failure. our finding regarding the presence of petechial haemorrhages in larynx and thymus as well as severe congestion of liver, spleen and lungs in birds dead after ibv experimental infection is in agreement with [23, 24] who confirmed the presence of ibv viral antigens in such organs. evaluation of the immune response to ibv vaccination is based on several criteria including: clinical signs, tracheal nucleotides identities of egypt/f/03 with commonly used vaccine strains sequences figure 3 nucleotides identities of egypt/f/03 with commonly used vaccine strains sequences. dots indicate residues identical to egypt/f/03. bold letters denotes codon areas. shaded letters denote sites of differences. histological lesion, virus neutralization, virus re-isolation from trachea, antigen detection in trachea and/or kidney by immunofluorescent or immunoperoxidase techniques [25] [26] [27] [28] [29] . in this study we used tracheal histological lesion and virus re-isolation as parameters for tracheal protection. kidney histopathology and antigen detection by ifa were used as indicators of kidney protection. complete protection is expected upon using closely related vaccine strain as the degree of cross protection among ibv strains generally reflects the similarities between the s proteins [12, 30] . the re-isolation of egypt/f/03 from the trachea of vaccinated birds and the presence of tracheal and renal microscopic lesions as well as viral antigen in kidneys (by ifa) in h120 vaccinated birds denote lack of complete protection afforded by h120 vaccination. h120 is a mild vaccine and it is possible that the challenge virus was too virulent for the level of immunity that the vaccine produced in these young chickens. other possible consideration includes that baby chickens are not fully immunocompetent at one-day of age, the time that they were vaccinated for the protection study experiment. however, commercial broiler chickens possess maternally derived antibodies, are routinely vaccinated at one-day of age [31, 32] without apparent interference by the maternal derived antibodies in the development of active immunity, at least in the respiratory tract that measured by challenge [33] . on the other hand, variable results were recorded regarding homologous protection of ibv. cavanagh et al. [12] inoculated groups of 10 chickens with the virulent uk/6/82 isolate and challenged with isolates that differed by up to 4% of s1 amino acids. challenge with two variants (98% s1 identity with uk/6/82) resulted in challenge scores virtually the same as with the homologous challenge however, challenge with two others iso-lates (96% and 98% s1 identity, respectively), resulted in less cross-protection, although the numbers were not statistically significantly different. in the s1 subunit, three hvrs are located within amino acids 38-67, 91-141 and 274-387 [4] [5] [6] . hvr1 and hvr2 contain sequences that have been associated with specific ibv serotypes [34, 35] as well as serotype specific neutralizing epitopes [4, 5, 14] . ibv serotypes commonly differ by 20 to 25% in s1 [11, 36] but some serotypes differ in s1 by as little as 2% [13] . although h120 showed 97% amino acid and nucleotide identity to egypt/f/03, it possesses 34 different nucleotides that resulted in 14 amino acid substitutions. among such amino acids, one is located in hvr1, four in hvr2 and one in hvr3 (fig.4) . the region between amino acid residues 123-152 has been previously identified as a possible region involved in the differing pathogenicity of gray and non-virulent jmk strains [37] . egypt/f/03 possesses different amino acid; phenylalanine within this region at positions 130 and 141 instead of serine and leucine in h120 respectively. it is apt to mention that some serotypes differ in s1 by as little as 10 amino acids [13] , suggesting that only a few epitopes may induce most of the vn antibody [38] . egypt/f/03 is a nephropathogenic ibv strain closely related to mass serotype. vaccination by h120 did not provide satisfactory protection against challenge with egypt/ protective capabilities of such vaccine(s) versus recently recovered field isolates in order to ensure optimum control of ibv. embryonated chicken eggs spf ece obtained from nile spf (koom oshiem, fayoum, egypt) were used for isolation of the field isolate, serial passages, titration of the seed stocks of egypt/f/03 and vaccine strain (h120), as well as virus re-isolation attempts following challenge in the protection study. sixty nine commercial 1-day-old chickens (el-waddi co, egypt) were reared under strict hygienic conditions in sep-arate rooms and used in both virulence test and protection study. rabbit anti-ibv polyclonal antiserum raised against vero adapted h120 vaccine was prepared previously in our lab [17] and used for detection of ibv antigens in both dot-elisa and indirect immunofluorescent antibody technique. infectious bronchitis was diagnosed during augest 2003 in fayoum governorate, egypt. the outbreak occurred in 25-day-old commercial broiler farm with no previous ibv vaccination. the flock was vaccinated against newcastle disease and infectious bursal disease viruses at 14 and 18 days of age respectively. the total flock density was 3000 birds. the first signs were depression and respiratory distresses including sneezing, coughing and rales. other signs included conjunctivitis and watery eyes. within a period of 10 days after the appearance of the disease, the mortality rate increased to 10% of the flock density. postmortem examination of dead birds revealed increased tracheal mucus, severe renal congestion, urates filled ureters as well as congestion in liver and spleen. egypt/f/03 was isolated from 25-day-old broiler chickens suffering from both respiratory and renal distresses from fayoum governorate in 2003. a kidney homogenate (10% in sterile pbs) and a tracheal scraping suspension were pooled, centrifuged at 500 × g for 10 min. the supernatant fluid was inoculated into chorioallantoic sac of 10day-old spf ece. allantoic fluid was harvested after 48 h and was used for re-passage into ece. five eggs of the 4 th egg passage were incubated till being 18-day-old and examined for typical lesions of ibv (stunting, curling and urates deposition in ureters). a dot-elisa was performed according to [39] . briefly, ncm of convenient size was cut, marked with waterproof ink for identification and then soaked for 10 min. in distilled water. ncm was laid on absorbent paper and airdried for 5 min. approximately 2 μl of the extracted viral rna was used to synthesize cdna. amplification of the s1 gene was performed using s1oligo3' (5'-cataactaacataag-ggcaa-3') and s1oligo5' (5'-tgaaactgaacaaaagac-3') primers [10, 37] . pcr of s1 gene was performed as described [11] with the exception that extension was performed at 60°c. s1 pcr product was cut from 1.8% agarose gels, purified with the qiaquick gel extraction kit (qiagen, inc.) and the dna was quantitated as described [11] . purified rt-pcr product was sequenced in the forward and reverse directions using the same primers. sequencing was performed as described [11] . a blast ® analysis [40] was initially performed using the s1 sequence of egypt/f/03 (dq487085) to establish its identity to genbank accessions. a comparative analysis of s1 sequences was performed using the clustal w multiple sequence alignment program, version 1.83 [41] . the tree was constructed using the neighbour-joining program [41] . ibv s1 sequences representative to genotypes used for the alignments were obtained from the genbank and embl database. forty 1-day-old chickens were used. thirty chickens were infected by intraocular instillation of 10 5 eid 50 /100 μl of infectious bronchitis virus taxonomy coronaviruses in poultry and other birds amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus analysis of the serotypespecific epitopes of avian 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immunity against infectious bronchitis virus diagnosticmethods in clinical virology egypt/f/03 according to [21] while other birds were kept as control uninfected group. clinical signs and gross postmortem lesions as well as mortalities were recorded. microscopic examinations of both tracheae and kidneys were performed at 5 and 7 days post infection. twenty nine commercial 1-day-old chickens were used to evaluate the protection provided by h120 vaccination against challenge with egypt/f/03. birds were divided into three groups; a (n = 12), b (n = 5), c (n = 12). vaccination was performed at day 1 by eye drop application. single dose of h120 vaccine (nobilis, intervet, the netherlands bv) was used for each bird in groups a and c according to manufacturer's instructions while birds in group b were kept as unvaccinated control. four weeks post vaccination, chickens in group a and b were challenged by eye drop with egypt/f/03 (10 5 eid 50 per bird) while birds in group c were not challenged and kept as vaccinated unchallenged control. tracheae of all birds from all groups were collected four days post challenge for virus reisolation attempts and histopathological examination. tracheal scrapings were emulsified in 2 ml of sterile pbs and centrifuged at 500 × g for 3 min. virus reisolation attempts were performed by inoculating 2-3, 10-day-old spf ece by the supernatant fluid of each sample as described [44] . embryos were examined for typical lesions of ibv. for histopathological examination, tracheae were fixed in formalin, processed routinely for histopathology and stained with haematoxylin and eosin. the trachea from each bird was examined microscopically and assigned lesion scores of 0-3 with 0 = none, 1 = focal, 2 = multifocal, 3 = diffuse. tracheae were scored for the amount of mucous, loss of cilia, epithelial hyperplasia, necrosis, lymphocyte and heterophil infiltrations as well as the extent of tissue reaction. the scores for each bird were added and the mean score for the birds in each group was calculated [45] . kidney samples were also taken 4 days post challenge and examined microscopically for tubular degeneration and inflammation consistent with interstitial nephritis. focal, multifocal and diffuse were used to assign kidney histopathology. the presence of viral antigens in kidneys was screened by immunofluorescent antibody technique. it was performed according to [46] to detect viral antigens in the kidneys of birds after challenge with egypt/f/03 in protection study. briefly, deparaffinized slides were incubated with rabbit anti-ibv antibodies (1:5) for 1 h and subsequently with fitc-conjugated goat anti-rabbit antibody (kirkegaard and perry laboratories, gaithersburg, md.) (1:1000) for 1 h. both primary and secondary antibodies were diluted in pbs. slides were rinsed three times (10min./single wash) with pbs after each step. slides were then mounted using glycerol/pbs (without allowing the slides to dry) and examined under fluorescent microscopy.abbreviations cam, chorioallantoic membrane; ece, embryonated chicken eggs; eid, egg infective dose fifty; fitc, fluorescien isothiocynate; hvr, hypervariable region; ibv, infectious bronchitis virus; ncm, nitrocellulose membrane; pbs, phosphate buffer saline; rt, reverse transcriptase; spf, specific-pathogen-free. the author(s) declare that they have no competing interests. asa isolated and serologically characterized egypt/f/03 virus and performed virulence test as well as protection study. he also performed multisequence alignment, phylogenetic analysis and drafted the manuscript, mfe provided sample for isolation, helped in performing virulence test and protection study and reviewed the manuscript, jgjr helped in performing rt-pcr, s1 gene sequence of egypt/f/03 and critically reviewed the manuscript, bsl made rt-pcr and s1 gene sequence of egypt/ f/03. key: cord-266585-jfjrk9gy authors: fang, shouguo; chen, bo; tay, felicia p.l.; ng, beng sern; liu, ding xing title: an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells date: 2007-02-05 journal: virology doi: 10.1016/j.virol.2006.08.020 sha: doc_id: 266585 cord_uid: jfjrk9gy genetic manipulation of the rna genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of rna viruses. during construction of an infectious clone from a vero cell-adapted coronavirus infectious bronchitis virus (ibv), we found that a g–c point mutation at nucleotide position 15526, causing arg-to-pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of ibv on vero cells. when the in vitro-synthesized full-length transcripts containing this mutation were introduced into vero cells, no infectious virus was rescued. upon correction of the mutation, infectious virus was recovered. further characterization of the in vitro-synthesized full-length transcripts containing the g15526c mutation demonstrated that this mutation may block the transcription of subgenomic rnas. substitution mutation of the arg132 residue to a positively charged amino acid lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg132 residue to leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. on the contrary, a g–c and a g–a point mutations at nucleotide positions 4330 and 9230, respectively, causing glu–gln and gly–glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses. coronaviruses cause severe diseases affecting human and other animal species. in 2003, a novel coronavirus (sars-cov) was identified as the causative agent of severe acute respiratory syndrome (sars) (marra et al., 2003; rota et al., 2003) . the potential risk to public health posed by sars-cov and other coronaviruses and the lack of specific antiviral agents and vaccines have triggered a global research effort to characterize this family of viruses at the molecular and cellular levels. major advances in studies of the biological functions of individual viral proteins and replication mechanisms are currently being made by genetic manipulation of coronaviral genomes using reverse genetics and targeted recombination approaches (almazan et al., 2000; casais et al., 2001 casais et al., , 2003 casais et al., , 2005 coley et al., 2005; hodgson et al., 2006; koetzner et al., 1992; masters and rottier, 2005; sanchez et al., 1999; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 yount et al., , 2003 yount et al., , 2005 . avian infectious bronchitis virus (ibv), a group 3 coronavirus, causes an acute and contagious disease in chickens with a significant impact on the poultry industry worldwide. ibv contains a 27.6 kb single-stranded, positive-sense rna genome. in the virus-infected cells, six mrna species, including the genome-length mrna 1 and five subgenomic mrnas (mrna 2-6), are produced by a discontinuous rna transcription mechanism. each mrna species possesses a 64 nucleotides leader sequence derived from the 5′ end of the virology 358 (2007) 136 -147 www.elsevier.com/locate/yviro genome (boursnell et al., 1987) . subgenomic mrnas 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (s), envelope protein (e), membrane protein (m), and nucleocapsid protein (n). the 5′ two-third region of mrna 1 comprises two large orfs, 1a and 1b, and encodes two polyproteins. the two polyproteins are proteolytically cleaved by two virus-encoded proteinases, the papain-like and 3c-like proteinases, into 15 functional proteins (nsp2-nsp16) liu, 1998a, 1998b; lim et al., 2000; liu et al., 1995 liu et al., , 1997 liu et al., , 1998 ng and liu, 1998 xu et al., 2001) . compared to other coronaviruses, nsp1 is absent in ibv but nsp2 is considerably larger liu, 1998a, 1998b; liu et al., 1995) . in general, ibv shares close similarities in the genome organization, gene expression, and rna replication with other coronaviruses but is non-infectious to human. these properties make ibv an attractive model system for studying the biology and pathogenesis of coronavirus. in construction of an infectious ibv clone by in vitro assembly of five cloned rt-pcr fragments from a vero celladapted ibv beaudette strain, a g-c (g15526c) point mutation at nucleotide position 15,526 was found to be lethal to the infectivity of ibv on vero cells. no infectious virus could be rescued from vero cells electroporated with in vitro-synthesized full-length transcripts containing this mutation. as this mutation causes arg132-pro mutation in a domain with unknown function within the helicase protein (nsp13), it implies that this region might play certain roles in the functionality of the helicase protein. on the contrary, a g-c (g4330c) and a g-a (g9230) point mutations at nucleotide positions 4330 and 9230, respectively, causing glu-gln and gly-glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) proteinases, did not impair the infectivity of the in vitrosynthesized transcripts containing these mutations. further characterization of the in vitro-synthesized full-length transcripts containing the g15526c mutation demonstrated that this mutation blocks the transcription of subgenomic rnas. substitution mutation of the arg132 residue to a positively charged amino acid (lys) affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg132 residue to a leu, which is conserved in most of other coronaviruses at the same position, reduced the viral recovery rate of the in vitrofig. 1 . in vitro assembly of full-length cdna clone derived from a vero cell-adapted ibv beaudette strain. (a) diagram of the genome organization of ibv. the regions coding for the replicase polyproteins, the structural proteins s, e, m, and n, the accessory proteins 3a, 3b, 5a, and 5b, and the 5′-and 3′-utr are shown. also shown are the regions of the five rt-pcr fragments, the t7 promoter at the 5′-end of fragment a, and the 30 as at the 3′-end of fragment e. (b) preparation of the five cdna fragments, assembly of the five fragments into a full-length cdna clone, and in vitro transcription of the full-length transcripts. the five cdna fragments covering ibv sequences from nucleotides 1-5751 (lane 2), 5752-8693 (lane 3), 8694-15,520 (lane 4), 15,521-20,900 (lane 5) , and 20,901-27,608 (lane 6), respectively, were obtained by digestion of corresponding plasmid dna with either bsmbi or bsai, purified from agarose gel, and analyzed on a 0.8% agarose gel (lanes 2-6). equal amounts of the purified fragments were ligated using t4 dna ligase (lane 7) and analyzed on a 0.4% agarose gel. the in vitro assembled full-length cdna was used as templates for generation of the full-length in vitro transcripts, which were analyzed on a 0.8% agarose gel (lane 10). lanes 1, 7, and 9 show dna markers, and numbers on the left indicate nucleotides in kilobases. synthesized full-length transcripts. the mutant virus showed much smaller-sized plaques. this study reveals the essential role of a domain with previously unassigned functions within the helicase protein in coronavirus replication. construction of a full-length cdna clone derived from a vero cell-adapted ibv beaudette strain by in vitro assembly of five rt-pcr fragments to construct a full-length ibv clone, five fragments (a to e) spanning the entire ibv genome were obtained by rt-pcr of total rna extracted from vero cells infected with a vero cell-adapted ibv beaudette strain (p65) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) . to facilitate the assembly of the full-length cdna in vitro, restriction sites for either bsmbi or bsai were introduced into both the 5′ and 3′ ends of the fragments (fig. 1a) . in fragment a, a 19-nucleotide sequence corresponding to the t7 rna promoter (table 1) was inserted into the 5′ end of the ibv genome to facilitate in vitro transcription using the t7 polymerase (fig. 1a) . the primers used to amplify these fragments are listed in table 1 . the pcr products were purified from agarose gel and cloned into either pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. for the convenience of digestion using the restriction enzyme bsmbi, the nhei-and ecori-digested fragment a was subcloned into pkt0 which contains a bsmbi site 400 bp upstream of the t7 promoter sequence (fig. 1a) . two to three independent clones for each construct were selected for sequencing. the complete sequences of the five fragments, determined by automated nucleotide sequencing, are summarized in table 2 . the five fragments were then prepared by digestion of the corresponding constructs with either bsmbi or bsai and purified (fig. 1b, lanes 2-6) . the full-length clone was made by ligation of the purified fragments in vitro (fig. 1b, lane 8) and used as the template for in vitro transcription. the fulllength in vitro-synthesized transcripts were generated using the mmessage mmachine t7 kit (ambion, austin, tex) (fig. 1b , lane 10). as coronavirus n gene transcripts were shown to enhance the recovery of the rescued virus from the in vitrosynthesized full-length transcripts (casais et al., 2001; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 , the n transcripts were generated from a linearized pkt0-ibvn construct containing ibv n gene and the 3′-utr region. the full-length transcripts together with the n transcripts were introduced into vero cells by electroporation. however, it was consistently observed that no infectious virus could be recovered from cells transfected with the full-length transcripts together with the n transcripts. sequencing comparison of the five fragments with the vero cell-adapted ibv strain (p65, accession no. dq001339) shows nucleotide changes at 16 positions (table 2 ). among them, 11 a unique mutations found only in the cloned fragments. b same as the published sequences but different from p65. caused unique amino acid changes (table 2) . to assess the deleterious effects of these mutations on the infectivity of the full-length clone, correction of some of the mutations was carried out by site-directed mutagenesis. three point mutations, g4330c, g9230a, and g15526c, were chosen based on the fact that they are located either in or near the catalytic centers of the two viral proteinases or in a region of the rna helicase protein with undefined functions. four full-length cdna clones either with correction of the mutations at all three positions (ribv) or combination of two positions (e.g., g4330c containing g-c mutation at nucleotide position 4330 but without mutations at the other two positions) were constructed. rna transcripts generated from the four full-length cdna clones were introduced into vero cells together with the n transcripts by electroporation. at 2 days post-electroporation, a typical cpe of the vero cell-adapted ibv, the formation of giant syncytial cells (fang et al., 2005) , was observed in cells transfected with transcripts generated from cdna clones ribv, g4330c, and g9230a. cpe was extended to almost the whole monolayers at 3 days post-electroporation ( fig. 2a) . no cpe was observed in cells transfected with transcripts generated from clone g15526c (fig. 2a) . rt-pcr analysis of the subgenomic mrna 5 was performed to confirm if cpe observed is caused by the replication of ibv. sequencing of the rt-pcr fragment generated from cells transfected with ribv showed correct sequence in the leader/body junction region of the subgenomic mrna (fig. 2b ). further sequencing of the rt-pcr fragments covering regions with unique amino acid mutations confirmed the recovery of ibv (ribv) from the in vitro-synthesized fulllength transcripts. compared to the parent ibv strains, ribv contains 8 amino acid mutations (table 2 ). to test if these mutations may affect the growth properties and genetic stability of the rescued virus, ribv was propagated on vero cells for 5 passages, and the plaque sizes and growth kinetics were determined and compared with wild type ibv p65. in cells infected with ribv, the average plague size is 0.56 ± 0.028 mm, which is slightly smaller than the average plaque size of 0.68 ± 0.034 mm in cells infected with wild type ibv (fig. 3a) . analysis of the growth curves demonstrated that ribv exhibited very similar growth properties as the wild type virus (fig. 3a) . further characterization of ribv was subsequently carried out by analysis of viral rnas and structural proteins. northern blot and western blot analyses showed the detection of very similar amounts of viral rnas (fig. 3b ), and s, n, and m proteins ( fig. 3c ) in cells infected with wild type and ribv, respectively, at 24 h post-infection. when probing with anti-n protein antibodies, other species migrating faster than the fulllength products on sds-page were also observed ( fig. 3c ). they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and ribv (fig. 3c ). the significance of these variations is unclear at the moment. taken together, these results confirm that ribv is stable and possesses very similar growth properties as wild type ibv. as no infectious virus was recovered from cells transfected with g15526c mutant transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in these transfected cells. total rna was extracted from vero cells transfected with wild type and g15526c mutant transcripts at 24 and 48 h post-electroporation, respectively. reverse transcription was performed by using equal amount of rna and the sense-primer ibv14931-f (5′-14,931 gct-tatccactagtacatc 14,949 -3′), and pcr was carried out by using the sense-primer ibv14931-f and the antisense-primer ibv15600-r (5′-15,600 cttctcgcacttctgcacta-gca 15,578 -3′). if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 4a , rt-pcr fragments amplified from negative strand rna templates were obtained from cells transfected with both wild type (lanes 2 and 4) and the mutant transcripts (lanes 3 and 5). sequencing of the pcr fragments confirmed that they represent the correct sequences. as a negative control, the in vitro-synthesized transcripts were mixed with total rna extracted from normal vero cells and were used as a template for rt-pcr. no rt-pcr fragment was detected (fig. 4a, lane 6) , confirming that the detection of negative strand rna from cells transfected with mutant transcripts is due to the replication of viral rna. further quantitation of the negative strand rna transcription in cells transfected with wild type and g15526c mutant transcripts was carried out by real-time pcr. at 24 and 48 h post-electroporation, transcription of the negative rna in cells electroporated with wild type transcripts was 24.76-and 945.54-fold, respectively, higher than that in cells electroporated with the g15526c mutant transcripts. these results confirm that transcription of the negative strand rna has taken place in cells transfected with the mutant transcripts, but with much lower efficiency. fig. 3 . analysis of the growth properties of wild type (p65) and ribv. (a) plague sizes and one-step growth curves of wild type and ribv. monolayers of vero cells on a 6-well plate were infected with 100 μl of 1000-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of wild type and ribv, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24, and 36 h post-inoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with wild type and ribv. ten micrograms of total rna extracted from vero cells infected with wild type and ribv, respectively, was separated on 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 nucleotides of the ibv genome. total rna extracted from mock-infected cells was included as negative control. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and ribv. vero cells infected with wild type (lane 1) and ribv (lane 2) were harvested, lysates prepared and separated on sds-10% polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with antiactin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. rt-pcr amplification of subgenomic mrnas was carried out to check if a low level of subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts. total rna prepared from the transfected cells 2 days postelectroporation was used in the rt reaction with the sense-primer ibv-leader (5′-26 ctattacactagccttgcgct 46 -3′) for the detection of negative-stranded sgrna, and the antisenseprimer ibv24803-r (5′-24,803 ctctggatccaataacc-tac 24,784 -3′) for the detection of positive-stranded sgrna. the two primers were then used for pcr. if transcription of subgenomic mrnas did occur, a 415 bp pcr product corresponding to the 5′-terminal region of the subgenomic mrna 4 and a 1010 bp fragment corresponding to the 5′terminal region of the subgenomic mrna 3 would be expected. as shown in fig. 4b , a dominant 415 bp band and a weak 1010 bp band were observed in cells electroporated with wild type full-length transcripts at 2 days post-electroporation (lanes 2 and 5). sequencing of the pcr fragments confirmed that they represent the correct sequences of the corresponding regions of the subgenomic mrnas 3 and 4, respectively. however, the same pcr products were not detected in cells electroporated with the mutant transcripts (fig. 4b, lanes 3 and 6) . as a negative control, the amplified fragments were not detected in cells without electroporation (fig. 4b, lane 4) . the failure to detect both negative-and positive-stranded sgrnas in cells transfected with the mutant transcripts show that the g15526c mutation leads to the disruption of subgenomic rna transcription. to further demonstrate that the failure to rescue infectious virus from the g15526c mutant transcripts is due to a defect in subgenomic rna transcription, the full-length clones with and without the g15526c mutation were used to generate recombinant ibv expressing the enhanced green fluorescent protein (egfp) by replacing the 5a gene with egfp. full-length transcripts containing egfp were synthesized in vitro and introduced into vero cells together with the n transcripts by electroporation. at 2 days post-electroporation, single cpe with the expression of egfp was observed in cells transfected with the full-length transcripts without the g15526c mutation vero cells electroporated with in vitro-synthesized transcripts derived from the in vitro assembled full-length clones containing egfp either with or without g15526c mutation. phase-contrast and fluorescent images were taken 2, 3, and 5 days post-electroporation, respectively. (fig. 4c) . gradually increased cpe and fluorescent cells were observed from 3 to 5 days post-electroporation (fig. 4c) . at 5 days post-electroporation, cpe and fluorescent cells were extended to almost the whole monolayer (fig. 4c) . however, it was consistently observed that much less infectious virus was recovered from cells transfected with this construct. furthermore, the recombinant virus rapidly lost infectivity when passaged on vero cells; the recovered virus maintains minor infectivity only for one passage. in cells transfected with the fulllength transcripts containing the g15526c mutation, neither cpe nor cells expressing egfp were observed (fig. 4c) , demonstrating that g15526c mutation led to total demolition of the egfp expression. as egfp could be expressed only if subgenomic rnas were synthesized but can be observed even if a single cell was transfected and expressed the protein to a certain level, these results reinforce the conclusion that the g15526c mutation blocks subgenomic rna transcription. mutational analysis of the r132 residue of the helicase protein g15526c mutation resulted in the substitution mutation of the r132 with a pro (r132p) of the helicase protein. sequence comparison of the ibv helicase protein with other known coronaviruses showed that r132 residue is located adjacent to a conserved motif (fig. 5a) . in all sequenced coronaviruses, only ibv has a charged amino acid (r132) at this position (fig. 5a) . to assess if a positive charge amino acid at this position is essential for the function of the protein, mutation of r132 to a lys (r132k) was carried out. meanwhile, a conserved leu residue (ile in the case of tgev) was found at this position in all other coronaviruses, mutation of r132 to a leu (r132l) was also included. in vitro full-length transcripts containing the r132k and r132l mutations were electroporated into vero cells. as shown in fig. 5b , transcripts generated from wild type (r132) and r132k mutant constructs showed very similar infectivity after introduction into vero cells. typical cpe was observed in large areas of the monolayers at 3 days post-electroporation (fig. 5b) , and recombinant viruses were recovered. however, r132l transcripts were found to be less infectious. in cells electroporated with transcripts generated from this mutant, typical cpe was observed in much restricted areas of the monolayer at 3 days post-electroporation (fig. 5b) . the growth properties of the r132k and r132l mutant viruses on vero cells were tested by analysis of plaque sizes and growth curves of passage 5 mutant viruses. compared to cells infected with wild type recombinant virus (average plaques size is 0.56 ± 0.028), plaques with similar size were observed in cells infected with the r132k mutant virus (fig. 6a) . the average plaque size in cells infected with r132k mutant virus is 0.59 ± 0.029 mm. in cells infected with the r132l mutant virus, much smaller-sized plaques were observed (fig. 6a) . the average plaque size in cells infected with this mutant virus is 0.24 ± 0.017 mm. however, analysis of the growth curves of wild type and mutant viruses demonstrated that the mutant viruses exhibited very similar, or even better, growth properties as the wild type recombinant virus (fig. 6a) . the r132k and r132l mutant viruses were subsequently characterized by analysis of viral rnas and structural proteins. northern blot analysis showed the detection of very similar amounts of genomic and subgenomic rnas in cells infected with ribv and the two mutant viruses at 24 h post-infection (fig. 6b) . similarly, western blot analysis of cells infected with ribv, r132k, and r132l mutant viruses showed that similar amounts of s, n, and m proteins were detected at 24 h post-infection (fig. 6c, lanes 1-3) . when probing with anti-n protein antibodies, other species migrating faster than the full-length products on sds-page were also observed (fig. 6c) . they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and different mutants (fig. 6c) . the genetic stability of r132k and r132l mutant viruses was tested by propagation of the viruses on vero cells for 5 passages. sequencing analysis of the fifth passages of the two mutant viruses showed that the mutations are stable. no reversion to the original sequences or mutation to other nucleotides was found in the position. in vitro assembly of full-length coronavirus clones, generation of full-length transcripts in vitro using a bacteriophage dna-dependent rna polymerase, and recovery of infectious viruses by introduction of the in vitro-synthesized transcripts into cells, first used by yount et al. (2000) , are a rapid and reliable approach to construct infectious clones from large rna viruses. it has been successfully used to construct infectious fig. 6 . analysis of the growth properties of ribv, r132k, and r132l mutant viruses. (a) plague sizes and one-step growth curves of ribv, r132k, and r132 mutant viruses. monolayers of vero cells on a 6-well plate were infected with 100 μl of 100-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of ribv, r132k, and r132l mutant viruses, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24 , and 36 h postinoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with ribv, r132k, and r132l mutant viruses. ten micrograms of total rna extracted from vero cells infected with ribv, r132k, and r132l mutant viruses, respectively, was separated on 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 nucleotides of the ibv genome. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and r132 mutant viruses. vero cells infected with wild type recombinant ibv (lane 1), r132k (lane 2), and r132l (lane 3) were harvested, lysates prepared, and separated on sds-10% polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with anti-actin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. clones for several coronaviruses, including transmissible gastroenteritis virus (tgev), mouse hepatitis virus (mhv), sars-cov, and ibv (youn et al., 2005a (youn et al., , 2005b yount et al., 2000 yount et al., , 2003 . in the process of developing an infectious ibv clone from a vero cell-adapted ibv beaudette strain using this approach, a g-c point mutation at nucleotide position 15,526 was found to be lethal to the infectivity of the in vitrosynthesized full-length transcripts on vero cells. no infectious virus could be rescued from vero cells electroporated with transcripts containing this mutation. this mutation causes arg132-pro substitution in a domain within the helicase protein (nsp13) with undefined functions, indicating that this domain may be essential for the functionality of the helicase protein. multiple enzymatic activities have been assigned to the nsp13 helicase protein. these include rna and dna duplex-unwinding activities, ntpase and dntpase activities, and an rna 5′-triphosphatase activity that might be involved in the formation of the 5′-cap structure of viral rnas (ivanov et al., 2004) . the protein is comprised of two domains: a putative n-terminal zinc binding domain, which spans the nterminal region of the protein from approximately amino acids 1 to 77, and a c-terminal helicase domain covering the c-terminal part of the protein from amino acids 279 to the cterminal end (ivanov et al., 2004) . a ser-pro substitution located immediately downstream of the putative zinc binding domain of nsp10, the equivalent rna helicase protein in equine arteritis virus (eav), caused defect in subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 . more detailed analysis of the zinc binding domain of nsp10 from eav and nsp13 from human coronavirus 229e by mutagenesis studies showed that this domain could modulate the enzymatic activities of the helicase domain (seybert et al., 2005) . through this regulatory role and some yet to be discovered mechanisms, the zinc binding domain is shown to be critically involved in the replication and transcription of coronavirus rna. in this study, introduction of the in vitro-synthesized fulllength transcripts containing the g15526c (r132p) mutation was shown to be totally defective in subgenomic rna transcription, a phenotype similar to, but appears to be much more severe than, the ser-pro mutation in eav (van dinten et al., 1997 (van dinten et al., , 2000 . mutation of the arg132 residue to a positively charged amino acid (lys) does not affect the infectivity of the in vitro-synthesized transcripts as well as the growth properties of the rescued virus. however, mutation of the arg132 residue to leu, a conserved residue at the same position in most of other known coronaviruses, impaired the recovery rate of the in vitrosynthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. the r132 residue is located 57 amino acids downstream of the last his (h75) residue in the putative zinc binding domain (fig. 5a) and 147 amino acids upstream of the helicase motif 1 (seybert et al., 2005) . so far, no functional domain has been found in this region of the helicase protein. why the r132p substitution, located outside of the two functional domains of the helicase protein, shows severe phenotypic defect in subgenomic rna transcription and the infectivity of ibv is not clear at the moment. three possibilities were considered. first, r132 may be part of the n-terminal zinc binding domain. as no studies have been attempted to define the boundary of the two domains, it is not even certain that an independent domain with unique function may exist in this region. r132p mutant virus shares certain phenotypic similarity to the ser-pro mutant eav, such as the absence of subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 , suggesting that the two mutations might disrupt a similar function of the helicase protein. however, as biochemical characterization of the effect of r132p mutation on the enzymatic activities of ibv nsp13 helicase protein is currently lacking, it would be difficult to draw a conclusion that the two mutants share mechanistically similar characteristics. second, r132 is located in a region with a high degree of amino acid conservation in all known coronaviruses. since the main defect of r132p mutant virus is in the subgenomic rna transcription, one possibility is that this region may be involved in the subgenomic rna synthesis by interacting with host or other viral functional proteins. alternatively, as a positively charged amino acid is required to maintain the full function of the protein, it would be possible that this region may be involved in binding of the helicase protein to viral rna during rna replication and transcription. finally, mutation to a pro would lead to the disruption of the three dimensional structures of the two domains. biochemical characterization of the involvement of this region in the enzymatic activities of the protein and determination of its three dimensional structures are underway to address these possibilities. the g433c (e-q) point mutation near the catalytic center of the plp domain in nsp3 affects neither the recovery of infectious virus from the full-length synthesized in vitro transcripts nor the infectivity of the rescued virus. similarly, efficient recovery of infectious virus from the in vitro transcripts containing the g9230a (g-e) point mutation in the 3clp was obtained. the two mutant viruses are genetically stable and show similar growth properties with the wild type recombinant virus. even though the two mutations are located in or near the catalytic centers of the two viral proteinases, the mutations did not alter their enzymatic activities. this relatively high degree of tolerance to mutations in non-essential regions of important functional proteins would minimize the occurrence of lethal mutations during the replication cycles of rna viruses and increase the adaptability of these viruses to a changed environment. vero cells were cultured at 37°c in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml), and streptomycin (100 μg/ml). a vero cell-adapted ibv beaudette strain (65 passages on vero cells (p65)) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) was propagated in vero cells in fbs-free mem. five fragments spanning the entire ibv genome were obtained by rt-pcr from vero cells infected with the vero cell-adapted ibv p65 at a multiplicity of approximately 1. briefly, total cellular rna was extracted from the infected vero cells with tri reagent (molecular research center, inc.), according to the manufacturer's instructions. reverse transcription was performed with expand reverse transcriptase (roche) using reverse primers ibv-5753r, ibv-8694r, ibv-15532r, ibv-20930r, and ibv-27608r (table 1) . each cdna fragment was amplified from rt products by pcr using kod hot start dna polymerase according to the manufacturer's instructions (novagen). the pcr products were purified from agarose gels and cloned into pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. subsequently, fragment a was removed from pcr-xl-topo by digestion with nhei and ecori and subcloned into pkt0 vector. two to three independent clones of each fragment were selected and sequenced by automated sequencing using specific primers and the abi dye termination sequencing method. sequence comparison, assembly, and analysis were performed by using blast and dna star software. mutations were introduced into the corresponding fragments by using quickchange site-directed mutagenesis kit (stratagene) and confirmed by sequencing of the whole fragments. in vitro assembly of full-length cdna clones plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d, and e). the digested plasmids were separated on 0.8% agarose gels containing crystal violet. bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). fragments a and b, and fragments c, d, and e were first ligated with t4 dna ligase at 4°c overnight. the two reaction mixtures were then mixed and further ligated at 4°c overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.4% agarose gels. full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, 30 μl of transcription reaction with a 1:1 ratio of gtp to cap analog was sequentially incubated at 40.5°c for 25 min, 37.5°c for 50 min, 40.5°c 25 min and 37.5°c for 20 min. the n transcripts were generated by using a linearized pkto-ibvn containing ibv n gene and the 3′-utr region as templates. a 1:2 ratio of gtp to cap analog was used for the transcription of ibv n gene. the in vitro-synthesized full-length and n transcripts were treated with dnasei and purified with phenol/chloroform. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 μl of vero cell suspension in an electroporation cuvette and electroporated with one pulse at 450 v, 50 μf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 mm dish or a six-well plate and further cultured in mem without fbs. analysis of the negative strand and subgenomic rnas by rt-pcr, real-time pcr total rna was extracted from vero cells electroporated in in vitro-synthesized transcripts after treatment with dnasei, using tri reagent (molecular research center, inc.) at 24 or 48 h post-electroporation. reverse transcription was performed with expand reverse transcriptase (roche) using equal amount of rna. after optimization, real-time pcr was performed using lightcycler faststart dna master sybr green i kit according to the manufacturer's instructions (roche). vero cells were infected with wild type and mutant viruses at a multiplicity of approximately 1, and total rna was extracted from the infected cells. ten micrograms of rna was added to a mixture of 1× mops, 37% formaldehyde, and formamide and incubated at 65°c for 20 min before subjected to gel electrophoresis. the segregated rna bands were transferred onto a hybond n+ membrane (amersham biosciences) via capillary action overnight and fixed by uv crosslinking (stratalinker). hybridization of dig-labeled dna probes was carried out at 50°c in hybridization oven overnight. membranes were washed 3 times for 15 min each with the probe buffer, before proceeding to detection with cdp-star (roche) according to the manufacturer's instructions. vero cells were infected with wild type and mutant viruses at a multiplicity of approximately 1. total proteins extracted from the infected vero cells were lysed with 2× sds loading buffer in the presence of 200 mm dtt plus 10 mm of iodoacetamide and subjected to sds-page. proteins were transferred to pvdf membrane (stratagene) and blocked overnight at 4°c in blocking buffer (5% fat free milk powder in pbst buffer). the membrane was incubated with 1:2000 diluted primary antibodies in blocking buffer for 2 h at room temperature. after washing three times with pbst, the membrane was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 h at room temperature. after washing three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl, amersham biosciences) according to the manufacturer's instructions. confluent monolayers of vero cells on six-well plates were infected with 100 μl of 100-fold diluted virus stock. after 1 h of incubation at 37°c, cells were washed twice with pbs and cultured in 3 ml of mem containing 0.5% carboxymethy cellulose for 3 days. the cells were fixed and stained with 0.1% toluidine. vero cells were infected with wild type and recombinant ibv and harvested at different times post-infection. viral stocks were prepared by freezing/thawing of the cells three times. the 50% tissue culture infection dose (tcid50) of each sample was determined by infecting five wells of vero cells on 96-well plates in duplicate with 10-fold serial dilution of each viral stock. engineering the largest rna virus genome as an infectious bacterial artificial chromosome completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus reverse genetics system for the avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant mouse hepatitis virus strain a59 from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vitro selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells neither the rna nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination characterization of a papain-like proteinase domain encoded by orf1a of the coronavirus ibv and determination of the cterminal cleavage site of an 87 kda protein characterization of the two overlapping papainlike proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification, expression and processing of an 87k polypeptide encoded by orf1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products the genome sequence of the sars-associated coronavirus coronavirus reverse genetics by targeted rna recombination identification of a 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein characterization of a novel coronavirus associated with severe acute respiratory syndrome targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence a complex zinc finger controls the enzymatic activities of nidovirus helicases emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated 3b gene: functional characterization of the 3b gene in pathogenesis and replication a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice this work was supported by the agency for science technology and research, singapore, and by a grant from the biomedical research council (bmrc 03/1/22/17/220), agency for science, technology and research, singapore. key: cord-253695-tjdw2uta authors: winter, christine; herrler, georg; neumann, ulrich title: infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent date: 2007-12-28 journal: microbes infect doi: 10.1016/j.micinf.2007.12.009 sha: doc_id: 253695 cord_uid: tjdw2uta avian infectious bronchitis virus (ibv) is a coronavirus that infects chickens via the respiratory epithelium as primary target cells. the binding of coronaviruses to the cell surface is mediated by the viral surface protein s. recently we demonstrated that α2,3-linked sialic acid serves as a receptor determinant for ibv on vero cells and primary chicken embryo kidney cells. here we analyze the importance of the sialic acid binding activity for the infection of tracheal organ cultures (tocs) by different ibv strains. our results show that α2,3-linked sialic acid also serves as a receptor determinant on chicken tocs. infection of tocs by ibv results in ciliostasis. desialylation induced by neuraminidase treatment of tracheal organ cultures prior to infection by ibv delayed the ciliostatic effect or resulted in partial loss of ciliary activity. this effect was observed with both respiratory and nephropathogenic strains. inhibition of ciliostasis was also observed when tocs were pretreated with an α2,3-specific neuraminidase. analysis of the tracheal epithelium for reactivity with lectins revealed that the susceptible cells in the epithelium abundantly express α2,3-linked sialic acid. these results indicate that α2,3-linked sialic acid plays an important role for infection of the respiratory epithelium by ibv. infectious bronchitis is one of the most significant diseases of chickens in the commercial poultry industry. the etiologic agent is infectious bronchitis virus (ibv), a member of the family coronaviridae [1] . the pathology of the disease associated with ibv infection can show different characteristics depending on the tissue where virus replication occurs. ibv enters its host via the oro/oculo-nasal route and the first site of replication is the respiratory epithelium. from there the virus infection can spread to several organs, including kidney and reproductive tract (reviewed in [2] ). secondary pathogens can complicate the disease resulting in increased morbidity and mortality. the tissue tropism of viruses may be determined by several factors including the distribution of the cellular receptor for the virus. in the case of coronaviruses, the surface protein s is responsible for attachment to cells. the s protein has not only receptor-binding activity, it also mediates the fusion of the viral lipid envelope with the cellular membrane. for several coronaviruses specific proteins have been described that serve as cellular receptors for the initiation of infection. angiotensin-converting enzyme 2 has been identified as a receptor for sars coronavirus [3, 4] and several group 1 coronaviruses including transmissible gastroenteritis virus require aminopeptidase n to enter their host cells [5, 6] . a protein receptor has not been identified so far for ibv. recently we demonstrated that a2,3-linked sialic acids serve as receptor determinants on vero-cells and primary chicken embryo kidney cells [7] . here we show that sialic acids also play a role in the infection of the avian respiratory epithelium by ibv. tracheas were prepared from 20-day-old spf chicken embryos (lohmann, cuxhaven, germany) and, after removing connective tissue, were cut manually into approximately 1 mm thick rings by using a microtome blade. individual rings were transferred into 5 ml tubes (sarstedt, nümbrecht, germany) containing 0.5 ml of medium 199 with hanks salts (biochrom, berlin, germany) and incubated at 37 c on a rotator. the next day, tocs were screened for 100% ciliary activity (see section 2.5). stock virus of the ibv strains m41 and b1648 were obtained by inoculating embryonated spf chicken eggs. following incubation at 37 c, the allantoic fluid was collected, clarified by low speed centrifugation and stored at à80 c. strain beaudette of ibv was propagated in vero cells. supernatants of infected cell cultures were harvested, clarified by low speed centrifugation and stored at à80 c. all ibv strains were kindly provided by dave cavanagh, institute for animal health, compton, uk. avian metapneumovirus subtype a, employed as a control virus, was kindly provided by silke rautenschlein, university of veterinary medicine, hannover, germany. tocs were washed with pbs prior to addition of neuraminidase from clostridium perfringens (type 6) or streptococcus pneumoniae (sigmaealdrich, st. louis, mo, usa) using mes (2[n-morpholino]ethanesulfonic acid) buffer as a diluant. if not otherwise indicated tocs were incubated with 50 mu neuraminidase per ring. after incubation at 37 c for 1 h, the tocs were washed three times with pbs and infected by ibv-beaudette (10 4 pfu/ring) or any of the other viruses for 1 h at 37 c. following three washes with pbs, the tocs were incubated with medium at 37 c on a rotator. for all experiments groups of four tocs were used to estimate the mean ciliary activity. all experiments were performed in triplicate. each ring of toc was suspended in 100 ml medium 199 containing 50 mu neuraminidase of clostridium perfringens and in some cases 2.5 mg of the neuraminidase inhibitor dana (2,3-didehydro-2-deoxyneuraminic acid). after incubation for 1 h at 37 c tocs were washed and infected by the beaudette strain with 10 4 pfu/ring. tocs were analyzed daily with a microscope to estimate the ciliary activity. rings were virtually divided into 10 parts and each part was monitored for ciliary movement. only rings with a starting ciliary activity of 100% were used for the experiments. groups of five tocs were infected in triplicate by 2 â 10 4 pfu of ibv. at 24 h post infection, the supernatants were collected and titrated on primary chick kidney cells as described previously [7] . plaques were visualized by immunofluorescence using a polyclonal anti-ibv serum raised in rabbits. tracheas were prepared from three-week old spf chickens (lohmann, cuxhaven, germany). they were cut into rings approximately 1 cm thick. the rings were washed with pbs and either infected by ibv-beaudette (1 â 10 5 pfu/ring) for 1 h at 37 c. or subjected to neuraminidase treatment and lectin staining. infected tocs were incubated in medium 199 (biochrom, berlin, germany) at 37 c. after 24 h, they were mounted on small filter papers with tissue freezing medium (jung, heidelberg, germany) and frozen in liquid nitrogen. neuraminidase treatment was performed by incubation with 500 ml medium199 containing 200 mu neuraminidase from clostridium perfringens for 1 h at 37 c, before freezing in liquid nitrogen. the frozen organs were stored at à20 c until they were cut with a cryostat. cryosections were fixed with ice-cold acetone for 10 min followed by air drying for another 10 min. the antibodies or lectins were diluted in 1% bovine serum albumin and sections were incubated with antibodies or lectins for 1 h at room temperature in an incubation chamber. after three washing steps with pbs, the sections were incubated with appropriate second antibodies for 1 h at room temperature in the dark. virus antigen was stained with polyclonal anti-ibv beaudette serum raised in rabbits. sialic acids were detected with lectins from maackia amurensis (binding to a2,3 linked sialic acids) or sambucus nigra agglutinin (binding to a2,6 linked sialic acids) labeled with digoxigenin (dig glycan differentiation kit, roche, basel, switzerland). mucus producing goblet cells were stained with anti muc-5ac antibody (acris, hiddenhausen, germany). bound antibodies or lectins were visualized by fitc-and cy3-labeled anti-rabbit (sigmaealdrich), anti-mouse (acris) or anti-digoxigenin antibodies (roche). cilia were detected by cy3 labeled anti-b-tubulin antibody (sigmaealdrich). fluorescence microscopy was performed with a leica inverted-2 confocal microscope. after having shown recently that sialic acid serves as a receptor determinant for ibv on cultured cells, we were interested to find out whether this type of sugar is also important for an infection in vivo. to address this question we chose tocs as a model for the upper respiratory epithelium. the trachea of chicken embryos was cut into pieces about 1 mm thick. tracheal rings chosen for this analysis showed ciliary activity along the whole contact area of the epithelium with the lumen of the trachea, i.e. 360 of the tracheal ring. infection of tocs by ibv results in ciliostasis that can easily be detected microscopically. as shown in fig. 1 , infection by ibv-beaudette reduced the portion of the epithelium with ciliary activity. at 2 days post infection (d.p.i.), complete ciliostasis was observed at this experimental setting. to analyze the importance of sialic acids we treated tocs with neuraminidase from clostridium perfringens, which releases the sialic acids from the cell surface. as a result desialylated tocs were less sensitive to the ciliostatic effect of the ibv infection. at 5 d.p.i., about 25% of the epithelium still showed ciliary activity. this effect is accounted for by the neuraminidase itself rather than by a contaminant enzyme, because in the presence of a neuraminidase inhibitor the enzyme was unable to protect the tracheal epithelium from the ciliostatic effect of the ibv infection (fig. 1) . the infection of cultured cells by ibv was found to be dependent on a2,3-linked sialic acid. to find out whether there is also a linkage specificity in the infection of the tracheal epithelium, tocs were treated with neuraminidase from streptococcus pneumoniae, which has a high preference for cleaving a2,3-linked sialic acid. as shown in fig. 2 , incubation with this enzyme protected the epithelial cells from the ciliostatic effect of the ibv infection in the same way as was observed with the neuraminidase from clostridium perfringens (fig. 1) . from this result we conclude that a2,3-linked sialic acid serves as a receptor determinant for the infection of avian tracheal epithelial cells by the beaudette strain of ibv. the beaudette strain of ibv has been adapted to grow in cultured cells of non-avian origin, such as vero cells. to demonstrate that the sialic acid-dependent infection of tracheal epithelial cells is a general feature of ibv, we included two other strains. the m41 strain causes respiratory disease, whereas the b1648 strain has a nephropathogenic potential [8] . after application of an infectious dose of 10 4 pfu per tracheal ring, the ibv strains caused ciliostasis by day 3 (m41), 4 (b1648) or 5 (beaudette) after infection (fig. 3aec) . strain m41 was more pathogenic than the other two strains resulting in a loss of ciliary activity at 2 d.p.i. on 80% of the epithelium in the microscopic field (fig. 3c) . a comparable reduction was observed with the two other strains only 3 days following infection of tocs (fig. 3a and b) . for all three strains, a protective effect of neuraminidase treatment was found. in the case of m41, ciliostasis was delayed for 1 day; in the case of the other two strains, the ciliary activity was retained in about 60% of the desialylated epithelium even on day 5 following infection. ciliostasis was also found after infection by avian metapneumovirus. however, in contrast to ibv infection, neuraminidase treatment did not prevent the ciliostatic effect of the metapneumovirus infection. this result confirms that the sialic acid dependence of the toc infection is a characteristic feature of ibv. in the experiments described above, the ciliary activity was used to monitor the course of infection. to determine the effect of neuraminidase treatment on virus production, the amount of infectious virus released from the epithelial cells was measured. groups of five toc rings were treated with neuraminidase prior to infection by either of the ibv strains, beaudette and m41. in parallel, toc were infected that had been incubated in the absence of neuraminidase. at 24 h p.i. the supernatants were collected and titrated on primary chicken kidney cells. with both virus strains, desialylation resulted in a decrease of the virus titer by about 60% (fig. 4) . there was a clear difference in the amount of virus released from infected tocs. for the beaudette strain, the titer of infectious virus in the supernatant was 50e100 fold higher than that determined for strain m41. the respiratory epithelium is the primary target for an ibv infection. cryosections of toc were prepared and ciliated cells were visualized by staining with antibodies directed against btubulin. mucus-producing goblet cells were stained with an antibody recognizing muc5ac. as shown in fig. 5 (top two rows), both ciliated and mucus-producing cells are infected by ibv. cryosections were also stained for sialic acid expression using the lectin maa which binds to a2,3-linked sialic acids and sna which recognizes a2,6-linked sialic acids. the epithelial cell layer lining the surface of the trachea shows bright fluorescence after staining with maa, indicating that these cells abundantly express a2,3-linked sialic acid. they colocalize with ibv-infected cells (fig 5, third row) . this result is consistent with a role of a2,3-linked sialic acid as a receptor determinant for ibv. staining with sna indicates that basal cell layers of the tracheal epithelium express a2,6-linked sialic acid. these cells are not infected by ibv (fig. 5, bottom row) . to visualize the effect of neuraminidase treatment, we prepared cryosections of neuraminidase-treated tracheal rings and stained the sections with the maa lectin. as shown in fig. 6 , neuraminidase treatment resulted in a reduced reactivity of maa with the apical membrane of the epithelial cells. the residual fluorescent signal indicates that the neuraminidase did not release all sialic acids from the epithelial cells. the binding to receptors on the cell surface is an important determinant of cell tropism and viral pathogenesis. recently we demonstrated that a2,3-linked sialic acid serves as a receptor determinant for ibv on cultured cells [7] . this binding activity of ibv has been shown for strains that only grow in avian cells as well as for the beaudette strain that has been adapted to grow also in cultured cells of mammalian origin. for the latter strain it has been recently reported that it also recognizes glycosaminoglycans of the heparan sulfate type [9] . the acquisition of this additional binding activity may have allowed the adaptation to new host cells as has been shown for other viruses, e.g. foot and mouth disease virus [10] and tick-borne encephalitis virus [11] . by contrast, the sialic acid binding activity has been detected on all ibv strains analyzed including respiratory and nephropathogenic variants. here we have demonstrated that this also holds true for epithelial cells of the trachea, which are among the primary target cells for this virus. the importance of sialic acid for ibv infection was evident from the delayed appearance of ciliostasis on desialylated tocs. for the m41 strain, the ciliostatic effect was delayed only by 1 day, whereas for the two other strains analyzed, some ciliary activity was retained even 5 days after infection. this difference in the protective effect of neuraminidase treatment is probably accounted for by the difference in the cytopathogenicity of these viruses. this may also explain the larger amount of virus that is released from tocs infected by ibv-beaudette compared to m41-infected tracheal rings. despite these differences, the relative effect of desialylation was similar, i.e. the amount of released virus was reduced by about 60%. the partial protection of tocs from ibv infection may be attributable to the difficulties in removing all sialic acid residues from the cell surface of the tracheal epithelium. staining by maa revealed that some sialic acids are still present on the apical membrane of the epithelial cells after neuraminidase treatment. whether this low level of sialic acid by itself is sufficient for a low level of infection is not known. an alternative explanation is that binding to surfacebound sialic acids is only a first attachment step in the infection cycle that precedes the binding to a second receptor, though such a receptor has not been identified for ibv so far. in favor of the latter explanation is the fact that ibv recognizes sialic acid with lower affinity when compared to influenza virus and sendai virus [7] . because of the higher affinity for sialic acids, a second receptor may be dispensable for influenza viruses; however, it may require the presence of a neuraminidase (receptor-destroying enzyme) to allow passage through the mucus layer covering the respiratory epithelium [12] and to enable virus release from infected cells [13] . on the other hand, a receptor-destroying enzyme may be dispensable for ibv because of the lower affinity for sialic acids. so far, two groups of coronaviruses with sialic acid binding activity have been described. on one hand, there is bovine coronavirus and related viruses that resemble influenza c virus, because they recognize n-acetyl-9-o-acetylneuraminic acid and contain an acetylesterase that acts as a receptor-destroying enzyme comparable to the neuraminidase of influenza a and b viruses [14, 15] . these viruses depend on sialic acid for infection of cells. on the other hand, there is transmissible gastroenteritis virus, a porcine coronavirus. this virus uses aminopeptidase n as a cellular receptor and it does not require sialic acid for successful infection of cultured cells. however, binding to sialylated surface components may increase the efficiency of infection, because mutants lacking the sialic acid binding activity have lost the enteropathogenicity [16, 17] . ibv takes an intermediate position; sialic acid is important for infection of cultured cells but it lacks a receptor-destroying enzyme. as mentioned above, ibv maydin addition to sialylated surface moleculesdbind to a specific receptor similar to the interaction of tgev with aminopeptidase n. though it fig. 6 . effect of neuraminidase treatment on the staining of the tracheal epithelium by the lectin maa. tocs were incubated in the presence (þna) or absence (àna) of neuraminidase. after incubation, cryosections were prepared and subjected to maa staining. arrows point to the apical membrane of the tracheal epithelium. it should be noted that the enzyme had access only to the apical side of the epithelium. has been suggested that feline aminopeptidase n may be used by ibv to infect cells [18] , recently it has been shown that aminopeptidase n does not serve as a receptor for ibv [19] . our analysis of toc revealed that both ciliated and mucusproducing cells are susceptible to infection. from this result we conclude that in vivo both cell types can act as primary target cells for ibv. the expression of a2,3-linked sialic acids and the absence of a2,6-linked sialic acids is consistent with the preference of ibv for the former linkage type ( [7] and this report). in this respect ibv appears to have developed a similar strategy to avian influenza viruses which also use a2,3-linked sialic on the cell surface to initiate an infection. however, there are also major differences between avian influenza and avian coronaviruses. a major determinant of the pathogenicity of influenza viruses is the proteolytic cleavage of the hemagglutinin. the hemagglutinins of highly pathogenic viruses have a multibasic cleavage site that is susceptible to furin-like enzymes present in many cell types. viruses of low pathogenicity become fusion-active by a protease that is secreted by respiratory cells. the s protein of all ibv strains has a multibasic cleavage site and is cleaved in all cell types into the subunits s1 and s2. therefore, proteolytical activation of s is not a major determinant of the pathogenicity of ibv. why some ibv strains are predominantly respiratory pathogens whereas others affect other organs to cause disease, e.g. the renal system or the reproductive tract, is not known. if there exists a second receptor for this virus as discussed above, such a protein may be responsible for the host specificity as well as the tissue or organ tropism of ibv. future work should clarify whether a second receptor exists. furthermore, the sialoglyconjugates used for primary attachment should be analyzed. the available lectins and neuraminidases only allow a differentiation between a2,3and a2,6-linked sialic acids. different oligosaccharides exist that contain a2,3linked sialic acid, and the sialoglycoconjugates recognized by ibv may be different from those that are preferred by avian influenza viruses. such differences may also contribute to a different course of infection. expression of a2,3-linked sialic acid on epithelial cells in chickens has been analyzed for the trachea and the intestine [20, 21] . as far as other target organs of ibvare concerned, e.g. kidney and the reproductive system, a2,3-linked sialic acid has been shown to be present on primary kidney cells [7] . it will be interesting in the future to find out whether infection of the reproductive system is also mediated by sialic acid. coronavirus avian infectious bronchitis virus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression cloning of functional receptor used by sars coronavirus aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev human aminopeptidase n is a receptor for human coronavirus 229e sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus vandenbroeck, incidence, characterisation and prophylaxis of nephropathogenic avian infectious bronchitis viruses heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette efficient infection of cells in culture by type o foot-and-mouth disease virus requires binding to cell surface heparan sulfate adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo neuraminidase is important for the initiation of influenza virus infection in human airway epithelium inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action the e3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity bovine coronavirus uses n-acetyl-9-o-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review feline aminopeptidase n is not a functional receptor for avian infectious bronchitis virus differences between influenza virus receptors on target cells of duck and chicken lectin histochemical investigations of the distal gut of chicks with special emphasis on the follicle-associated epithelium we thank martina kaps for technical assistance. this work was supported by a grant from deutsche forschungsgemeinschaft (ne221/5-1 and sfb 587 tp a1). key: cord-260145-grz0fe9l authors: liu, shengwang; zhang, xiaonan; gong, liyang; yan, baolong; li, chengren; han, zongxi; shao, yuhao; li, huixin; kong, xiangang title: altered pathogenicity, immunogenicity, tissue tropism and 3′-7 kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos date: 2009-07-23 journal: vaccine doi: 10.1016/j.vaccine.2009.05.072 sha: doc_id: 260145 cord_uid: grz0fe9l in this study, we attenuated a chinese lx4-type nephropathogenic infectious bronchitis virus (ibv) strain, ck/ch/lhlj/04v, by serial passage in embryonated chicken eggs. based on sequence analysis of the 3′-7 kb region, the ck/ch/lhlj/04v virus population contained subpopulations with a mixture of genetic mutants. the titers of the virus increased gradually during serial passage, but the replication capacity decreased in chickens. the virus was partially attenuated at passage 40 (p40) and p70, and was fully attenuated at p110. it lost immunogenicity and kidney tropism at p110 and p70, respectively. amino acid substitutions were found in the 3′-7 kb region, primarily in the spike (s) protein. substitutions in the s1 subunit occurred between p3 and p40 and all subpopulations in a virus passage showed the same substitutions. other substitutions that occurred between p70 and p110, however, were found only in some subpopulations of the virus passages. a 109-bp deletion in the 3′-utr was observed in most subpopulations of p70 and p110, and might be related to virus replication, transcription and pathogenicity. the changes described in the 3′-7 kb region of the virus are possibly responsible for virus attenuation, immunogenicity decrease and tissue tropism changes; however, we cannot exclude the possibility that other parts of the genome may also be involved in those changes. coronaviruses belong to the family coronaviridae and the order nidovirales, and are classified into three groups based on the absence of genetic and antigenic relationships between the species of the different groups [1, 2] . they are known to cause upper and lower respiratory diseases, gastroenteritis, and central nervous system infection in a number of avian and mammalian hosts, including humans [3] . the infectious bronchitis virus (ibv) belongs to the group 3 coronaviruses. it primarily causes respiratory disease in domestic fowl, although it also replicates on epithelial surfaces of the alimentary tract, oviduct, and kidney, and is one of the most economically important pathogens in the poultry industry [4] . ibv has four essential structural proteins: the phosphorylated nucleocapsid (n) protein, and the three membrane proteins spike (s), integral membrane (m), and small envelope (e). although the s1 subunit of the s protein carries virus-neutralizing and serotype-specific deter-embryonated eggs, recombination between virus subpopulations, and accumulation of mutations in the s1 region can lead to the formation of attenuated viruses [19] . the importance of s1 in determining cell and tissue tropism has been demonstrated for several coronaviruses, such as murine hepatitis virus [20] [21] [22] [23] [24] [25] [26] [27] [28] , porcine transmissible gastroenteritis virus [29, 30] , and severe acute respiratory syndrome coronavirus [31] . in the case of ibv, the s1 subunit of the s protein determines the serotype of ibv and is responsible for viral attachment to cells. furthermore, it has been shown that s1 is a major determinant of cell tropism in culture [32] , and the majority of changes accumulated during the adaptation of ibv to vero cells are in the s gene [13] . however, differences in one or more other genes are responsible for the highly attenuated phenotype of the beaudette ibv laboratory strain [13] , and the roles of these gene products in the attenuation process have yet to be determined. in spite of the extensive use of vaccines, nephrotropic ibv outbreaks are frequent in china [33] [34] [35] [36] . lx4-type has been the predominant ibv type in china in recent years [34] [35] [36] , and appears to have become widespread in several countries in europe, causing severe losses to both the layer and broiler industries [6] . in addition, this type of ibv has increased in recent years in both china and european countries; thus, the development of an efficacious live-attenuated vaccine against lx4-type ibv is important. we are developing an ib vaccine by serial passage of the ibv strain ck/ch/lhlj/04v, which represents the lx4-type, in embryonated eggs. evaluating the attenuation, the growth of viruses in embryos, the efficacy in poultry populations, and the changes in molecular characteristics after serial passage were the primary focus and objectives of the present study. in a future study, practical considerations regarding the development of such a vaccine will be examined. we used a virulent ibv strain, ch/ck/lhlj/04v, which was previously isolated during an outbreak of ib in 2004 at a broiler farm in the heilongjiang province in china. clinical signs and lesions observed during the outbreak included nephritis and mortality. the virus was isolated from the kidney of a dead broiler using 9-dayold embryonated specific pathogen-free (spf) chicken eggs. the ibv strain was identified by means electron microscope examination, reverse transcriptase-polymerase chain reaction (rt-pcr) and sequencing of the entire s1 protein gene, as described previously [34] . an analysis of the molecular characteristics showed that this virus exhibited limited homology (not more than 83% of amino acids) to genotypes representing the vaccine strains h120 and w93 [34] . phylogenetic analysis showed that this virus was an lx4-type [34] . the virus stock for this study was produced by inoculating the virus into embryonated spf chicken eggs via the allantoic cavity and collecting the infectious allantoic fluid 72 h post-inoculation. the allantoic fluid was clarified by centrifugation at 3000 × g for 10 min and filtered with a teflon membrane. in addition, three ibv strains, ck/ch/ldl/04ii, ck/ch/lxj/02i and ck/ch/lshh/03i, were used as references for the pathogenicity study; their backgrounds and types were reported previously [34] . fertile white leghorn spf chicken eggs, and white leghorn spf chicks were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. the birds were maintained in isolators with negative pressure, and food and water were provided ad libitum. fifty-one-day-old white leghorn spf chickens were used to assess the pathogenicity of the ck/ch/lhlj/04v strain. five groups of 10 chickens were kept in isolators with negative pressure. at the age of 15 days, groups 1-4 were inoculated intranasally with 0.1 ml per chick containing 10 4.7 -10 4.8 median embryo infectious doses (eid 50 ) at passage level 3 of strains ch/ck/lhlj/04v, ck/ch/ldl/04ii, ck/ch/lxj/02i and ck/ch/lshh/03i. group 5 was mock-inoculated with sterile allantoic fluid and served as a control ( table 1 ). the chicks were examined daily for signs of infection for 30 days after inoculation. tracheal swabs and blood samples were collected from all 10 birds in each treatment groups at 4, 8, 12, 16 and 20 days post-inoculation. serum was stored at −70 • c until elisa testing was performed. the tracheal swabs were used for virus recovery attempts in embryonated chicken eggs. the ch/ck/lhlj/04v strain was serially passaged 110 times by inoculating 9-day-old spf chicken eggs by the allantoic cavity route as described previously [19] . inoculated eggs were incubated for 48-72 h at 37 • c in an egg incubator (heraeus, germany). the chorioallantoic fluids were harvested and stored at −70 • c or used directly for subsequent passage. at every 15th passage starting with passage 30, the virus was examined for viability by inoculation of two to three additional eggs for 7 days and observation of the embryos for clinical signs consistent with ibv infection. in addition, these selected passages were examined by negative contrast electron microscopy (jem-1200, ex) for the presence of coronavirus, and by reverse transcriptase-polymerase chain reaction (rt-pcr) and sequencing [34] to verify the virus type. passage 3 (p3) for the pathogenic strain, and p40, p70 and p110 were examined in more detail. the viruses of these four passages were propagated once in 10-day-old embryonated spf chicken eggs as described for field isolates [34] , to obtain titers of 10 6 -10 8 eid 50 per 0.1 ml. before use, viruses from the allantoic fluids of inoculated eggs were confirmed by negative contrast electron microscopy and by rt-pcr and sequencing. one hundred fifteen, 1-day-old spf white leghorn chicks were housed in different isolators and divided into five groups. groups 1-4 had 25 birds each, and group 5 included 15 birds. chickens in groups 1-4 were inoculated with p3, p40, p70 and p110, respectively, by oculonasal application at 15 days of age with a dose of log 10 4.7 -log 10 4.8 eid 50 per chick (table 2 ). birds in group 5 were mock-inoculated with sterile allantoic fluid and served as the control. five birds from each group were killed humanely 5 days post-inoculation. the trachea and kidney were collected for virus titration. blood samples from 10 birds in each group were collected at 4, 8, 12, 16 and 20 days post-inoculation. the serum was stored at −70 • c for elisa testing. the chicks were examined daily for signs of infection for 30 days after inoculation. ninety, 1-day-old spf white leghorn chicks were housed in different isolators and divided into five groups. groups 1-3, and the positive control group, had 20 birds each, and the negative control group included 10 birds. chickens in groups 1-3 were inoculated with p40, p70 and p110, respectively, by oculonasal application at 15 days of age with a dose of log 10 4.7 -log 10 4.8 eid 50 per chick (table 3) . birds in the positive and negative control groups were mock-inoculated with sterile allantoic fluid. at 20 days post a ten chicks per group. b dose per chick, 100 l. c two procedures were used for virus recovery after challenge. first, lesions in embryos that had been inoculated with pooled samples (tracheal swabs) were observed. second, rt-pcr using oligonucleotide primers n(+) and n(−) on rna recovered from allantoic fluid of the same eggs was conducted. the results from the two procedures were identical. d number seroconverted/number inoculated. e days after challenge. f ck/ch/lhlj/04v passage 3 (p3) was used for the virulence study. inoculation, birds in groups 1-3 and in the positive control group were challenged by oculonasal application with 10 4.8 eid 50 /0.1 ml of pathogenic ch/ck/lhlj/04v virus, while the birds in the negative control group were mock-inoculated again with sterile allantoic fluid. ten birds each from groups 1-3 and the positive control group, and 5 birds from the negative control group were killed humanely 5 days post-challenge. the trachea and kidney were collected for virus recovery. blood samples from 10 birds in each group were collected at 4, 8, 12, 16 and 20 days after challenge. the serum was stored at −70 • c for elisa testing. the chicks were examined daily for signs of infection for 30 days after inoculation. the virus stocks used for the pathogenicity study, and the tissue samples of tracheas and kidneys collected 5 days post-inoculation from experiment 1, were used for rt-pcr amplification and virus titration. tissue samples were homogenized individually and rt-pcr was conducted using primers n(+) and n(−) as described previously [9] . virus titrations were performed in 9-day-old embryonated chicken spf eggs via the allantoic cavity route of inoculation, and titers were expressed as 50% (median) embryo infectious doses (eid 50 ) [9, 37] . serial l0-fold dilutions were used for titrations. at each dilution, five embryos received 0.1 ml inoculum. the embryos were candled daily and examined for one week; those showing characteristic ibv lesions, such as dwarfing, stunting, or curling of embryos, were recorded as infected by ibv. the 10 swab samples taken in the pathogenicity study from each group at each time point were pooled, and the tissue samples of tracheas and kidneys collected 5 days post-challenge from experimental 2 were homogenized individually for virus isolation. for virological examination, the pooled samples were clarified by centrifugation at 300 × g for 5 min and filtered with a teflon membrane. for virus isolation from the trachea and kidney, individual samples the morbidity and mortality were those of p40, p70 and p110 vaccinated chickens after challenge. c number seroconverted/number inoculated. d two procedures were used for virus recovery after challenge. first, lesions in embryos that had been inoculated with individual tissue samples (trachea or kidney) were observed. second, rt-pcr using oligonucleotide primers n(+) and n(−) on rna recovered from allantoic fluid of the same eggs was conducted. the results from the two procedures were identical. e days after challenge. sequence and position of the oligonucleotides used in rt-pcr. oligonucleotide sense a sequence (5 -3 ) gene position in genome b ibv-257 + tattgattagagatgtgg s1 20356-20373 s1oligo3 − cataactaacataagggcaa s1 22002-22021 ibv-167 + gcttcttgagaa (t/c) were homogenized, diluted 1:10 with pbs, clarified by centrifugation at 300 × g for 5 min and filtered with a teflon membrane. the filtered samples were inoculated into at least four spf embryonated eggs via the allantoic cavity (0.2 ml per egg). the eggs were candled daily to record embryo mortality, and allantoic fluid from two of the inoculated embryos was collected 72 h post-inoculation for rt-pcr amplification. after 7 days, the remaining embryos were chilled at 4 • c and examined for characteristic ibv lesions such as the dwarfing, stunting, or curling of embryos. embryo mortality recorded in the first 24 h post-inoculation was considered nonspecific. samples were considered negative if the embryos did not show lesions after three blind passages of 7-day duration. a positive sample was recorded if the specific lesions were observed and the rt-pcr amplification was positive. serum samples were assayed using a commercial total antibody elisa (idexx corporation, westbrook, maine, usa) according to the manufacturer's instructions. each sample was usually tested in triplicate. serum-to-positive ratios (s/p ratios) were calculated as described previously [38, 39] . individual serum titers were calculated from these s/p ratios, evaluated as positive or negative, and expressed as od 650 nm values according to the manufacturer's instructions. the strategy for cloning the 3 -7 kb region of ck/ch/lhlj/04v strains was described previously [9] . briefly, four fragments spanning the 3 -7.8 kb region of the ibv genome were obtained by rt-pcr from each of the four virus passage levels. the sequences and locations of the primers used in this study are in table 4 . briefly, viral rna was extracted from 200 l of allantoic fluid from p5, p40, p70 and p110 virus stocks using trizol reagent (invitrogen, grand island, usa) according to the manufacturer's instructions. rna was air-dried for 2-10 min, re-dissolved in 30l rnase-free water and stored at −70 • c until use. reverse transcription (rt) was performed with m-mlv reverse transcriptase (invitrogen, grand island, usa) using the reverse primer n(−). rt procedures were performed using 20 l of rna in a 40 l reaction volume as previously described [40] . each cdna fragment was amplified from rt products by pcr as previously described [9] . pcr products were purified from agarose gels using a dna extraction kit (boehringer mannheim, germany) and sequenced directly or cloned into the pmd-18t (takara, dalian, china) vector following the manufacturer's instructions. rna extraction, cdna generation, pcr amplification and gene fragment cloning and sequencing were independently conducted 4 times for each of the four passages, p3, p40, p70 and p110. the viral stocks used were from independently inoculated embryos. in total, 20 clones of each gene fragment were selected and sequenced for each of the ck/ch/lhlj/04v passages. five clones were selected for sequencing each time. sequences were compiled and orfs were determined using the gene runner program, version 3.00 (http://www.generunner.com). nucleotide and amino acid sequences for the 3 -7.8 kb fragments were assembled and aligned using the megalign program (dnastar). the genomic sequences of the 3 -7 kb region of ibv ck/ch/lhlj/04v p5, p40, p70 and p110 have been submitted to the genbank database and have been assigned the accession number fj641062 and fj821732 to fj821773. in the pathogenicity study, all four ibv strains produced typical ib-induced disease. all chicks exhibited respiratory clinical signs at about 4-15 days post-challenge with all four ibv strains. clinical signs included tracheal rales, watery eyes, nasal mucus, and sneezing, similar to those caused by other ibv strains with affinity for the respiratory tract [39] . all ibv strains caused death 3-12 days post-challenge; however, strain ck/ch/lhlj/04v caused the highest mortality. gross lesions of the dead chickens were mainly confined to the kidneys. the kidney parenchyma of the affected birds was pale, swollen and mottled; tubules and urethras were distended with uric acid crystals. hemorrhagic lesions of the cecal tonsil and respiratory tract were also observed in some of the affected chickens. the clinical signs of the inoculated birds tended to disappear gradually after 20 days of challenge. no clinical signs and gross lesions were observed in the negative-control group (data not shown). all of the challenge ibv strains could be detected in the trachea at 4 days post-challenge by the recovery of the virus using 9-day-old embryos and subsequent rt-pcr; however, strains ck/ch/lhlj/04v and ck/ch/ldl/04ii could be detected in the trachea of the birds at day 12 post-challenge. the virus was not detected in the trachea of the unchallenged negative-control birds. as summarized in table 1 , most of the chicks challenged with the four ibv strains showed no seroconversion at 4 days post-challenge, but antibodies were detected by elisa in most of the birds after 8 days postchallenge. nine-day-old embryonated eggs were used to determine the growth ability of p3, p40, p70 and p110 in vitro. equal doses (10 2 eid 50 ) of each virus at each passage level were used to inoculate three 9-day-old embryos. the inoculated embryos were incubated at 37 • c, and allantoic fluid was harvested at 72 h for virus titration. based on movement and the extent of bleeding, curling, and dwarfing, all of the inoculated embryos were determined to be infected but alive after 72 h. eid 50 was determined for each sample. the titers of ck/ch/lhlj/04v increased gradually from p3 to p110, indicating an increase during serial passage in spf embryonated chicken eggs (fig. 1) . clinical signs, mortality and gross lesions were used to assess the attenuation of ibv ck/ch/lhlj/04v p3, p40, p70 and p110 using spf chickens. as summarized in table 2 , all birds given p3 and p40, and 50% of birds given p70 by oculonasal application showed overt disease, as did the birds challenged with virulent ck/ch/lhlj/04v in the pathogenicity study, in comparison with p110-inoculated and negative control chicks. clinical signs were observed from day 3 or 4, to day 15 post-inoculation. eight chicks inoculated with p3 and two chicks inoculated with p40 died during the experiment. gross lesions of dead chicks were mainly confined to the kidneys, and were similar to those in the birds in the pathogenicity study. in addition, mucous exudate was observed at 5 days post-challenge in the tracheas of all 5 birds inoculated with p3 and p40, while only one of the chickens in the p40-inoculated group exhibited a respiratory lesion. chickens in both the p110-inoculated and negative control groups showed no clinical signs, death or gross lesions. based on the clinical response, mortality and gross lesions, we can conclude that ibv ck/ch/lhlj/04v p40 and p70 were partly attenuated, but p110 was fully attenuated by serial passages in embryos. two criteria were used for evaluating altered immunogenicity of selected ibv ck/ch/lhlj/04v passages. first, serum igg antibodies specific for ibv ck/ch/lhlj/04v were measured with an indirect elisa test. as shown in table 2 and fig. 2 , none of fig. 2 . humoral immune responses in spf chickens inoculated with ibv ck/ch/lhlj/04v passages evaluated by indirect elisa. ten chickens were tested in each inoculated group at 4, 8, 12 and 16 days after inoculation. dashes show the s/p ratios, calculated as described in section 2. serum samples with s/p ratios equal or above the dashes were considered positive, and those below were considered negative. the serum sample s/p ratios of chickens in the negative control group were all below the dashes and are not indicated in figures. the chickens inoculated with the four selected ck/ch/lhlj/04v passages showed seroconversion at 4 days post-inoculation. at 8 days post-inoculation, 80%, 30% and 40% of the p3-, p40and p70-inoculated chickens showed seroconversion, respectively. however, none of the p110-inoculated chickens showed seroconversion at that time. all chickens inoculated with p3 and p40 showed seroconversion from 12 days on, and only 70% and 40% of the p70-and p110-inoculated chickens, respectively, showed seroconversion at 12 days post-inoculation. fewer than 40% of the p110-inoculated chickens showed seroconversion at 16 days postinoculation. however, almost all chickens inoculated with p3, p40 and p70 showed seroconversion at that time point. second, the vaccination-challenge test was used to evaluate the immunogenicity of ibv ck/ch/lhlj/04v after serial passage in spf embryonated eggs. as summarized in table 3 , none of chickens inoculated with p40 or p70 showed clinical signs or death after challenge with the virulent p3 level strain, indicating good clinical protection provided by vaccination with p40 or p70. however, 30% of the p110vaccinated chickens showed clinical signs, and 2 of 10 vaccinated chickens died after p3 challenge. parallel to the clinical protection results, vaccination with p40 and p70 also offered good trachea and kidney protection against virulent p3 challenge, although one of the p70-vaccinated chickens was positive for virus recovery in the trachea after p30 challenge. in contrast, more than 50% of the p110vaccinated chickens were positive for virus recovery from both tracheas and kidneys after challenge with the virulent p3 strain, indicating poor trachea and kidney protection after p110 vaccination. based on these results, the partly attenuated p40 and p70 viruses were considered capable of stimulating systemic immunity in chicks; however, immunogenicity against the fully attenuated p110 decreased as expected. viruses were identified by rt-pcr at 5 days post-inoculation in both the tracheas and kidneys of the p3-, p40-, p70-and p110inoculated chickens. all trachea samples, and kidney samples from p3-and p40-inoculated chickens, were positive by pcr; however, only one kidney sample from p70-and p100-inoculated chickens was positive, respectively. all trachea and kidney samples were titered at that point, and as shown in fig. 3 , viral titers steadily decreased with passage. p40 had almost the same titer in the trachea as p3; however, the titers of p70 and p110 in the trachea were lower than those of p3 and p40, as expected. it is puzzling that viral titers in the kidneys rapidly decreased and were eventually lost when the corresponding virus adapted to embryonated eggs. compared to the virulent p3 virus, p40 showed a lower titer in the kidneys, while p70 and p110 had already lost kidney tropism after serial passages in spf embryonated chicken eggs (fig. 4) . the ibv ck/ch/lhlj/04v strain was propagated in embryonated eggs and passaged 110 times. to investigate whether nucleotide and/or amino acid sequences changed during passage, p3, p40, p70 and p110 were chosen for rt-pcr amplification and sequence analysis of the 3 -7 kb region. all rt-pcr products were analyzed, and a single band of expected size was visible after ethidium bromide staining of the products on a 1.0% agarose gel. twenty independent clones of each fragment from four independent rt-pcrs were selected and sequenced, so the sequence profiles represented all genetic diversity within the populations of the viral rnas from a given passage. as summarized in table 5 , no sequence changes were observed in orf 3a or gene 5 (orf5a and orf5b) after 110 passages. only one to two nucleotide mutation(s) occurred in the orf 3b, 3c (e), m and n genes after 110 passages, and all amino acid substitutions in those regions occurred between p70 and p110. however, most nucleotide mutations were found in the s region between p3, p40, p70 and p110 and all of the nucleotide mutations resulted in amino acid substitutions. significantly, a majority of the sequence changes in the s1 region of the s protein occurred between p3 and p40. most of the sequence changes in the s2 region were observed between p70 and p110, however. in addition, a single base mutation (g → t) was observed in 10 out of 20 clones of p3, and this mutation was located at the 3 -terminal region of the s gene, at nucleotide 3469 from the aug start codon of the s1 gene. this caused a frameshift that would result in a c-terminally truncated product, if synthesized. the truncated product would contain 1157 residues instead of the 1166 residues of the normal s protein (fig. 5) . furthermore, the s proteins of p40, p70 and p110 all contained the mutation for truncated s. to investigate whether serial passage of ibv ck/ch/lhlj/04v in embryonated eggs can cause sequence changes in the 3untranslation region (3 -utr), the 3 -utr sequences of p3, p40, p70 and p110 were amplified and sequenced. surprisingly, a 109-bp deletion, located 8 nucleotides downstream of the stop codon of the n protein gene, was found in the 3 -utr of 70% and 85% clones of p70 and p110, respectively, as shown in fig. 6 . interestingly, both the deleted and non-deleted 3 -utr sequences of the secreted offspring viruses were detected in the respiratory tracts of the chickens inoculated with ibv ck/ch/lhlh/04v p110 at 5 days post-inoculation (data not shown). nucleotide and amino acid changes in the 3 region of ibv ck/ch/lhlj/04v and embryo-passaged derivatives. position (nt) a nucleotide change codon change amino acid change ibv ck/ch/lhlj/04v is a nephropathogenic ibv strain of the lx4type that is highly pathogenic to spf chickens, with 100% morbidity and approximately 40% mortality. lx4-type ibv has been one of the major types of ibv circulating both in china and european countries in recent years [34, [41] [42] [43] [44] . however, both experimental infections and field results have shown that available commercial vaccines provide poor protection against the lx4-type ibv [39] . hence, we selected the lx4-type ibv ck/ch/lhlj/04v strain for attenuation by serial passage in spf chicken embryonated eggs. coronaviruses, including ibv, have been shown to exist as a mixture of genetic mutants within an isolate [45] [46] [47] . this is also the case for the ibv isolate ck/ch/lhlj/04v. based on sequence data from cloned rt-pcr products, 50% of the sequences in the low-level virus populations (p3) showed a 9-bp deletion upstream of hypervariable region 1 (hvr1) of the s1 gene compared to the other sequences in the same population. however, this deletion was not observed in all of the s1 sequences in the high-level virus populations (p40, p70 and p110). we cannot determine if this reversion of the s1 gene sequence was due to a recombination event or the selection of a subpopulation in the process of serial passage in chicken eggs. in addition, a stop codon was observed in 50% of the s protein sequences of p3 at residue 1157 due to a mutation changing a glu codon to a stop codon, resulting in an s protein with 9 amino acids missing at its carboxy-terminal end. all sequences in p40, p70 and p110 showed this mutation. this indicates that this region is not necessary for virus formation. the above mentioned s gene sequence heterogeneity indicated the presence of different proportions of subpopulations in ck/ch/lhlj/04v. most genetic changes occur in the s1 gene during adaption to the host [19, 47, 48] . however, until now, it was not clear if mutations or the selection of a fit subpopulation was responsible for the changes observed when coronaviruses were attenuated or adapted to a particular host system [48] . routinely, ib attenuated vaccines are developed by multiple passages (generally 52 or more) of a field isolate, in embryonated domestic fowl eggs, until the desired blend of non-pathogenicity and immunizing capacity has been achieved. the mutations that cause the attenuation of pathogenicity are not known. we found 13 amino acid substitutions between pathogenic ck/ch/lhlj/04v p3 and its embryo-passaged, attenuated derivative p110. of these substitutions, 10 were in the s protein (5 substitutions each in the s1 and s2 regions). importantly, a common substitution at residue 132 in the s1 protein (his to tyr), found in the d207 and tw2296/95 ibv strain and related to antibody attachment [49] , was also found in the s1 protein of ck/ch/lhlj/04v after 40 passages. this substitution was maintained for p70 and p110, indicating that this residue was likely to be important for virus pathogenicity. however, other amino acid substitutions in the s1 protein were not observed between the m41 vaccine strain and m41 challenge strain [50] , the 4/91 pathogenic and 4/91 attenuated strains [9, 51] , the tw1171/92 pathogenic and tw1171/92 attenuated strains, the tw2296/95 pathogenic and tw2296/95 attenuated strains, the tw2575/98 pathogenic or tw2575/98 attenuated strains [48] , or the arkdpi strain [52] . investigations with other coronaviruses have shown that the s protein is a determinant of pathogenicity; however, the replacement of the s protein gene of the apathogenic beaudette strain with that of the pathogenic m41 strain resulted in a recombinant virus that was still not pathogenic [53] . thus proteins other than, or in addition to, the s protein must affect pathogenicity. our findings were consistent with this result. we found that nearly all of the amino acid substitutions in the s1 subunit were between p3 and p40, and other substitutions were between p70 and p110. the virus was still pathogenic to chickens after 40 passages and it was fully attenuated after 110 passages in embryonated eggs. the structural e gene in sars-cov is a virulence factor, and a sars-cov that lacks the e gene is attenuated in vitro and in vivo [54] . in this study, a synonymous mutation in the e gene was found between p3 and p40, and three substitutions in the 3a, m and n proteins were also observed between p70 and p110. nonetheless, the relationship between those substitutions and virus pathogenicity is unknown. a single mutation, tyr6398his, in mouse hepatitis virus mhv-a59 nsp14 resulted in attenuated virus pathogenesis in mice [55] . sequence changes in the 5 -two-thirds of the genome, which contains two overlapping replicase genes, were not investigated in this study and should be further studied. an additional orf, detected within the 3 -utr of several ibv isolates, and with the potential to encode hydrophobic proteins is referred to as orf7 [48] . it was also observed in the ibv ck/ch/lhlj/04v strain. in other coronaviruses, such as porcine transmissible gastroenteritis virus (tgev) and fcovs, a relationship between gene 7 and virus virulence has been observed [49] , although it is difficult to compare the hydrophobic proteins of these coronaviruses directly with that of ibv. until now, the corresponding protein and its ibv function have not been clear. however, it is hypothesized that the sequences in the ibv 3 -utr are involved in regulating viral rna replication and transcription [56] . in addition, sapats et al. [57] reported that shorter forms of the 3 -utr in the australian n1/88, q3/88, and v18/91 strains are associated with a decrease in virulence. huang and wang [48] found a 49-bp deletion in the 3 -utr immediately downstream from the n protein at passage 76 of strain tw2296/95, that is not present in the pathogenic parent. in this study, we observed a longer (109-bp) deletion at the same position in the 3 -utr in most subpopulations of p70 and p110 (70% and 85%, respectively), indicating that not only this deletion, but also the size of the deletion may be correlated with ibv attenuation. in addition, both the deleted and non-deleted sequences in the 3 -utr of the offspring viruses were detected in the respiratory tracts of chickens inoculated with p110 at 5 days postinoculation, implicating that this deleted region is not necessary for viral replication in the chicken respiratory tract. although multiple passage of a field isolate in embryonated domestic fowl eggs is the usual method for development of ib attenuated vaccines, not all ibv strains stimulate the immunity after serial passages. the beaudette strain is apathogenic in chickens after many serial passages in embryonated chicken eggs [58] . in addition, this embryo-passaged virus is considered to be poorly immunogenic [58] , and consequently, has never been used as a vaccine strain. this is also the case with the ibv ck/ch/lhlj/04v strain. the immunogenicity of the virus has been gradually decreased by serial passage in embryonated eggs. the ck/ch/lhlj/04v p110 did not confer immunity to spf chickens when compared to p3, p40, p70 and the negative control. the reduced immunogenicity of the attenuated virus may correlate with its reduced replication efficiency and infectivity in chickens. the s glycoprotein induces protection against virulent challenge, and several epitopes that induce virusneutralizing antibodies have been mapped within the s protein [59] [60] [61] [62] [63] [64] . these epitopes showed the importance of inducing the ctl response in primary infections and neutralizing the antibody response against secondary exposure to the same virus [65, 66] . in this study, 3 amino acid substitutions were observed in the abovementioned epitopes of s1 protein, and no substitutions were found in the epitopes residues of the s2 protein. in addition, n is another important protein that induces protection in ibv. in this study, a substitution (tyr → the) was observed in the n protein between p70 and p110 viruses. this substitution was located in an identified epitope in the ibv n protein that induces a t-cell response and protection [67, 68] . the amino acid substitution at residue 188 (thr to ile/ala) in the m protein, which was observed to be related to antigenicity and/or virulence of ibv strains h52/h120, tw2296/95 and arkansas [48, 69] , was not observed in our ck/ch/lhlj/04v strain. the ibv strains, as a group, infect a large range of epithelial surfaces, literally from the top to the bottom of the chicken. isolates differ in their extent of replication in non-respiratory tissues, and some produce clinical disease in non-respiratory tissues, most notably the kidney and proventriculus. ck/ch/lhlj/04v is a nephropathogenic strain; however, it lost kidney tropism after 70 serial passages in embryonated eggs. using a reverse genetics system, the s glycoproteins for a group 2 coronavirus (mhv), a group 1 coronavirus (tgev) and a group 3 coronavirus (ibv) were demonstrated to be involved in the tropism of these coronaviruses [26] [27] [28] [29] [30] 68, 70] . for the tgev, several amino acid changes at the nterminus of the s protein resulted in the loss of enteric tropism [29, 30] . in this study, an amino acid substitution was found at residue 581 (leu → phe) between p70 and p110; however, loss of kidney tropism of ck/ch/lhlj/04v occurred between p40 and p70. the only amino acid difference between p40 and p70 was at residue 58; however, this residue is not likely to be a determinant of tissue tropism of ck/ch/lhlj/04v, because some viral subpopulations in p70 and p110 showed this change. several other substitutions were found in s and other proteins in this study. further investigation by reverse genetics and animal studies is needed to verify the exact function of substitutions. in this study, the titers of the embryo-adapted ibv ck/ch/ lhlj/04v strain increased gradually with the serial passage in embryonated eggs, indicating that the virus had a high replication capacity in vitro; however, its capacity for in vivo replication decreased dramatically. the n, m and e proteins of ibv play a role in viral replication and assembly [2] . it is difficult to conclude that the decreased replication of the virus in vitro in this study was due to substitutions in m and n proteins. a point mutation in the coronavirus hcov-229e and the arterivirus eav nendou (nsp15) resulted in a lack of viral genome replication and transcription, indi-cating that this rnase mostly affected viral production [71] [72] [73] . it is unclear whether this is the case for ibv ck/ch/lhlj/04v. the embryo-adapted ibv strains appeared to contain a mixture of genetic variants, and selection and mutations occurred in the viral populations during the passages in the embryos. in the process of serial passage, almost all of the amino acid substitutions in s1 proteins occurred between p3 and p40, and all the subpopulations in the virus passages showed those substitutions; however, other substitutions were found between p70 and p110 and only parts of the subpopulations in the virus passages showed those substitutions. the exact roles of different subpopulations in changes in virus replication, pathogenicity, antigenicity, immunogenicity and tissue tropism are unknown; we have not succeeded in isolating the different subpopulations from the virus population by limited dilution (data not shown). understanding the molecular mechanism of ibv attenuation, tissue tropism and immunogenicity changes is important, because not only is this virus of economical importance to the poultry industry, but it also shows antigenic and biological similarities and differences to other coronaviruses. although it is reasonable to conclude that some of the few sequence changes described in this study in the 3 -7 kb region are responsible for virus attenuation, decrease in immunogenicity and tissue tropism changes, we cannot conclude that they are the only predictors for these changes. we also cannot completely exclude the possibility that other parts of the genome are responsible for the observed changes, because ib coronavirus has a large genome (27.6 kb) . in addition, similar to other reports [19, 47, 48, 59] , we found that none of the sequence changes were shared by all pathogenic ibv strains and their attenuated derivatives, indicating that there may be many factors and pathways that affect virus replication, pathogenicity, antigenicity, immunogenicity and tissue 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the arterivirus life cycle key: cord-260042-cs0wp99n authors: khan, samiullah; roberts, juliet; wu, shu-biao title: genes involved in mitochondrial biogenesis and function may not show synchronised responses to mitochondria in shell gland of laying chickens under infectious bronchitis virus challenge date: 2019-04-01 journal: bmc mol cell biol doi: 10.1186/s12860-019-0190-7 sha: doc_id: 260042 cord_uid: cs0wp99n background: egg formation takes place in the oviduct of laying hens over a 24 h period. infectious bronchitis virus (ibv) causes pathological lesions in the chicken oviduct. in the current study, mitochondrial counts were determined in three different segments of the oviduct during egg formation in laying chickens challenged with ibv t strain. nuclear dna encoded genes that are involved in mitochondrial biogenesis, fission and function were studied in the shell gland of the oviduct undergoing virus multiplication. results: in the shell gland, the mitochondrial count was significantly lower (p < 0.05) in the challenged group, compared with the control group. however, it did not vary in response to ibv challenge in the isthmus and magnum regions of the oviduct. the gene succinate dehydrogenase complex, subunit a, flavoprotein variant (sdha) was down-regulated in the shell gland by ibv challenge (p < 0.05), while other genes being studied did not show responses to the challenge (p > 0.05). differential expression of the genes was observed at different time-points of egg-shell formation. the expression levels of citrate synthase (cs), cytochrome c, somatic (cyc, s) and sodium-potassium adenosine triphosphatase (na(+)-k(+)atpase) genes were significantly higher, while those of sdha and dynamin related protein 1 (drp1) genes were significantly lower, at 15 h compared with 5 h following oviposition of the previous egg. the expression level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (pgc-1α) did not show significant change at different time-points. conclusions: it was concluded that ibv t strain infection in laying hens reduced mitochondrial counts only in the shell gland region of the oviduct. the genes involved in mitochondrial biogenesis or function may not show synchronised responses to that of mitochondria in the shell gland of chickens under t strain of ibv challenge. the infundibulum, magnum, isthmus and shell gland (uterus) contribute to egg formation in laying chickens. egg-shell formation takes place in the isthmus and shell gland regions, where the former contributes only to shell membrane formation. egg formation involves the production of steroid hormones, which regulate the proliferation and growth of oviducal epithelial cells throughout the oviduct. for example, the administration of estrogen and/or progesterone leads to massive growth of the epithelia in the oviduct of juvenile hens [1] [2] [3] . an egg is composed of egg internal contents and egg-shell. among the internal contents, albumen is secreted by the magnum and is composed mainly of ovalbumin, muco-proteins and globulins [4] . the shell membranes are synthesized in the isthmus region of the oviduct and contain collagen proteins in its composition [5] . egg-shell synthesis occurs in the shell gland and is composed mainly of calcium carbonate [6] and shell matrix proteins, such as the ovocalyxin family [5] . mitochondrion acts as a powerhouse of the cell, where it plays a vital role in cellular metabolism, calcium handling, heme biosynthesis, cell differentiation, apoptosis and aging [7] [8] [9] . mitochondrial count in a cell varies in different cell types [10, 11] , different organs, the sex and age of an organism [12, 13] and physiological and disease conditions [10, 14] . a higher mitochondrial respiratory capacity and mitochondrial counts have been recorded in more physically active humans [15, 16] . similarly, increases in mitochondrial dna (mtdna) have been observed during myogenesis in rats [17] and in brown adipocytes of various mammals in response to low temperature [18] . during the cell cycle, mitochondria undergo changes in shape, count and location; however, it is not known how quickly mitochondria divide in metabolically active and inactive cells. in the reproductive track of laying chickens, it is unclear how mitochondria respond to the need of the cells for atp by either inducing mitochondrial division or accelerating mitochondrial function without division. cells can respond to alterations in mitochondrial function by up-or down-regulations in the expression of nuclear-dna encoded gene [19] . mitochondrial transcription factor a (mttfa) encodes a protein that in conjunction with nuclear-dna polymerase γ (polγa) control mtdna replication in a cell [20, 21] . mtdna replication machinery synthesizes different proteins that include the single-stranded dna binding protein (mtssb), the catalytic subunit of dna polγa and processivity factor, the mitochondrial rna polymerase and the mitochondrial replicative helicase twinkle [22, 23] . dynamin related protein 1 (drp1 also called dnm1l) is the main regulator of mitochondrial division in a cell, which is oligomerized by mitochondrial division protein 1 (mdv1) bound to the outer membrane of mitochondria via mitochondrial fission 1 protein (fis1) [24] [25] [26] [27] . nuclear genome of the cell encodes proteins essential for mtdna maintenance and replication [23] . the synthesised proteins are translocated into the mitochondria after their packaging in the cytoplasm. therefore, expression of genes coded by mtdna and nuclear genomes are accurately coordinated for regulating the electron transport linked phosphorylation capacity in response to changes in physiological demands of cells [28] . studies in a single celled green algae (chlorella) [29] , hela cells (strain-f315) [30] and the opportunistic pathogenic dimorphic yeast (candida albicans) [31] indicate that increase in total mitochondrial count per cell occurs continuously during the cell cycle. this increase in mitochondrial counts is usually positively correlated with the increase in cell volume. in hela and dimorphic yeast cells, mitochondrial count is comprised of approximately 10% of the total cell volume while, in algae, this value is about 3% but is constant throughout cell division [29] [30] [31] . citrate synthase (cs) gene encodes an enzyme that is localised in the mitochondrial matrix after being shaped in the ribosome [32] . in mammals, the cs gene has been used extensively as a marker for intact mitochondrial density [33] [34] [35] ; however, its role has been questioned in studies of developmental stages [36] , age of animal [37] and physical activity [38] . cs has been shown to be positively correlated with acute exercise activities in mammalian muscles [39, 40] . succinate dehydrogenase complex, subunit a (sdha) gene, encodes a major catalytic subunit of succinate-ubiquinone oxidoreductase, a complex of the mitochondrial respiratory chain. sdha gene provides instructions for synthesizing one of four parts (subunits) of the succinate dehydrogenase (sdh) enzyme. sdh then participates in both the electron transport chain and the krebs cycle. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (pgc-1α/ppargc1a) is the master regulator in mitochondrial division [41] . in vitro studies in muscle cells [42] , c2c12 myoblasts [43] or in mice cardiac myocytes [44] have shown that pgc-1α is capable of activating the expression of a cascade of genes involved in mitochondrial synthesis and respiratory function in different types of cells. therefore, pgc-1α is mainly involved in mitochondrial energy metabolism and mitochondrial biogenesis. cytochrome c somatic (cyc, s) gene encodes cytochrome c enzyme that plays a role in the electron transport chain complex in mitochondria and during apoptosis. na + -k + atpase gene encodes an enzyme that is an integral membrane protein which consists of α and β subunits [45] . this enzyme plays an essential role in maintaining the transmembrane gradient of na + and k + ions in cells [46] . infectious bronchitis virus (ibv) is a highly contagious avian mucosal pathogen that not only affects layer and broiler chickens but also other avian species worldwide [47] . similar to other coronaviruses, ibv is composed of a small membrane protein (e), the integral membrane protein (m), the nucleoprotein (n) and the spike glycoprotein (s) [48, 49] . the s protein is composed of two subunits, the s1 (head) and the s2 (stalk) that is responsible for binding to the target cell receptor in host [50, 51] , while the n protein induces cell mediated immunity [52] . there is not much information about whether ibv entry and fusion to host cells occurs following endocytosis or at the cell membrane [51, 53] . host cell neutral ph is considered lethal for fusion of the virus particle [54] . among other host cell surface receptors, sialic acid has been shown to act as a receptor for binding of ibv spike proteins in respiratory, kidney and oviduct epithelia [55] [56] [57] [58] . ibv can infect any age of birds; however, the mortality is higher in very young chicks compared with older birds [59] . mortality rates depend upon viral strain, birds age and immune status of the bird [60] . among all the field strains of australian ibv, t (n1/62) is considered the most virulent in inducing pathological changes in the tracheal, kidney and oviduct epithelia of laying hens. infection with ibv in the oviduct leads to various degrees of pathogenesis in the oviduct and reduction in egg production [61] [62] [63] [64] [65] . the present study aimed to: a) determine mitochondrial counts in the cells of oviduct segments in laying hens at different time-points of egg formation in relation to the requirement of energy for egg production during ibv challenge; b) to determine the expression of nuclear dna encoded genes in the shell gland to gain insights into their responses to ibv infection and time-points of egg-shell formation. day-old isa-brown laying chickens were vaccinated with rispens vaccine for marek's disease but were not vaccinated for infectious bronchitis. at the university of new england, the chickens were raised in ibv free isolation sheds following strict biosecurity protocol. the birds were reared as per the guidelines of the isa general management guide 2009-10. from the isolation sheds, pullets (18-week old) were moved to cages in an isolated poultry house. during the rearing period, no morbidity or mortality was recorded until the birds were challenged with ibv. before ibv challenge, an elisa (elisa kit, idexx laboratories, inc., westbrook, ma, usa) was performed on blood serum of all the chickens. at 35-week of flock age, eggs were processed for egg quality parameters following the method of samiullah et al. [66] . chickens were allocated into treatment groups (table 1) by 2 × 2 factorial design based on egg-shell colour (l*) and egg weight (g) that were not significantly different (p > 0.05) among the treatment groups (data not shown). the hens selected for the virus inoculation were transferred to a separate layer cage house one week before the challenge in order for the chickens to adapt to the new shed and recover from the translocation stress. in both the virus challenge and the control groups, five chickens from each time-point at one time were inoculated intra-occularly with either 10 7 embryo infective dose (e.i.d 50 )/bird of allantoic fluid or mock infected (pbs). the challenged chickens were closely observed for the development of clinical signs of ib [65] and loss of egg-shell colour until days 9-10 post-infection (p.i.). in a separate experiment, the e.i.d 50 dose was calculated from virus being titrated in embryonated spf-eggs. the incubated eggs were inoculated at day-9 with 10 − 3 to 10 − 8 serial dilutions of ibv t strain (n1/62) [67] . on day-16 of incubation, the eggs were opened and the number of dead, live or ibv affected embryos recorded after the method of reed and muench [68] . the virus infection in the chickens was confirmed through quantitative pcr (in oviduct tissue) and by elisa of blood plasma samples. for accuracy of sampling, the oviposition times for individual hens were recorded by video camera. at specific post-oviposition times (5 or 15 h), hens were euthanized with co 2 gas and approximately 500 mg pieces of tissue from each of the shell gland, isthmus and distal magnum were aseptically collected into rnalater. the tissue samples were stored in a − 20°c freezer until processed for dna/rna extraction. for the total genomic (including mitochondrial) dna extraction from the oviduct tissue (all tissue layers), the trisure (bioline, australia) protocol was slightly modified for obtaining pure nucleic acids. briefly, a 100 mg of tissue was thoroughly homogenized in 1 ml of trisure. for obtaining quality dna/rna, the trisure added samples were maintained on ice and the homogenisation step was also performed in a tube containing ice. for total rna extraction, after the chloroform step, the transparent upper layer was used (see below). for dna during dna extraction, the upper layer after chloroform treatment was processed for total rna extraction according to the manufacturer's instructions (trisure protocol). the rna was precipitated with 0.5 ml chilled iso-propanol and the pellet washed with 1.5 ml of 75% ethanol. the rna pellet was dissolved in 100 μl of pcr grade water (rnase/dnase free) and processed for rna purification using an rneasy mini kit (qiagen, australia) as per the manufacturer's instructions. the purified total rna was analysed in a nanodrop as described earlier. rna quality and concentration were further analysed in an agilent 2100 bioanalyzer using the agilent rna-6000 nano kit as per the manufacturer's protocol. all the samples showed distinct 18s and 28s bands with an average rin of ≥9.1. to quantify the viral rna from the oviduct segments, epithelial scrapings were also processed for total rna extraction following a partial modification of the method described by chousalkar et al. [69] . approximately 1 g of oviduct scrapings (magnum, isthmus and shell gland segments) was mixed with 1 ml of sterile pbs, shaken vigorously and centrifuged at 4800×g for 10 min at 4°c. from the supernatant, 200 μl was mixed with 1 ml of trisure and total rna was extracted as per the manufacturer's protocol. the extracted total rna was used for viral rna quantification from oviduct tissue scrapings. the primer sequences shown in table 2 were designed using ncbi-primer software with the design of at least one primer sequence spanning the exon-intron junction or the amplicon of the primers spanning over two exons with the intron in-between at size of at least 500 bp. for sequence specificity, the primers were cross checked in ensemble chicken galgal4, ncbi database using blastn and ucsc's chicken (gallus gallus) genome browser gateway. prior to real-time qpcr analysis, the primer specificity and amplification efficiency were determined in serial dilutions (10 − 1 to 10 − 8 ) of the purified rna/dna. the primer amplification efficiency (%) was analysed based on the mathematical equation [70] : e = 10 (1/slope) -1. for mtdna copy number quantification through qpcr, the 137 bp fragments of each of the gapdh and nd4 genes were ligated into plasmid vector using the rapid one shot chemical transformation protocol of the topo ta cloning kit for sequencing (thermofisher scientific, australia) as per the manufacturer's protocol. next, the recombinant plasmid was transformed into one shot chemically competent escherichia coli cells. a 50 μl of the cells was plated on difco luria-bertani (lb) agar (bacto laboratories, australia) and incubated overnight at 37°c. from the grown colonies, a single colony was enriched in lb broth and incubated overnight at 37°c. from the cultured broth, 1 ml was used to extract recombinant plasmid dna using the purelink quick plasmid miniprep kit (thermofisher scientific, australia) as per the manufacturer's protocol. the eluted recombinant plasmid dna purity and concentration were analysed in the nanodrop as described earlier. the recombinant plasmid dna samples were stored at − 20°c until used for qpcr assays. to check the amplification efficiencies for the standard curve construction of gapdh and nd4, qpcr was run on the recombinant plasmid dna (10 − 2~1 0 − 8 ) in a 20 μl pcr reaction. the master-mix preparation and qpcr cycling conditions were as per the protocol of the sensifast sybr no-rox kit (bioline, australia). for standard curve construction, eight different serial dilutions (10 − 2~1 0 − 9 ) of the recombinant plasmid dna were prepared and qpcr amplified. to construct the standard curves for gapdh and nd4, quantification cycles (cq) were plotted against log 10 copy number of plasmids calculated according to the molecular weight of recombinant plasmids [71] . the amplified pcr products were sequenced by the australian genome research facility, australia for confirmation of gapdh and nd4 fragment inserts into the plasmid vector. for mitochondrial count per cell enumeration, mtdna copies in a cell were normalised with genomic dna copies [71, 72] . the sybr green method of the sensifast sybr no-rox kit (bioline, australia) was followed for qpcr as per the manufacturer's protocol. the qpcr was performed in a rotor-gene q thermocycler (qiagen, australia) in a total volume of 20 μl master-mix reaction. the reaction consisted of 10 μl 2× sensifast sybr no-rox mix, 6.4 μl rnase-free pcr grade water, 0.8 μl each of the primers and 2 μl of the dna (10 − 2 dilution of the extracted dna). to rule out external contamination, a negative control reaction with no dna template was included in each qpcr run. for standard curve construction and mitochondrial count per cell calculation, recombinant plasmid dna dilutions (10 − 2~1 0 − 9 ) were included in the respective qpcr runs. the conditions for a 2-step qpcr were: denaturation at 95°c for 3 min, 40 cycles of denaturation at 95°c for 5 s and annealing and extension at 60°c or 63°c for 30 s ( table 2 ). fluorescent data were acquired at the end of each annealing/extension step during qpcr cycles (40) . the mtdna copy number per cell in the magnum, isthmus and shell gland was calculated according to the equation: (mtdna copies)/(gdna copies/2) [71] . for the relative expression of gene studies, shell gland rna samples were run in duplicate with the inclusion of appropriate internal controls. the qpcr master-mix was prepared as per the manufacturer's protocol of sen-sifast sybr lo-rox one-step rt-pcr kit (bioline, australia). the pcr final 20 μl volume reaction contained 10 μl of 2× sensifast sybr low-rox one-step mix, 0.4 μl of ribosafe rnase inhibitor, 0.2 μl of reverse transcriptase, 0.8 μl of each of the forward and reverse primers, 3.8 μl rnase-free pcr grade water and 4 μl of rna template (10 − 2 dilution). using a qiagility robotic, the reaction volume was distributed into rotor-gene disc 100 (qiagen, australia) and run in a rotor-gene q thermal cycler. the two-step pcr conditions were: reverse transcription at 45°c for 10 min, polymerase activation and denaturation at 95°c for 2 min, 40 cycles of denaturation at 95°c for 5 s and annealing and extension at 60°c, 61°c or 63°c (according to table 2 ) for 20 s. the fluorescent data collection, melting curve analysis and amplification efficiency calculation were performed as described previously. viral rna quantification from oviduct tissue ibv t strain (kindly provided by csiro, geelong, australia) was cultured in 11-day-old spf embryonated eggs and allantoic fluid was harvested at day-16 of incubation. viral rna was extracted from the allantoic fluid using trisure as per the manufacturer's protocol. a 181 bp fragment of viral rna was amplified using the sensi-fast sybr lo-rox one-step rt-pcr kit as per the manufacturer's instructions and cloned (using rapid one shot chemical transformation protocol of topo ta cloning kit for sequencing) into a plasmid vector for standard curve construction. details of the cloning method have been described in a previous section. a standard curve was constructed from 10-times serial dilutions (10 − 2~1 0 − 9 ) of recombinant plasmid dna cloned with 181 bp fragment of ibv t strain. approximately 1 μg rna from individual samples extracted from the magnum, isthmus and shell gland whole tissue (all tissue layers) or mucosal scrapings was reversely transcribed into cdna using the quantitect reverse transcription kit (qiagen, australia). quantitative pcr was performed with the sybr green method using the sensifast sybr no-rox kit. quantitative pcr reaction was performed in a total volume of 20 μl in duplicate on a rotor-gene q thermal cycler and cloned plasmid dna was included in the same run as standards. the cloned plasmid dna with insert was calculated as number of copies per μl in six different dilutions for standard curve construction. plasmid copy number was calculated based on the concentration of plasmid dna and its molecular weight [71] . individual sample amplicons from all the three segments of the oviduct were run in the bioanalyzer as described earlier, to assess the specificity and size of the virus nucleic acid fragment. viral load was calculated as cdna copies per μl of pcr reaction volume multiplied by the total reaction volume, rna elution volume and weight of the tissue used for rna extraction. viral load was expressed as viral cdna copy number per gram of oviduct tissue. to determine the mitochondrial count per cell, the mtdna copies per cell were analysed in statview software (sas institute inc., version 5.0.1.0) taking the time-point and virus challenge as main effects. the statistical significance (p < 0.05) between mean values was determined by the tukey-kramer test. for gene expression data analysis, raw cq values were analysed in qbase+ software version 3.0 against tbp and ywhaz as reference genes [73] . the analysis was based on relative expression (2 -δδcq ) using gene specific amplification efficiencies [74] [75] [76] . to determine the effect of time-point and ibv challenge, from the qbase+, normalised relative quantities (nrq) were exported and analysed in statview version 5.0.1.0 (sas institute inc., 1998) using one-and two-way anova. level of significance (p < 0.05) between the mean values was determined by tukey-kramer test. all the primers used in this study were specific in amplifications of the expected products (fig. 1) . the amplification efficiency of the primers ranged from 94 to 104% (table 2) . ibv t rna was not detectable in any of the three segments of the oviduct (all tissue layers) samples processed for real-time qpcr. however, viral rna was detected in the shell gland epithelial scrapings. all samples from challenged birds were positive with a mean viral load of approximately 3.55 × 10 6 copies per gram of shell gland tissue. viral rna was not detected in the epithelial scrapings of the magnum and isthmus segments of the oviduct of any of the challenged birds. a significantly higher titre of antibodies for the challenged birds in the elisa test confirmed that the virus multiplied and caused a significant immune response in the challenged birds (fig. 2) . during the post-challenge period, all infected birds showed characteristic clinical signs of ib. no viral rna was detected in the control birds as expected. the mitochondrial count in the shell gland region of the oviduct was significantly lower (p < 0.05) in the ibv t strain challenged group compared with the control group (fig. 3a) . ibv t challenge did not significantly affect mitochondrial counts in the isthmus and magnum regions of the oviduct (fig. 3b, c) . the mean mitochondrial count (per cell) was not significantly affected (p > 0.05) by time-points of egg formation in any of the three segments of the oviduct (fig. 3) . there was no significant interaction between the time-point and ibv t strain challenge for mitochondrial counts in the shell gland, isthmus and magnum regions of the oviduct. significant interactions (p < 0.05) between ibv challenge and time-point of egg-shell formation were observed for the expression of sdha and na + -k + atpase (table 3 ; fig. 4) . the relative expression level of sdha was significantly lower in the virus challenged groups compared with the control group at the 5 h time-points, whereas this effect was not observed at the 15 h time-point of egg-shell formation. the relative expression level of na + -k + atpase was significantly lower in the virus challenged compared with the control group at the 15 h time-point, whereas at the 5 h time-point, this effect was not significant (fig. 4a, b) . the relative expression levels of all of the genes except the sdha were not significantly affected (p > 0.05) by ibv challenge ( table 3 ). the expression levels of all the genes except pgc-1α were significantly affected by time-point of egg-shell formation. the expression levels of cs, cyc, s and na + -k + atpase genes were significantly higher, while those of sdha and drp1 genes were significantly lower, at the 15 h compared with the 5 h time-point of egg-shell formation (table 3 ). we investigated the mitochondrial counts in three different segments of the oviduct of laying hens challenged with ibv t strain, at two stages of egg formation. a significantly lower mitochondrial count in the shell gland of challenged hens suggests that the virus had a greater effect on this region, as compared with the isthmus and magnum segments of the oviduct. a greater severity of permeability transition pore, oxidative balance, mitochondrial membrane potential, electron transport and energy production at the cellular level [78] . it is not clear how ibv t challenge led to the reduction of mitochondrial counts in the cells; however, in previous studies, degenerated mitochondria in ibv infected cells of shell gland tissue of laying hens have been recorded [64] . in the current study, it seems that the viral load in the shell gland tissue was very low at days 9-10 p.i. and therefore was not detectable in the shell gland tissue containing all tissue layers. however, the mean viral load was 3.55 × 10 6 copies per gram in the epithelial mucosa (epithelial scrapings) of the shell gland region of the oviduct, indicating that shell gland had succumbed to viral infection particularly in the epithelial layer. based on the lack of significant differences in mitochondrial count in the cells between the challenged and control groups for the magnum and isthmus, we focused further on shell gland tissue and studied the expression level of genes involved in mitochondrial density, biogenesis and fission. the lack of any significant difference in the relative expression levels of all of the genes except sdha, between the control and ibv t challenged groups, may indicate that mitochondrial function may have been enhanced and thus overall egg quality may not have been affected by fewer mitochondria in the shell gland cells of ibv t infected hens. sdha functions in mitochondrial complex ii, a part of the citric acid cycle and electron transport chain. a significantly lower expression level of the sdha in the infected group correlates with the lower number of mitochondria. however, to confirm this correlation, further research is needed as the remaining genes studied were not affected by the virus challenge. it also seems that the virus effect on the expression level of sdha was not consistent, as the mrna was significantly lower in the virus challenge group at the 5 h but not the 15 h time-point. cs enzyme activity has been widely used as a marker for intact mitochondrial density. the activity of cs was 42% higher in the leg muscle of dominant versus subordinate male red jungle fowl, with no differences in overall muscle mass [79] . in the current study, there was a significantly higher level of cs mrna at the 15 h time-point implying that mitochondrial density was higher at this time-point; however, this was not the case as the mitochondrial counts were not significantly different between the 5 and 15 h time-points of egg-shell a b pgc-1α protein is a nuclear encoded protein that is localised both in the nucleus and cytoplasm [80] . in mammals, the expression of pgc-1α is prominent in tissues with high demands for energy [81, 82] and the mrna level is induced in conditions such as physical exercise, fasting and exposure to cold [82] . increase in pgc-1α protein is sufficient to induce cellular pathways important for mitochondrial biogenesis and energy metabolism [43, 44] . mitochondrial content and oxidative capacity are different in different cells and are regulated by energy demand of a cell [83] . in a study of adenovirus infected saos-2 cells, the mitochondrial proteins induced by pgc-1α regulation resulted in increased mitochondrial content in the cells [83] measured 48 h after infection. in the current study, the expression level of pgc-1α was not affected by viral infection and time-points of egg-shell formation. the expression level of pgc-1α cannot be correlated with mitochondrial counts as the virus challenge reduced the mitochondria at both 5 and 15 h time-points of egg-shell formation. it seems that a lower number of mitochondria may not necessarily induce the up-regulation of pgc-1α in cells undergoing virus multiplication. drp1 is a nuclear encoded protein that plays an important role in the fission of the outer mitochondrial membrane. in healthy cells, fission and fusion events occur to regulate mitochondrial morphology [84] . once again, the higher expression level of drp1 at 15 h compared with the 5 h time-point cannot be clearly linked with higher activities of mitochondrial fission and/or fusion as most of the genes studied did not show a clear pattern of change with lower mean mitochondrial counts in ibv t challenged groups. also, the mitochondrial counts were significantly lower at both 5 and 15 h time-points in the challenge groups, whereas no expression change of drp1 was observed upon virus challenge during egg-shell formation. this suggests that having a lower number of mitochondria in a cell may not necessarily relate to the expression level of drp1. we consider that a spatio-temporal investigation is needed to understand the response of nuclear dna encoded genes to mitochondrial biogenesis and fission in cells undergoing virus multiplication possibly through immunohistochemistry assays. the objective of na + -k + atpase quantification was to understand cell membrane potential level in the control and ibv t challenged groups and at two different timepoints of egg-shell formation. na + -k + atpase is involved in maintaining membrane excitation potential, tight junction polarity and vectorial transport in cells (reviewed in rajasekaran et al., [85] ). a higher expression level of na + -k + atpase at 15 h compared with the 5 h time-point is an indication of higher cellular activity of the shell gland tissue during the formation of the egg-shell. a lack of significant difference in the expression level of na + -k + atpase between the control and ibv t challenged groups indicates that viral multiplication in the cells did not alter the normal function of na + -k + atpase and that the cells in both the groups were functioning similarly. however, an interaction of time-point and virus challenge indicated that the expression level of na + -k + atpase was significantly affected by viral multiplication when the egg-shell formation was at its peak in the shell gland. a significant interaction between the time-point and virus challenge for sdha and na + -k + atpase indicates that the virus challenge downregulates these genes only at certain stages of egg-shell formation when the expression of the genes is high, at 5 h for sdha but at 15 h for na + -k + atpase. taken together, the results of the current study show that the ibv t strain challenge in laying hens significantly lowered the mean mitochondrial counts only in the shell gland part of oviduct. furthermore, the expression levels of the nuclear dna encoded genes that are involved in mitochondrial biogenesis and/or function could not be clearly correlated with lower mean mitochondrial count and mitochondrial biogenesis. a significant difference in the expression levels of most of the genes at two different time-points of egg-shell formation showed that the mitochondrial function was more affected by egg-shell formation stages than by the viral multiplication in the cells. further investigation is required to determine the actual turnover of mitochondria in metabolically active organs, such as laying hen oviduct, in normal and pathological conditions, and spatio-temporal relationship between mitochondrial count and expression of genes coding mitochondrial biogenesis related proteins. abbreviations cs: citrate synthase; cyc, s: cytochrome c, somatic; drp1: dynamin related protein 1; gdna: genomic dna; ibv: infectious bronchitis virus; mtdna: mitochondrial dna; na + -k + atpase: sodium-potassium adenosine triphosphatase; pgc-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; sdha: succinate dehydrogenase complex, subunit a, flavoprotein variant availability of data and materials all the data have been presented in this article. access to the raw data can be provided through request. this study did not involve the generation of ngs sequences. authors' contributions s.k. and j.r. framed the hypotheses, designed and performed the animal trial. s.k. analysed and interpreted data and drafted the manuscript. j.r. monitored the overall research project and critically revised the manuscript. s-b.w. designed gene expression experiments, analysed and interpreted the data and drafted the manuscript. all the authors read and approved the manuscript for publication. the animal trial was approved by the university of new england, animal ethics approval committee under authority no. aec15-118. the experimental protocol complied with the guidelines specified in the australian code for the care and use of animals for scientific purposes 8th edition 2013. this study did not involve the use of human data or tissue. the authors declare no consent for publishing the data originated from this study. the authors declare that they have no competing interests. interaction of estrogen and progesterone in chick oviduct development iii. tubular gland cell cytodifferentiation estrogen-induced cytodifferentiation of the ovalbumin-secreting glands of the chick oviduct interaction of estrogen and progesterone in chick oviduct development ii. effects of estrogen and progesterone on tubular gland cell function achieving sustainable production of eggs achieving sustainable production of eggs calcium transport in strongly calcifying laying birds: mechanisms and regulation mitochondrial function controls proliferation and early differentiation potential of embryonic stem 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metabolic regulator the estrogen-related receptor α (errα) functions in pparγ coactivator 1α (pgc-1α)-induced mitochondrial biogenesis the role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis multiple functions of na, k-atpase in epithelial cells evaluation of the suitability of six host genes as internal control in real-time rt-pcr assays in chicken embryo cell cultures infected with infectious bursal disease virus investigating reference genes for quantitative real-time pcr analysis across four chicken tissues upregulation of genes encoding digestive enzymes and nutrient transporters in the digestive system of broiler chickens by dietary supplementation of fiber and inclusion of coarse particle size corn we thank dr. sandra sapats at the australian animal health laboratory, csiro, geelong, australia for providing the infectious bronchitis virus t (n1/62) strain. this study was funded by the australian egg corporation limited, under grant number aecl 1un121 to juliet roberts. the funding body was not involved in the collection, analysis and interpretation of data and in writing the manuscript. key: cord-264716-igl25jhg authors: koo, b.s.; lee, h.r.; jeon, e.o.; han, m.s.; min, k.c.; lee, s.b.; mo, i.p. title: molecular survey of enteric viruses in commercial chicken farms in korea with a history of enteritis date: 2013-11-01 journal: poult sci doi: 10.3382/ps.2013-03280 sha: doc_id: 264716 cord_uid: igl25jhg several enteric viruses have increasingly received attention as potential causative agents of runting-stunting syndrome (rss) in chickens. a molecular survey was performed to determine the presence of a broad range of enteric viruses, namely chicken astrovirus (castv), avian nephritis virus (anv), chicken parvovirus (chpv), infectious bronchitis virus (ibv), avian rotavirus (avrv), avian reovirus (arv), and fowl adenovirus (fadv), in intestinal samples derived from 34 commercial chicken flocks that experienced enteritis outbreaks between 2010 and 2012. using techniques such as pcr and reverse-transcription pcr, enteric viruses were identified in a total of 85.3% of investigated commercial chicken flocks in korea. furthermore, diverse combinations of 2 or more enteric viruses were simultaneously identified in 51.7% of chicken farms positive for enteric viruses. the rank order of positivity for enteric viruses was as follows: anv (44.1%), castv (38.2%), chpv (26.5%), ibv (20.6%), arv (8.8%), avrv (5.9%), and fadv (2.9%). additionally, other pathogens such as escherichia coli, salmonella spp., eimeria spp., and fadv were detected in 79% of chicken flocks positive for enteric viruses using pcr, bacterial isolation, and microscopic examination. the results of our study indicate the presence of several enteric viruses with various combinations in commercial chicken farms that experienced enteritis outbreaks. experimental studies are required to further understand the roles of enteric viruses in rss in commercial chickens. enteritis is one of the most economically important diseases in poultry industry because it directly decreases feed absorption, resulting in growth retardation, impaired feed efficiency, immunosuppression, and sometimes increased mortality due to secondary infection (hoerr, 1998; yegani and korver, 2008) . genetic improvements in broiler chickens to increase feed conversion have made intestinal health particularly important, because fast-growing broilers have a tendency to be hyperphagic and can be severely affected by decreased feed absorption (dekich, 1998) . various factors can adversely affect intestinal health, including microorganisms, suboptimal environments, and feed, because the intestines have an extensive surface that is exposed to the external environment (smart et al., 1988; goodwin et al., 1993) . moreover, the intestines have a delicate and balanced bacterial microenvironment referred to as the microflora that can also be adversely affected by factors such as feed, infectious agents, antibiotics, and the environment (yegani and korver, 2008 ). an imbalanced microflora could result in decreased vitamin production, immunosuppression, and increased growth of harmful bacteria in the intestines of chickens (yegani and korver, 2008) . therefore, enteric diseases are a common occurrence in the poultry industry worldwide. among various factors affecting intestines, enteric viruses have been a focus of interest because enteritis in broiler chickens can lead to runting-stunting syndrome (rss; guy, 1998; ono et al., 2001; otto et al., 2006; nili et al., 2007; pantin-jackwood et al., 2008; palade et al., 2011) . several enteric viruses have been identified in a high proportion of chickens suffering from rss in the fields using molecular surveys, including chicken astrovirus (castv), avian nephritis virus (anv), chicken parvovirus (chpv), infectious bronchitis virus (ibv), avian rotavirus (avrv), avian reovirus (arv), and fowl adenovirus (fadv; yu et al., 2001; otto et al., 2006; smyth et al., 2009; hewson et al., 2010; palade et al., 2011; canelli et al., 2012) . however, it is usually difficult to isolate enteric viruses in vitro, especially castv, avrv, and chpv for virus purification; furthermore, the severity of enteritis in experimental infections is less than that of enteritis in the field (yason and schat, 1986; guy, 1998; songserm et al., 2003; baxendale and mebatsion, 2004) . enteric viruses such as castv, anv, avrv, and arv are also present in a high proportion of healthy broiler flocks (pantin-jackwood et al., 2008) . therefore, the exact role of enteric viruses in rss in chickens has not been clarified. in experimental infections, significant growth retardation has been observed in broiler chickens inoculated with intestinal contents and litters collected from broiler farms showing signs of rss (smart et al., 1988; songserm et al., 2000; kang et al., 2012) . additionally, broiler chickens inoculated with bacteria-free intestinal contents filtered with 0.22 μm pore filters experienced a significant reduction in weight compared with nontreated chickens, although the severity of weight depression in these chickens was less than that of broilers inoculated with nontreated intestinal contents (nili et al., 2007; sellers et al., 2010) . based on these findings, enteric viruses are suspected to be the etiologic agents of rss, but other pathogens, such as bacteria and protozoa, may also cause intestinal damage in broilers infected with enteric viruses. a molecular survey was performed for a broad range of enteric viruses, namely castv, anv, ibv, avrv, arv, and fadv, in commercial chicken flocks suffering from enteritis. additionally, other viral and bacterial infections were documented, because secondary infections need to be identified in rss cases due to immunosuppression. between 2010 and 2012, a total of 34 commercial chicken flocks including 32 broiler and 2 layer flocks from various provinces in korea were selected for molecular examination of the following enteric viruses: castv, anv, chpv, ibv, avrv, arv, and fadv (table 1) . flocks ranging in age from hatching to 4 wk old that had a history of growth retardation (5 cases), increased mortality (21 cases), and both of them (5 cases) were submitted to the avian disease laboratory (college of veterinary medicine, chungbuk national university) for clinical diagnosis. during routine necropsy procedures, one or more pathologic lesions were observed in the intestines, including watery and undigested food contents, intestinal dilatation with thin wall and gaseous contents in the ceca, and we aseptically collected these intestines for molecular studies. anatomical regions from the duodenum to the cecum were collected. in addition to intestinal pathologic lesions, various other gross lesions were observed in submitted chicken flocks (data not shown). intestinal samples collected from each flock were pooled and diluted with 10 volumes of pbs and homogenized at 30 hz for 5 min using tissuelyser ii (qiagen, valencia, ca). the homogenized intestines were then centrifuged at 2,095 × g at 4°c for 10 min in an allegra x-15r (beckman coulter, fullerton, ca), and the supernatant was harvested into 1.5-ml eppendorf tubes and stored at −4 or −80°c for subsequent procedures. viral dna/rna was extracted from 300 μl of intestine sample supernatant using the viral genespin dna/rna extraction kit (intron biotechnology, seongnam, korea) according to the manufacturer's instructions. polymerase chain reaction targeting a partial region of the nonstructural (ns) gene of chpv and the hexon gene of fadv was performed using a commercial dna kit (intron biotechnology, seongnam, korea) as per the manufacturer's instructions (raue and hess, 1998; zsak et al., 2009) . briefly, 20-μl reaction mixtures contained 10 pmol/μl of forward and reverse primers specific for the target gene, 2 μl dna template, taq dna polymerase, distilled water, and other reaction solutions including deoxyribonucleoside triphosphates. the thermal cycling conditions for amplifying the chpv gene were as follows: one cycle of denaturation (94°c for 2 min) followed by 35 cycles of amplification (94°c for 20 s, 50°c for 20 s, and 72°c for 35 s), and finally one cycle of extension (72°c for 5 min). the thermal cycling conditions for amplifying the fadv gene were the same as above except for the annealing temperature (50 to 52°c) and extension time (35 to 80 s). cycling reactions were conducted using a takara pcr thermal cycler dice gradient (takara, shiga, japan). all primers used in this report are summarized in table 2 . reverse-transcription (rt) pcr targeting a partial region of open reading frame (orf) 1b gene of castv and anv was performed using a commercial one step rt-pcr kit (takara, shiga, japan) according to the manufacturer's instructions. briefly, reaction mixtures with a total volume of 30 μl contained forward and reverse primers (10 pmol/μl), 2 μl dna template, 2× reaction buffer, 1.2 μl m-mlv rt-polymerase, and distilled water. to amplify castv and anv target genes, rt-pcr was performed using 2 different primer sets described below because of the relatively high possibility of primer template mismatch caused by the high nucleotide (nt) sequence diversity of astroviruses (tang et al., 2005a; day et al., 2007) . for simultaneous amplification of castv and anv with the primers tapg-l1 and r1, the thermal cycling conditions were as follows: one cycle of reverse transcription (50°c for 30 min), one cycle of initial denaturation (94°c for 2 min), followed by 40 cycles of amplification (94°c for 30 s, 50°c for 30 s, and 72°c for 45 s). to amplify genes from castv and anv viruses, the primer sets cas pol 1f and 1r and anv pol 1f and 1r were used, respectively. the thermal cycling conditions were as follows: one cycle of reverse transcription step (50°c for 30 min), one cycle of initial denaturation (94°c for 2 min), followed by 40 cycles of amplification (94°c for 30 s, 50°c for 30 s, and 72°c for 30 s). first, we performed rt-pcr targeting a partial region of ibv 3′ untranslated region (utr), which is known to be more conserved in ibv compared with the ibv spike gene (s; mardani et al., 2006) . for positive ibv 3′ utr samples, we conducted rt-pcr targeting part of the ibv s1 gene using 2 primer sets: ibv s1 and s2, and new s1 oligo5′ and ibv s2 (lee et al., 2000; mase et al., 2004) . to amplify the ibv 3′ utr gene, we used the following thermal cycling conditions: one cycle of reverse transcription (50°c for 30 min), one cycle of initial denaturation (94°c for 2 min), and 40 cycles of amplification (94°c for 30 s, 55°c for 30 s, and 72°c for 25 s). to amplify the ibv s1 gene, the thermal cycling conditions were one cycle of reverse transcription (50°c for 30 min), one cycle of initial denaturation (94°c for 2 min), and 40 cycles of amplification (94°c for 30 s, 50°c for 30 s, and 72°c for 45 s). reverse-transcription pcr was conducted targeting a partial region of the avrv nonstructural protein (nsp) 4 and arv small segment (s) 4 (day et al., 2007; pantin-jackwood et al., 2008) . for samples positive for the avrv nsp4 gene, we performed rt-pcr targeting avrv vp6 (ito et al., 1995) . the same procedure described above was used except for the thermal cycling conditions. thermal cycling conditions were as follows: one cycle of reverse transcription step (50°c for 30 min), one cycle of initial denaturation (94°c for 2 min), and 40 cycles of amplification (94°c for 30 s, 52°c for 30 s, and 72°c for 80 s). the pcr products were separated by electrophoresis on 1% agarose gels with 0.5 μg/ml of ethidium bromide and identified via uv transillumination. target size amplicons for each virus were purified using a commercial gel extraction kit (geneall, seoul, korea). the nt sequences were obtained by direct nt sequencing using an abi3730xl dna sequencer (perkin-elmer, foster city, ca). sequences were manipulated using bioedit software, version 7.0.9.0 (http://www. mbio.ncsu.edu/bioedit/bioedit.html), and assembled using clc sequence viewer 6.7 (clc bio a/s, aarthus, denmark). multiple alignments of nt sequences and the predicted amino acids (aa) were obtained using clustal w method. the phylogenetic trees were constructed using the neighbor-joining algorithm with 1,000 bootstrapping replicates in the software package molecular evolutionary genetics analysis (mega, version 5.01). sequence pairwise similarities of nt and aa sequences were calculated using default values in the lalign program (http://www.ch.embnet.org/ software/lalign_form.html). the nt sequences of the reference strains were obtained from the genbank database. for field cases of suspected bacterial septicemia during necropsy, liver samples were selected and cultured onto blood and macconkey agar plates, and then incubated at 37°c for 24 h. coccidial infection in the intestines was examined by both the direct smear method and microscopic examinations of the intestines under a light microscope. inclusion body hepatitis (ibh) was diagnosed using rt-pcr and microscopic examination (raue and hess, 1998) . all nt sequences of enteric viruses identified in this report were submitted to genbank. the genbank accession numbers are as follows: enteric viruses were identified in 85.3% of korean commercial chicken flocks. the breakdown of enteric viruses was as follows: anv (44.1%), castv (38.2%), chpv (26.5%), ibv (20.6%), arv (8.8%), avrv (5.9%), and fadv (2.9%). two or more enteric viruses were identified in 51.7% of chicken flocks positive for enteric viruses. in particular, castv, anv, and chpv were simultaneously identified in investigated flocks at a high rate (table 3) . additionally, bacterial septicemia, ibh, and coccidial infection were identified in 79% of chicken flocks positive for enteric viruses (data not shown). other bacterial and protozoa infections were identified in all cases of chpv infection. the castv, anv, and arv were identified in a high proportion of chickens under 2 wk old, whereas chpv was detected in chickens between 1 and 3 wk of age (table 4) . no geographic or seasonal patterns were identified. polymerase chain reaction products and corresponding nt sequences (330 bp) of a partial region of the orf1b gene of castv and anv were successfully obtained using rt-pcr. the nt and predicted aa sequences of the castv orf1b gene had low sequence identities ranging from 76.7 to 98.5% and 89.1 to 100%, respectively, among the castv identified in this report. phylogenetic analysis based on a 330-bp segment of the orf1b gene revealed 4 subgroups of korean castv strains that showed a 12% difference in nt sequences ( figure 1 ). the anv identified in this study were more similar to each other based on orf1b region than the castvs, with nt and aa sequence identities ranging from 91.2 to 99.2% and 94.7 to 100%, respectively. a fragment of the chpv ns gene (430 bp) was successfully amplified using pcr. the nt and aa sequence similarities of the chpv ns fragments identified in this study ranged from 96.7 to 100% and 96.5 to 100%, respectively (data not shown), consistent with previous studies (biđin et al., 2011; palade et al., 2011) . the nt sequences were successfully amplified from all ibv strains that showed positive rt-pcr results for the ibv 3′ utr gene using 2 primer sets. in the phylogenetic tree based on analysis of the nt sequence (549-589 bp) of the partial ibv s1 gene, most ibv were classified into the qx-like ibv group except for 2 ibv that did not group with any of the ibv subtypes previously identified in commercial chickens ( figure 2) . strain adl121459 had 85 to 88% nt sequence identities to ibv reference strains belonging to the k-i group, which is a korean indigenous ibv type (lee et al., 2008) . furthermore, the adl120942 strain clustered with turkey corona virus in the phylogenetic tree and showed nt sequence identities of 90 to 91% to turkey corona virus reference strains (maurel et al., 2011) . target genes of avrv, arv, and fadv were all successfully amplified. two avrv grouped into group a in a phylogenetic tree and shared a high nt identity (94 to 96%, 1,162 bp) with group a avrvs according to the nt sequence query in the national center for biotechnology information basic local alignment search tool program (figure 3 ). three arv identified in this report showed high sequence similarity (95.4 to 98.7%) to one another based on alignment of a 963-bp fragment of the arv s4 gene, and formed a cluster separated from the other avian reoviruses based on a 6% difference in nt sequences (figure 3 ). one fadv identified in this report was classified as a serotype 11, genotype d fadv based on sequence analysis of 630-bp of the hexon gene. various enteric viruses have come under the spotlight as potentially causative etiologic agents of rss in commercial chickens. using molecular methods, several enteric viruses have been identified in a high proportion of chickens suffering from rss in the field (yu et al., 2001; otto et al., 2006; smyth et al., 2009; hewson et al., 2010; palade et al., 2011; canelli et al., 2012) . moreover, poliovirus and reovirus have been shown to use microflora in the intestines of mice for replication and transmission, indicating that the infectivity of enteric viruses may be closely related to the enteric bacteria present (kuss et al., 2011) . a comprehensive approach is therefore required to further our understanding of rss. in this study, a molecular survey was performed for a broad range of enteric viruses including castv, anv, chpv, ibv, avrv, arv, and fadv in intestine samples from commercial chicken flocks suffering from enteritis. additionally, the presence of bacterial and protozoan infections was documented in each flock. at least one of the following enteric viruses was identified in 85% of the commercial chicken flocks in korea that we investigated: castv, anv, chpv, ibv, avrv, arv, and fadv. diverse combinations of 2 or more enteric viruses were identified in 51.7% of chicken flocks positive for enteric viruses (table 3) . concomitant infection with several enteric viruses has been shown to result in more severe enteritis than single infection with one enteric virus type in turkeys spackman et al., 2010) . therefore, the parfigure 1 . phylogenetic tree based on analysis of partial nucleotides sequences of the chicken astrovirus (castv) and avian nephritis virus (anv) nonstructural protein (nsp) 4 genes. molecular evolutionary genetics analysis version 5.01 was used for phylogenetic tree reconstruction using the neighbor-joining algorithm with 1,000 bootstrap replicates. accession numbers are shown in parentheses. circles indicate anv and castv identified in this report, respectively. phylogenetic tree based on analysis of partial nucleotide (nt) sequences of infectious bronchitis virus (ibv) s1. molecular evolutionary genetics analysis version 5.01 was used for phylogenetic tree reconstruction using the neighbor-joining algorithm with 1,000 bootstrap replicates. accession numbers are shown in parentheses. circles indicate ibv identified in this report. ticular combination of enteric viruses that a flock is infected with likely plays a major role in determining the severity of the enteritis. additionally, pathogens such as escherichia coli, salmonella spp., eimeria spp., and fadv were detected in 79% of the chicken flocks positive for enteric viruses (table 1) . impaired development of primary immune organs, including the bursa of fabricius and thymus, is a common finding in chickens suffering from rss. this impaired development is thought to be caused by nutritional deficiencies due to decreased feed absorption or direct infection of immune organs (guy, 1998; tang et al., 2006; nili et al., 2007) . enteric viruses may also impair mucosal immunity in the intestines (guy, 1998) . due to impaired immunity, other bacterial infections, mainly e. coli and salmonella spp. infection, are frequently identified in rss cases (guy, 1998) . the presence of enteric viruses needs to be determined in young chickens that show increased mortality, even though infection with other pathogens such as e. coli, salmonella spp., and ibh may also contribute to increased mortality. avian astroviruses, including castv and anv, were identified in a high proportion of chicken flocks in this report, although positive rates for these viruses were lower than those reported in a previous study (pantin-jackwood et al., 2008) . chicken astrovirus and anv commonly coinfect chickens (table 3 ). in an earlier report, castv and anv were simultaneously identified in intestines that showed significant pathological lesions collected from chickens inoculated with contaminated litter derived from flocks suffering from rss (kang et al., 2012) . furthermore, high quantities of castv and anv were found to be present in intestine samples of rss flocks using quantitative rt-pcr (smyth et al., 2010) . these data suggest that concomitant infection of chickens with these viruses may play an important role in rss outbreaks in korea. the castv identified in this report were classified into 4 subgroups based on phylogenetic analysis of a partial sequence of orf1b (figure 1 ). of the 4 subgroups, castv korean subgroup i showed large differences (87 to 89% nt similarity) to castv reported earlier canelli et al., 2012) . the anv were divided into 2 subgroups based on phylogenetic analysis of a partial fragment of the orf1b gene; the sequence divergence between these 2 subgroups was 4%. the capsid gene sequence of astroviruses is quite variable and is closely related to antigenicity (koci and schultz-cherry, 2002; tang et al., 2005b) . human and turkey astroviruses were classified into different serotypes based on amino acid sequence divergences of 95 and 82.8% of the astrovirus capsid gene, respectively (tang et al., 2005b) . the castv and anv identified in this report were therefore expected to show high nt sequence divergence for the capsid gene and distinct serotypes. additionally, the presence of castv was identified in hatched chicks, strongly indicating vertical transmission of castv in broiler chick-ens. this positive result is unlikely to be due to contamination given the distinctness of the nt sequence of this castv strain compared with other castv strains identified. chicken parvoviruses have also been identified in a high proportion of chicken flocks suffering from enteritis; this virus can cause growth retardation, bad feathering, and bone disorders in broiler chickens when experimentally infected (kisary, 1985; zsak et al., 2009; biđin et al., 2011) . in this report, fewer korean flocks were positive for chpv (26.5%) than reported from flocks from other countries (zsak et al., 2009; biđin et al., 2011) . chicken parvoviruses, except for one case, were identified simultaneously with other enteric viruses, especially castv and anv. moreover, e. coli, salmonella spp., eimeria spp., and fadv infections were present in all chpv-positive flocks. in an earlier report, chpv was detected in the epithelial and local inflammatory cells of the duodenum and jejunum in chickens suffering from enteritis, but pathological lesions in the small intestines did not always correspond to regions of chpv detection (palade et al., 2011) . this discrepancy between pathological lesions and chpv ihc positive regions in the intestines indicates that other viral infections and factors such as bacteria and protozoa are responsible for these lesions. considering the extent of coinfection with other pathogens in the chpv-infected flocks, chpv infection may only occur in immunosuppressed hosts. however, we cannot exclude the possibility that chpv itself may cause immunosuppression because turkey parvovirus was detected in the bursa of fabricius that showed atrophy (palade et al., 2011) . in this report, concomitant infection with chpv and anv was identified in submitted layer chickens that had generalized failure of ossification in the growth plate and pale intestines with watery contents on necropsy. although enteric viruses are not significantly problematic in layer flocks (kisary, 1985; dekich, 1998) , coinfection with these 2 viruses may have contributed to enteritis in this case. the pathogenicity and immunosuppressive abilities of chpv merit further study. most ibv were classified as qx-like type ibv; this is a prevalent and nephropathogenic ibv type in korea (lee et al., 2008) . although we did not identify enteric-type ibv that cause proventriculitis and mild hemorrhage of the intestines in this report (yu et al., 2001) , 2 novel ibv were identified by sequence analysis. first, the adl121459 strain represented sequence identity (89%) to k-i ibv isolated in korea (lee et al., 2008) . considering that several eimeria spp. were also observed in intestinal contents in this case, the contribution of this variant ibv to enteritis was unclear. the adl120942 strain was very different (sequence identity 44.4 to 48.2%) to ibv reported earlier but was relatively similar (90 to 91% sequence identity) to turkey coronavirus (maurel et al., 2011) . in phylogenetic analysis, this virus clustered with the turkey coronaviruslike group (figure 3) . gross lesions of chickens observed in this case included enlargement of the proventriculus, failure of muscular development of the ventriculus, and gaseous contents in the cecum. therefore, the pathogenicity of this turkey coronavirus-like ibv in the intestines should be evaluated in future studies. avian rotavirus, arv, and fadv were less prevalent in the korean chicken flocks that we examined than other enteric viruses. two avrv were identified as group a avrv by phylogenetic analysis (figure 3) . in contrast to group a avrv that have been identified primarily from normal intestines, group d avrv have been identified in intestines showing severe villous atrophy microscopically (otto et al., 2006) . therefore, the pathogenicity of the avrv identified in this report is not clear. three arv detected in this report formed a group distinct from other strains, including strains 1733 and 2408 that are known as pathological arv strains (rosenberger et al., 1989 ; figure 3 ). one fadv was assigned to serotype 11 by sequence analysis of the hexon gene. serotype 1 of fadv is associated with gizzard erosion and enteritis in commercial chicken flocks; furthermore, ibh and gizzard erosions have simultaneously been identified in chickens infected with fadv serotype 8 (ono et al., 2001; okuda et al., 2004) . severe enteritis and hepatitis without gizzard erosions was observed in the flock infected with fadv serotype 11. the role of this virus in enteritis is therefore not clear. to summarize, a molecular survey of various enteric viruses was performed in intestinal samples derived from commercial chicken flocks suffering from enteritis in korea. concomitant infections were identified by a variety of combinations of enteric viruses as well as secondary bacterial and protozoa infections. given primer template mismatches, the presence of pcr inhibitors in feces, and the lower sensitivity of conventional rt-pcr than quantitative rt-pcr (das et al., 2009; otto et al., 2012) , the actual prevalence of enteric viruses in korean chicken flocks is likely to be higher than what we reported. although the presence of various enteric viruses was identified in commercial chickens, their exact role was not clear. the roles of these enteric viruses in the pathogenesis of rss should be evaluated by experimental infection of chickens with individual viruses and various combinations of these viruses. the isolation and characterisation of astroviruses from chickens identification and phylogenetic diversity of parvovirus circulating in commercial chicken and turkey flocks in croatia 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chickens with avian rotaviruses factors affecting intestinal health in poultry characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses key: cord-260667-5aurua6o authors: falchieri, marco; lupini, caterina; cecchinato, mattia; catelli, elena; kontolaimou, maria; naylor, clive j. title: avian metapneumoviruses expressing infectious bronchitis virus genes are stable and induce protection date: 2013-05-24 journal: vaccine doi: 10.1016/j.vaccine.2013.03.055 sha: doc_id: 260667 cord_uid: 5aurua6o the study investigates the ability of subtype a avian metapneumovirus (ampv) to accept foreign genes and be used as a vector for delivery of infectious bronchitis virus (ibv) qx genes to chickens. initially the gfp gene was added to ampv at all gene junctions in conjunction with the development of cassetted full length dna ampv copies. after recombinant virus had been recovered by reverse genetics, gfp positions supporting gene expression while maintaining virus viability in vitro, were determined. subsequently, either s1 or nucleocapsid (n) genes of ibv were positioned between ampv m and f genes, while later a bivalent recombinant was prepared by inserting s1 and n at ampv mf and gl junctions respectively. immunofluorescent antibody staining showed that all recombinants expressed the inserted ibv genes in vitro and furthermore, all recombinant viruses were found to be highly stable during serial passage. eyedrop inoculation of chickens with some ampv-ibv recombinants at one-day-old induced protection against virulent ibv qx challenge 3 weeks later, as assessed by greater motility of tracheal cilia from chickens receiving the recombinants. nonetheless evidence of ampv/ibv seroconversion, or major recombinant tracheal replication, were largely absent. avian metapneumovirus (ampv) is a major endemic respiratory pathogen of global domestic poultry [1] , and secondary infections can exacerbate the disease [2] . ampv belongs to the subfamily pneumovirinae, genus metapneumovirus [3] of which four subtypes have been recognized (a, b, c and d) [4] [5] [6] . the genome has 8 genes, with genome sizes typically close to 13.5 kb [7] . reverse genetics (rg) systems for subtypes a and c have allowed their rational mutation [8, 9] . to date rg has produced viruses with deletions, gene modifications and reporter gene insertions [8, [10] [11] [12] . some studies have considered the replicative ability of recombinants in vivo [11, 12] but not foreign viral gene insertion, nor the genetic stability of expanded genomes. avian infectious bronchitis (ibv) is the major worldwide endemic respiratory pathogen of chickens (family coronavirus, genus gammacoronavirus) which, like ampv primarily infects the respiratory tract but can also infect the kidneys, intestine and reproductive system [13] . ibv epidemiology is characterized by the emergence of virulent variants which evade prevailing ibv immunity. in the last decade the qx genotype [14] has emerged, which predominantly affects the respiratory tract and kidneys [15] . it was first recognised in china and has continued to spread to much of europe and asia. in this study, novel qx candidate vaccines based on ampv vectors were constructed and tested. the major ibv surface protein spike s0 becomes cleaved into the outer, antigenically important s1, and inner membrane bound, s2 section [13] . previously inoculation of s1 expressed in baculovirus was shown to induce protection [16] . the internal nucleocapsid (n) protein can also induce protective immunity [17, 18] involving both t and b lymphocyte epitopes [18, 19] . here two different qx s1 genes and a qx n gene were inserted into ampv, both to assess the vector's acceptance of foreign genes, and its suitability for delivery of ibv genes to chickens. three ampv genomes acted as vectors. prior to introduction of s1 or n genes, a cloning site flanked by a transcription start and stop sequence, was added to intergenic regions of full length (fl) dna genome copies. to aid ibv recombinant optimization, green fluorescent protein (gfp) was initially added to these sites and recombinant viruses recovered; then ampv s1 and n recombinant viruses were constructed. the two s1 genes used differed by a 15 nucleotide deletion present in a proportion of the donor virus. recombinant viruses were tested in chickens in two experiments to determine their protective capacity. ibv inoculation of chickens leads to infection of and damage to the tracheal ciliated epithelium [20] . loss of cilial motility is readily observed [21] and protection is considered best assessed by the maintenance of motility following challenge [22] , as also adopted in european pharmacopoeia, ibv vaccine monographs. to assess replication of recombinants, real time rt pcr was performed on material from choanal swabs while induced specific antibody to ibv and ampv was measured by elisa and haemaglutination inhibition (hi). three subtype a ampv viruses were employed as vectors for gfp and later ibv genes. first was a german field isolate (virus a) passaged in vero cells [9] and found avirulent in turkeys [12] , second was virus avf which contained an f gene modification found to better induce protection in turkeys [12] and third was 309/04, a virulent field isolate deriving from a subtype a vaccine and arising in field conditions [23] . seven versions of virus a [12] fl cdna were made by site directed mutagenesis, as outlined in fig. 1 . an xho1 restriction endonuclease (re) site was added to each intergenic region, following the preceding gene stop signal and prior to the downstream gene start signal. a cloning cassette flanked by cut sal 1 re sites, allowing gene insertion and transcription, was ligated into each cut xho1 re site of the seven fls. after cloning into stb12 cells (invitrogen, according to manufacturer's recommendation (amr)), the seven fls were cut with sal 1. gfp genes flanked by xho1 sites were produced by high fidelity pcr using primers gfp ins+ and gfp ins neg (table 1 ) and these were added to the seven fl cdnas by ligation after xho1 digestion. colonies containing dna of correct orientation and sequence were selected by sequencing. fls containing gfp were recovered by rg [9] . gfp expression was assessed by fluorescence microscopy. virus viabilities were evaluated as maximum titres obtained following three vero passages. an ibv qx virus isolated in germany was grown in eggs, titrated in tracheal organ cultures (toc) and stored in aliquots at -80 • c. as the genome sequence was unknown, a range of ibv genomes were aligned to identify conserved regions within, and flanking, s1 and n genes. these were used for the design of rt-pcr and sequencing primers, as detailed in table 1 . sequencing of the s1 gene amplicon revealed two populations, one of which showed a 15 nucleotide deletion (del) (5 -uguuugauucugaua-3 ) between bases 59 and 73. 2.3. predictive computational comparison of s1 populations s1 genes with and without the deletion were compared. the protean program of the dnastar multiple program package (lasergene inc., usa) was used to estimate physicochemical properties, composition of the proteins and prediction of secondary structures. order-disorder prediction used the vl-xt predictor on the pondr server [24] . prediction of immunodominant helper tlymphocyte antigenic sites from primary sequence data was by analysis of the occurrence of amphipathic fragments using the amphi algorithm [25] . the predictive algorithms had been previously shown correct in more than 75% of cases. qx s1 1050+ ggtttaattccttgtcagtttctcttacttatgg s1 sequencing 15 qx s1 1380+ gctgctaattttagttatttagcagatggtgg s1 sequencing 16 qx s1 270 neg cctgaagaggtgctgtcatagc s1 sequencing 17 qx s1 400+ ggcatgattccacgtgatcatattcg s1 sequencing 18 qx s1 550 neg cagtagttttgttggaagtaaaaacaagatcacc s1 sequencing 19 qx s1 end neg cgaaccatctggttcaatacaaaatctgc s1 pcr amplification 20 qx s1 start+ ccagttgtgaatttgaagaaagaacaaaagaccgacttag s1 pcr amplification 21 rt qx s1 neg catctttaacgaaccatctgg s1 rt amplification 22 s1 1380+ gctgctaattttagttatttagcagatggtgg s1 primer for mrna pcr 23 s1 start xho+ ggtaaattattgctcgaggatgttggtgaagtcactgtttttagtg s1 amplification adding xhoi sites 24 s1 stop xho neg gttacgttttgctcgagttaacgcctacgacgatgtgagctattgg s1 amplification adding xhoi sites 25 sx 3+ taatactggyaatttttcaga s1 sequencing for gene insertion, xho i re sites were added to s1 and n gene extremities by rt-pcr using modifying primers s1 start xho+ s1 stop xho neg, n start xho+ and n stop xho neg (table 1) . ibv genes were inserted into fls a, avf [12] and fl 309/04 [23] with the cassette at the mf intergenic region. for insertion of a second gene, the cloning cassette was additionally added between g and l genes. after cloning into stb12 cells, seven recombinant cdnas were produced as detailed in table 2 . recombinant viruses were rescued from fls [9] then passaged in vero cells to produce sufficient virus for protection studies. viruses were titrated in 48 well plates containing vero cell monolayers. cytopathic effect end points were observed using low power microscopy and titres were calculated [26] . virus stocks of 1.5 ml aliquots were stored at −80 • c. to verify the transcription of inserted s1 and n genes in vero cell grown virus, a previously reported protocol [10] was used, except that primers within those genes were s1 1380+ and n 920+ for s1 and n genes respectively (table 1 ). s1 and n protein expression were assessed using immunofluorescence (if) on ampv recombinant infected vero monolayers, using a polyclonal chicken antiserum (gd) anti qx for s1 expression and a monoclonal mouse antibody (biozol) for n gene expression. appropriate fitc conjugated antibodies were used to visualize specific s1/n proteins (amr). approximately 70 one-day-old spf chickens were divided into seven groups, each containing ten animals. in groups one to four, birds were inoculated by eyedrop with 4 log 10 tcid50 a full s1 mf , avf full s1 mf , a del s1 mf and avf del s1 mf respectively. the ampv control group was inoculated with avf. at 21 days post vaccination (dpv), all birds, and half the unvaccinated control (c+) were challenged with 4 log 10 tcid50 qx ibv strain by eyedrop, while the other controls remained unvaccinated (c−). for sampling, of tracheas and kidneys, half the chickens per group were humanely killed at 4 days post challenge (dpc) and the remainder at 6 dpc. approximately 60 one-day-old spf chickens were divided into six groups of ten birds, of which four were inoculated with 4 log 10 tcid50 of viruses 309 full s1 mf , avf full s1 mf + n gl , avf n mf and avf full s1 mf virus by eyedrop. the remaining groups acted as controls. at 21 dpv, all vaccinated birds and half the unvaccinated controls (c+) were challenged with 4 log 10 tcid50 of ibv qx by eyedrop, while the other controls remained unvaccinated (c−). in each group, five birds were humanely killed at 4 dpc and five at 6 dpc and tracheas and kidneys were collected. chickens from each group were bled at 18 dpv for ibv and ampv serology. sera from birds receiving s1 recombinant ampvs were tested using an ibv qx hi test, while those receiving either recombinant containing the n gene were tested by ibv hi and elisa (biochek, amr). ampv elisa (idexx, amr). ten choanal swabs were collected from all groups at 3, 6 and 9 days post vaccination (dpv) for ampv real time rt-pcr [27] to assess recombinant replication. at 4 and 6 dpc in both chicken experiments, tracheas were collected and cut into 1 mm transverse sections. for each trachea, ten sections were collected (three upper, four middle and three lower) and examined under low power microscopy to determine activity of cilia. individual sections were classified as either containing beating cilia or beating being entirely absent. statistical analysis of cilial motility was performed using the chisquare test. a p value < 0.05 was considered statistically significant. birds were examined daily for the presence of nasal exudate and generally for overall health. the gfp gene was added to modified fl ampv copies and viruses were recovered. maximum tcid 50 titres per ml of vero cell lysate, following three vero cells passages, were low when the inserted gene was positioned at a gene junction close to the virus leader. hence with gfp placed between n and p genes, the titre was 2.1 while in all other positions it exceeded 4.0. at the mf position it was 5.0. for all constructs, strong fluorescence was observed when infected vero cells were viewed by uv microscopy for all constructs as illustrated for recombinant gfp mf (fig. 2) . protean suite software analysis predicted s1 proteins to share the same physicochemical properties and pondr predicted no differences to the disordered region. the protean suite predicted the proportion of alpha helix to remain constant while a difference of beta pleated sheets was evident. the amphi program predicted a t cell epitope present only in the complete s1 protein, at amino acid positions 15-19. however s1 containing the deletion possessed two additional predicted t cell epitope regions (amino acids 264-268 and 383-388). virus was rescued from ampv fl cdnas containing ibv genes as confirmed by cytopathic effect typical of ampv on vero cell monolayers. viruses were sequenced to after three passages. rt-pcr of virus mrnas confirmed transcription of the inserted ibv genes (data not shown). expression of ibv proteins was confirmed in all recombinant viruses by if (fig. 3) . ibv and ampv antibody responses were not generally detectable (table 3 ). for ibv in experiment 1, a single bird in group avf del s1 mf and two birds in group avf full s1 mf had detectably seroconverted by hi. for ampv elisa serology, two seroconversions were detected in the avf control group (experiment 1) and three in the 309 full s1 mf group (experiment 2). except for 309 full s1 mf , real time rt-pcr showed minimal ampv recombinant replication (table 3) . at 4 dpc, sections from all birds challenged with ibv were ciliostatic. at 6 dpc, some sections from birds previously inoculated with ampv qx recombinants showed cilial motility, with those birds given avf full s1 mf + n gl showing most, followed by avf n mf and with the least for avf viruses expressing only the s1 gene. in all cases the distribution of beating rings within any group was even. no differences were observed between recombinant ampvs expressing the full and deleted s1. comparing ampv vectors with identically positioned inserts, a led to greater cilial recovery than either 309/04 or avf. see table 3 for details. significantly increased cilial activity, calculated by comparing each group to the respective positive control, was found for birds vaccinated with a full s1 mf (p = 0.0002), a del s1 mf (p = 0.0008), avf full s1 + n gl (p = 0.00001) and avf n mf (p = 0.0002) and analysis between these groups did not reveal any significant differences. table 3 chickens experiments 1 and 2: post vaccination virus detection by real time rt-pcr, pre challenge serology and cilial activity post challenge. a del s1 mf 4/10 0/10 0/10 0/10 0/10 n.d. c 0 20 a full s1 mf 0/10 0/10 0/10 0/10 0/10 n.d. c 0 24 avf del s1 mf 0/10 0/10 1/10 0/10 1/10 n.d. c 0 0 avf full s1 mf 0/10 0/10 0/10 0/10 2/10 n.d. no signs were detected after vaccination. in experiment 1, clinical signs were observed in three birds at 6 dpc. one positive control bird and two avf control birds were humanly killed after displaying lethargy, ruffled feathers and reduced response to external stimuli. gross kidney lesions, typical of qx ibv infection, were detected at post-mortem examination in the same birds. no signs or lesions were seen in experiment 2. when ibv mf recombinants were used to inoculate one-day-old chickens, many induced ibv protection of the trachea, yet serology and real time rt pcr virus detection indicated poor tracheal replication. surprisingly, recombinants replicating least induced most protection whereby recombinants based on virus a protected best while virus 309/04 induced no detectable protection. despite computer analysis predicting t cell epitopes differences concerning the s1 15 nucleotide deletion, no protection differences were observed. the primary site of ampv and ibv replication in chickens is the upper respiratory tract, hence an ampv based ibv recombinant might be predicted to be ideal for inducing ibv protection. conventional wisdom suggests that the minimal levels of upper respiratory tract replication detected in our study, led to the observed protection. therefore any ampv recombinant replicating better might yield greater protection. growing worldwide commercial field evidence is indicating that subtype b ampv better infects commercial chickens than subtype a. if a subtype b rg system becomes available, protection afforded by any resultant ibv recombinants will be keenly compared to the current study, which counter-intuitively suggests that recombinant ampvs inducing the best protection replicate least well. if more protection inducing recombinants are found not to appreciably replicate in the respiratory tract or produce generalised seroconversion, it will become necessary to investigate whether replication might occur at another site or alternatively protection may result from a yet unrecognized mechanism. there is already some evidence that, ampv replication in the respiratory tract of turkeys does not imply induction of ampv protection [12] , protection following live vaccination does not require initial replication in the trachea [28] and ampv vaccination can lead to apparent protection without ampv seroconversion [29, 30] . for the first time, ampv recombinants are reported carrying foreign viral genes. previously ampvs were shown highly stable both in cell culture and during natural passage [20] as has also been reported for other members of the mononegavirales such as vesicular stomatitis and rabies viruses [31, 32] . this contrasts with single strand positive viruses such as ibv and feline calicivirus where minimal passage readily results in consensus sequence mutations [13, 33] . furthermore, the recombinant ampv genome carrying more than 3000 extra nucleotides (ibv s1 and n genes) was stable with respect to functionally irrelevant inserted genes, and their presence did not appreciably reduce virus viability, if distanced beyond the phosphoprotein gene. our study suggests that a range of ampv vectored candidate vaccines could be readily prepared. for ibv, this is in marked contrast to conventional attenuation where more than 100 egg passages is typically required. such ampv recombinants would have greater genetic stability in two key respects. firstly instability on simple passage implies that any live vaccine will be genetically heterogeneous. of greater significance is the ability of ibv live vaccines to recombine with other ibv genotypes during co-infection, via homologous recombination, thus potentially leading to new field genotypes. in contrast, ampv has been shown to be stable with respect to multiple passage while the absence (or extremely low frequency) of homologous recombination avoids recombinant generation. the nature of the protective immune response to ibv is not well understood [13] . while virus avf containing n gene alone or n + s1 genes together protected tracheas better than avf containing the s1 gene alone, the expression of the inserted genes would need to be quantified, before conclusions about the relative contribution of each gene to protection, could be drawn. previous studies indicate that the internal n protein is able to stimulate a cell mediated immune response (cmi) [18] whereas the exposed s1 protein would be more likely to stimulate antibody production [34] [35] [36] , hence our study adds weight to the notion that both a cmi and antibody response are important in protection. in conclusion, we report for the first time that ampv is able to accept genes from, express proteins of, and confer protection against, foreign viruses. those recombinant ampv-ibv viruses are genetically stable and able to incorporate inserted genes totalling at least 3000 nucleotides; though the upper limit is yet to be established. it is likely that further developments will lead to flexible vaccines of greater efficacy as well as providing a better understanding of the essential components for induction of effective protection against ibv as well as other avian viruses. failure to detect antibody to turkey rhinotracheitis virus in australian poultry flocks avian rhinotracheitis virus taxonomy -san diego nucleotide sequences of the f, l and g protein genes of two non-a/non-b avian pneumoviruses (apv) reveal a novel apv subgroup extensive sequence variation in the attachment (g) protein gene of avian pneumovirus: evidence for two distinct subgroups avian pneumovirus and emergence of a new type in the united states of america animal pneumoviruses: molecular genetics and pathogenesis recovery of avian metapneumovirus subgroup c from cdna: cross-recognition of avian and human metapneumovirus support proteins development of a reverse-genetics system for avian pneumovirus demonstrates that the small hydrophobic (sh) and attachment (g) genes are not essential for virus viability a single polymerase (l) mutation in avian metapneumovirus increased virulence and partially maintained virus viability at an elevated temperature deletion of the sh gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the g gene or m2-2 open reading frame charged amino acids in the ampv fusion protein have more influence on induced protection than deletion of the sh or g genes coronavirus avian infectious bronchitis virus a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus study of protection by recombinant fowl poxvirus expressing c-terminal nucleocapsid protein of infectious bronchitis virus against challenge the carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection a novel b-cell epitope of avian infectious bronchitis virus n protein characterization of strains of infectious-bronchitis virus isolated in chile relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of crossprotection in vivo immunigenicity testing of vaccines against avian bronchitis virus -comparison of several methods to demonstrate experimental infection demonstration of loss of attenuation and extended field persistence of a live avian metapneumovirus vaccine sequence data analysis for long disordered regions prediction in the calcineurin family prediction of immunodominant helper t cell antigenic sites from the primary sequence a simple method of estimating fifty percent end points development of a real time rt-pcr assay for the simultaneous identification, quantitation and differentiation of avian metapneuomovirus subtype a and b demonstration of a virulent subpopulation in a prototype live attenuated turkey rhinotracheitis vaccine a live attenuated turkey rhinotracheitis virus vaccine i. stability of the attenuated strain further studies on the development of a live attenuated vaccine against turkey rhinotracheitis the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus highly stable expression of a foreign gene from rabies virus vectors the challenge for the next generation of feline calicivirus vaccines coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus monoclonal antibodies to the s1 spike and membrane proteins of avian infectious bronchitis coronavirus strain massachusetts m41 there is no conflict of interest. key: cord-255870-gmq5zs2d authors: liu, shengwang; zhang, qingxia; chen, jianfei; han, zongxi; shao, yuhao; kong, xiangang; tong, guangzhi title: identification of the avian infectious bronchitis coronaviruses with mutations in gene 3 date: 2008-04-15 journal: gene doi: 10.1016/j.gene.2008.01.004 sha: doc_id: 255870 cord_uid: gmq5zs2d the sequence of a 6.0-kb fragment was compared in the 3′-encoding region of the genome in 27 infectious bronchitis virus (ibv) strains. all these strains have the same s-3-m-5-n gene order, as is the case for other ibvs. however, the sizes of the corresponding open reading frames (orfs) of some genes varied among the virus strains. phylogenetic analysis and sequence alignments demonstrated that recombination events had occurred in the origin and evolution of the strains ck/ch/lsd/03i and ck/ch/lln/98i and the possible recombinant junction sites might be located at the 3c and m genes, respectively. the normal product of orf 3a is 57 amino acids long, whereas a 43-bp deletion at the 3′-end of the ck/ch/lsd/03i 3a gene was detected, resulting in a frameshift event and c-terminally truncated protein with 47 amino acids. comparison of the growth ability in embryos and replication and pathogenicity in chickens with ibv carrying the normal 3a gene indicated that this deleted sequence in the 3a gene of ck/ch/lsd/03i was not necessary for viral pathogenesis and replication either in vitro or in vivo. occurrence of a mutation at the corresponding position of the ck/ch/lln/98i start codon in the 3a gene led to the absence of orf 3a in this virus, resulting in a novel genomic organization at the 3′-encoding regions: s-3b, 3c-m-5a, 5b-n. comparison with other viruses carrying the normal 3a gene revealed that ck/ch/lln/98i had replication and pathogenicity abilities in vivo similar to those of other ibvs; however, its growth ability in embryos was lower, although the relationship between the lower growth ability and the orf 3a defect requires further confirmation. coronaviruses belong to the family coronaviridae, a member of the order nidovirales, and are classified into 3 groups based on the lack of genetic and antigenic relationships between the species of different groups (gonzález et al., 2003; masters, 2006) . they have been known to cause upper and lower respiratory diseases, gastroenteritis, and central nervous system infection in a number of avian and mammalian hosts, including humans (weiss and navas-martin, 2005) . the etiological importance of coronaviruses has received much attention since the discovery of the newly emerged severe acute respiratory syndrome-associated coronavirus (sars-cov) in 2003. in particular, how coronaviruses break the host species barrier, cause interspecies infection, and become zoonotic are questions of interest to the public. the infectious bronchitis virus (ibv), an avian coronavirus, together with the genetically closely related turkey coronavirus guy, 2000) , pheasant coronavirus (cavanagh et al., 2002) , and viruses recently detected in 3 species of wild birds (cavanagh et al., 2001) , forms group 3 coronaviruses. ibv primarily causes respiratory disease in domestic fowl, although it also replicates at many epithelial surfaces of the alimentary tract, oviduct, and kidney (cavanagh, 2003) , and it is one of the most economically important pathogens in the poultry industry . coronaviruses have the largest rna viral genomes ranging from 26 to 32 kilobases (kb) in length (masters, 2006) . genetic diversity among coronaviruses is generated by point mutations, insertions, and deletions introduced into the viral genome by viral rna-dependent rna polymerase, which lacks proofreading capabilities, and by genetic recombination, which occurs by a genomic template-switching mechanism (masters, 2006) . two-thirds of the viral genome encodes replicase activity and the remaining one-third, structural proteins and small nonstructural (ns) accessory proteins (masters, 2006) . ibv has 4 essential structural proteins, the 3 membrane proteins being the spike (s), integral membrane (m), and small envelope (e) proteins, and a phosphorylated nucleocapsid (n) protein. although the s1 subunit of the s protein carries virus-neutralizing and serotypespecific determinants, the s2 subunit may also induce neutralizing antibodies, and the ibv serotypes can be grouped based on the s2 gene sequence (bosch et al., 2003) . the n gene and n-terminus of the ibv m protein also vary between strains (cavanagh, 2007) . furthermore, mutations and recombination events have been observed in multiple structural genes of ibv recovered from naturally occurring infections. fig. 1 . sequence alignment of orfs 3a and 3b of 4 ibv strains. deletion in gene 3 of ibv ck/ch/lsd/03i results in a 3′-end truncated 3a gene and the mutation in gene 3 of ck/ch/lln/98i results in the absence of orf 3a in this virus. sequences of the pf/ch/lkq3/03 and ck/ch/ljl/04i strains as representatives of the normal 3a gene were compared. the putative transcription regulatory sequence (trs), ctgaacaa, of gene 3 is indicated in gray. the atgs with a single underline in boldface are the start codons of genes 3a, 3b, and 3c. the single mutation at the first site of the start codon of gene 3 in the ck/ch/lln/98i strain (atg→att) results in the absence of orf 3a in this virus. the taas/tgas in boxes are the termination codons of genes s, 3a, and 3b, respectively. a 43-bp nucleotide sequence is deleted in the ck/ch/lsd/03i strain (represented as -). ns proteins associated with viral rna replication and transcription are encoded by gene 1. interspersed among the structural protein genes are small ns protein genes that vary in number, position, and sequence among the coronaviruses (cavanagh, 2007; masters, 2006) . ibv has 2 such genes, genes 3 and 5 (boursnell et al., 1987) . gene 3 is functionally tricistronic with 3 orfs, 3a, 3b, and 3c. orfs 3a and 3b of ibv encode 2 small ns proteins of unknown function, 3a and 3b, respectively, and structural protein e is encoded by orf 3c (fig. 1 ; smith et al., 1990) . it has been recently reported that neither the rna nor the proteins of orfs 3a and 3b of ibv are essential for replication (hodgson et al., 2006; shen et al., 2003) . however, in most cases, the reasons that these orfs have been maintained throughout evolution in natural conditions and the effects of these gene products on animal hosts are yet to be understood. the primary aim of this work was to compare and analyze the 3′ 6.0-kb encoding regions that bore the 4 structural protein genes s2, m, e, and n and the 4 ns protein genes 3a, 3b, 5a, and 5b, in 27 ibv strains. the second objective was to examine the replication and pathogenicity of the ibv strains with abnormal genes in gene 3 either in vitro or in vivo. as described in table 1 , 19 ibv field strains and 8 commercially available vaccine strains were used in the present study. all the field strains were isolated as previously described (liu et al., 2006b; liu and kong, 2004) and the vaccine strains were produced by different manufacturers in china (liu et al., 2006b) . these ibv strains were representatives of different types based on s1 gene analysis and comparison with the prototype (liu et al., 2006a (liu et al., ,b, 2007 ). all the ibv field and vaccine strains were propagated once in 9 to 11-day-old embryonated chicken-specific pathogen-free (spf) eggs (harbin veterinary research institute, china) and confirmed using negative contrast electronic microscopy (jem-1200, ex) in the allantoic fluids of inoculated eggs as described previously (liu and kong, 2004) , before being used in sequence analysis. 2.2. reverse transcriptase (rt)-polymerase chain reaction (pcr), cloning, and sequencing first, 5 fragments spanning the ibv 3′ 6.0-kb encoding regions of genome were obtained by rt-pcr from each of the 27 ibv strains. secondly, the other 2 sets of primers, , were subsequently used to amplify, sequence, and confirm the entire 6.0-kb fragment. the primers ibv-280 and ibv-281 were designed based on the consensus nucleotide sequences obtained above. the sequences and locations of the primers used in this study are presented in table 2 . viral rna was extracted from 200 μl of infectious allantoic fluid by trizol reagents (invitrogen, grand island, usa) following the manufacturer's protocol. rt was performed with m-mlv reverse transcriptase (invitrogen, grand island, usa) using the reverse primer n (−). the rt procedures were performed using 20 μl of rna in a 40-μl reaction volume as previously described (liu et al., 2005) . each cdna fragment was amplified from rt products by pcr as previously described (liu et al., 2005) . the pcr products were purified from agarose gels by using a dna extraction kit (boehringer mannheim, germany) and sequenced directly or cloned into pmd-18t (takara, dalian, china) vector by following the manufacturer's instructions. each region of the fragment of the 27 ibv strains was sequenced 4 to 5 times from pcr products or independent clones. sequences were compiled and orfs were determined using the gene runner program version 3.00 (http://www.generunner. com) (liu et al., 2005) . nucleotide and amino acid sequences of different genes of different ibvs in the 3′ 6-kb fragment were assembled, aligned, and compared with the ibv pf/ch/lkq3/ 03 strain (liu et al., 2005) as the reference strain by using the megalign program (dnastar). phylogenetic analysis of the nucleotide sequences of different genes in different ibv strains was performed by the clustal v method using dnastar software (liu et al., 2006b) . the sequences of the 27 ibv strains have been submitted to the genbank database, and their accession numbers are listed in table 1 . the sequence of the ibv pf/ch/lkq3/03 strain used as a reference strain for comparison was from the genbank database with the genbank accession number ay702085. each of the nine 9-day-old spf chicken embryos was inoculated with the isolates ck/ch/lln/98, ck/ch/lsd/03i, ck/ch/ljl/04i, ck/ch/ldl/97i, and ck/ch/lgd/04ii passage level 3 (the latter 3 virus strains, each representing different ibv serotypes in china, were used as controls) with 10 2 eid 50 per embryo in 0.1 ml inoculum into the allantoic cavity. the embryos were inoculated at 37°c in an egg incubator (heraeus, germany), and the allantoic fluid from the 3 embryos of each virus was harvested at 24, 48, and 72 h, after the embryos were chilled at 4°c. the allantoic fluid was stored at −70°c individually and subsequently used for virus titration. virus titrations were performed in 9-day-old embryonated chicken spf eggs via the allantoic cavity route of inoculation (yachida et al., 1979) , and the titers were expressed as 50% (median) embryo infectious doses (eid 50 ). serial l0-fold dilutions were used for titrations. at each dilution, 5 embryos received 0.1 ml inoculum. the embryos were candled daily and examined for 1 week; those showing characteristic ibv lesions, such as dwarfing, stunting, or curling of embryos, were recorded as infected by ibv. we housed 6 groups of 10 white leghorn spf chickens (harbin veterinary research institute, china) each in separate isolators under negative pressure. they were provided with food and water ad libitum. as listed in table 4 , 5 ibv strains were used for experimental infection at day 15 by oculonasal application. groups 1 to 5 were inoculated with each of the 5 ibv field strains, and group 6 was mock inoculated with sterile allantoic fluid and served as the control (table 4 ). the actual inoculation dose per bird, as determined by egg titration of the inocula, is summarized in table 4 . the chicks were examined daily for signs of infection up to 30 days after inoculation. cloacal swabs were taken on days 4, 8, 12, and 16 postinoculation. directly after sampling, the swabs were stored individually in 200 μl of virus isolation medium (50% glycerol; 50% phosphate-buffered saline [pbs]) at −70°c until virus isolation. blood samples were also collected on these days and stored at −70°c. swab samples that were taken at the same time point in each group post-inoculation were pooled for virus isolation. the pooled sample containing 10 000 u penicillin and 10 000 μg streptomycin was inoculated into at least 4 spf embryos via the allantoic cavity (0.2 ml per egg). the eggs were candled daily, and allantoic fluids from 2 of the inoculated embryos were table 2 sequence and position of the primers used in rt-pcrs collected 72 h post-inoculation for rt-pcr amplification, and the remaining embryos were examined 1 week later for characteristic ibv lesions such as dwarfing, stunting, or curling of embryos. for rt-pcr negative samples, another 2 passages were performed and rt-pcr was carried out. rt-pcr detection was performed as previously described (liu et al., 2006a) . briefly, rt was conducted using ibv primer n (−) and pcr was carried out using primers n (−) and n (+) to generate a product of approximately 1600 bp from the allantoic fluids inoculated with samples collected post-inoculation. the pcr products were analyzed on 1.0% agarose gel. 2.7. amplification and sequencing of the 3a gene in strains ck/ ch/lsd/03i and ck/ch/lln/98i from kidney tissues of experimentally infected chickens in order to further investigate the abnormal 3a gene sequences of the ibv strains ck/ch/lsd/03i and ck/ch/lln/98i, primers ibv-199 and ibv-200 were used to amplify gene 3 from the kidney tissues of the chickens experimentally infected with strains ck/ch/lsd/03i and ck/ch/lln/98i, respectively. rt-pcr amplification was performed as described above. briefly, rt was conducted using ibv primer n (−) and pcr was carried out using primers ibv-199 and ibv-200 to generate a product of approximately 1100 bp from the kidney tissues of chickens inoculated with strains ck/ch/lsd/03i and ck/ch/ lln/98i, respectively. the pcr products were purified from agarose gels by using a dna extraction kit (boehringer mannheim, germany) and sequenced directly. serum samples were assayed in single dilutions by using a commercial total antibody elisa (idexx corporation, westbrook, maine, usa) according to the manufacturer's instructions. serum-to-positive ratios (s/p-ratios) were calculated as described previously (de wit et al., 1998; liu et al., 2006a) . from these s/pratios, individual serum titers, expressed as log 2 values, were calculated according to the manufacturer's instructions. table 3 presents the orf sizes in the 3′ 6.0-kb encoding regions of the 27 ibv strains compared with ibv pf/ch/lkq3/ 03 as the reference strain. the primary structures of all the 3′encoding regions of our ibv strains containing the s protein gene, gene 3, m protein gene, gene 5, and n protein gene in this order from the 5′-end to the 3′-end were very similar to those detected in the corresponding genomic areas of the ibv pf/ch/ lkq3/03 strain. however, the sizes of corresponding orfs of some genes varied between our strains and in comparison with the pf/ch/lkq3/03 strain. the genes that were the most conserved in sizes were genes 5, n, s2, and 3a in our ibv strains, as compared with the pf/ch/lkq3/03 strain. in the ibv strains investigated, the orfs 3b and 3c contained 189 to 219 nucleotides and 309 to 330 nucleotides, respectively. similarly, different ibv strains had m genes of various sizes, and the orfs comprised 672 to 681 nucleotides. as illustrated in fig. 1 , a 43-bp nucleotide sequence was detected at the 3′-end of the 3a gene in the virus ck/ch/lsd/03i, as compared with the normal 3a gene, resulting in a truncated 3a gene. this deletion resulted in a frameshift event and therefore, if expressed, it results in a c-terminally truncated protein with 47 deduced amino acids. however, an interesting observation is that a single mutation occurred at the corresponding position of the start codon of ck/ch/lln/98i (atg→att). this mutation resulted in the virus lacking orf 3a and changed the primary structure of the 3′-encoding regions of ck/ch/lln/98i, leading to a novel genomic organization of the avian coronavirus that had the s-3b-3c-m-5a-5b-n gene order from the 5′-end to the 3′-end, instead of the typical gene order in the 3′-encoding regions of group 3 coronaviruses isolated from chicken (ibv) (boursnell et al., 1987) , turkey (breslin et al., 1999; cavanagh et al., 2001; lin et al., 2002) , and pheasants (cavanagh et al., 2002) . the sequence profiles of rt-pcr products would represent the populations of the viral rnas presented in a given passage. overlapping peaks at the same position, representing different populations of viral rnas, would be observed if several populations of viruses coexisted. however, only a single t mutation was detected in viral rna by rt-pcr amplification prepared from ck/ch/lln/98i-infected allantoic fluid. these abnormal sequences in orf 3a of ck/ch/lsd/03i and ck/ch/lln/98i were confirmed by sequencing the corresponding regions of the viruses from the kidney tissues of experimentally infected chickens. phylogenetic analysis was performed based on alignments of nucleotide sequences with each gene in the 27 ibv strains and the beaudette reference strain. as expected, phylogenetic analysis of s2, 3a, 3b, and 3c genes in the ibv strains in the present study revealed results parallel to its genotype (fig. 2) . however, the chinese ibv isolates exhibited close relationships by phylogenetic analysis of the m, 5a, 5b, and n genes. analysis of the s2, 3a, 5a, and 5b genes in the vaccine strain jaas, which was closely related to the australian ibv strains by s1 gene typing, revealed a greater diversity. interestingly, in the case of ck/ch/lsd/03i and ck/ch/ lln/98i, the results were varied. similar to the results of the s1 gene (liu et al., 2006b) , analysis of the s2, 3a, 3b, and 3c genes revealed that ck/ch/lsd/03i belonged to the lx4-type, whereas the analysis of the m, 5a, 5b, and n genes classified this strain into the mass-type group, suggestive of a recombinant event that occurred during the origin and evolution of ck/ch/lsd/03i. the possible recombinant junction site was determined in the 3c and m genes in this study. as illustrated in fig. 3 , the nucleotide sequence alignment of the 3c and m genes of ck/ch.lsd/03i with the pf/ fig. 2 . phylogenetic trees with each gene in the 27 ibv strains were generated using the megalign (dnastar) program. ibv pf/ch/lkq3/03 was used as the reference strain. all the orfs, except the s2 gene, which contained all the sequences downstream the cleavage recognition sites of the s gene, were used for phylogenetic tree construction. sequence distance indicated by the scale was calculated using the pam250 matrix in lasergene. the corresponding sequence of 3a was used for constructing the ck/ch/lln/98i phylogenetic tree. ch/lkq3/03 (mass-type) and ck/ch/shh/03i (lx4-type) strains revealed that it shared identical sequences with ch/ck/ lshh/03i for the first 80 bp of the 3c gene; however, it bore a striking homology with the pf/ch/lkq3/03 strain in the m gene. this shift in homology suggested that the recombinant site might be located at the 3c gene (fig. 3) . results of the phylogenetic analysis in strain ck/ch/lln/98i were similar to those in strain ck/ch/ lsd/03i. an exchange of genetic information between ck/ch/ ltj/95-like and j9-like strains resulted in ck/ch/lln/98i sequences in the 3′-encoding region. sequence alignment revealed that the recombinant site might be located at the beginning of the m gene (fig. 4) . given that ibv orf 3a has an important function in infection (hodgson et al., 2006) , we undertook the first experiment to investigate the potential effect of the mutations that occurred in ibv strains ck/ch/lsd/03i and ck/ch/lln/ 98i, which resulted in a truncated 3a gene and absence of the 3a gene, respectively, on the growth ability of virus in vitro. since no cell lines could sustain the growth of the ibv strains in this study (data not shown), the 9-day-old embryonated eggs were used to determine the growth ability of virus in vitro. an equal dose (10 2 eid 50 ) of each virus at passage level 3, using strains ck/ch/ljl/04i, ck/ch/ldl/97i, and ck/ch/lgd/04ii carrying the normal 3a gene as controls, was used to inoculate 9-day-old embryos. the inoculated embryos were incubated at 37°c, and the allantoic fluid from 3 embryos of each virus was harvested at 24, 48, and 72 h, respectively, for virus titration. based on their movement and the extent of bleeding, curling, and dwarfing, all the inoculated embryos were infected but alive within 72 h. eid 50 was determined as the virus titer with each sample. the titers of 5 ibv strains in this study were increased gradually after inoculation with each viral strain from 24 to 72 h (fig. 5) . the virus titer of ck/ch/lln/98i was obviously lower than those of the other 4 strains; the latter 4 viruses exhibited similar titers. the second experiment was extended to determine if the ibv strains with normal and mutant gene 3 have different pathogenicity to chickens. three days after incubation with ck/ch/lsd/03i and ck/ch/lln/98i, using ck/ch/ljl/04i, ck/ch/ldl/7i, and ck/ch/lgd/04ii carrying the normal 3a gene as controls, some chicks in each virus-inoculated group exhibited respiratory clinical signs and the diseased chicks were listless and huddled together, presenting ruffled feathers and a dark, shrunken comb. as summarized in table 4 , both ck/ch/ lsd/03i and ck/ch/lln/98i could infect chickens and caused obvious clinical signs, similar to those shown by the 3 control strains. in the case of the ck/ch/lln/98i-inoculated group, 2 chicks died between days 5 to 15 post-inoculation. gross lesions of dead chicks were mainly confined to the kidneys. the kidney parenchyma of the dead birds was pale, swollen, and mottled; tubules and urethras were distended with uric acid crystals. these gross lesions were similar to those caused by strains ck/ch/ljl/04i, ck/ch/ldl/97i, and ck/ ch/lgd/04ii in the present study. replication of the inoculated virus was assessed in the chicks of the 6 groups at days 4, 8, 12, and 16 after inoculation by virus recovery using 9-day-old embryos via the allantoic cavity and rt-pcr amplification. as shown in table 4 , virus was detected in birds of the 5 virus-inoculated groups (groups 1 to 5) and in none of the birds in the negative control group, suggestive of the similar propagation ability in chickens by all the ibv strains ck/ch/lsd/03i, ck/ch/lln/98i, ck/ch/ljl/04i, and ck/ ch/lgd/04ii, although we had not quantified the viruses. serum antibodies were detected in the chicks of all virusinoculated groups. the antibody was detectable from 8 days onwards in each of the virus-inoculated groups and in none of the negative control groups (table 4) , suggestive of the induction of immunity by the viruses. in addition, no obvious difference in the antibody titer induced by the 5 viruses was observed in this study (data not shown). the 27 ibv strains in this study have the same s-3-m-5-n gene order, as is the case for the other ibvs (boursnell et al., 1987) and coronaviruses from turkeys (breslin et al., 1999) , pheasants (cavanagh et al., 2002) , mallard ducks, greylag geese, pigeons (jonassen et al., 2005) , and quails (circella et al., 2007) . ibvs have 2 genes termed genes 3 and 5 that are interspersed amongst the structural protein genes. gene 3 has 3 orfs, encoding proteins 3a, 3b, and 3c, of which 3c is the e protein. gene 5 encodes 2 proteins, 5a and 5b. these genes are located as follows: -s-3a, 3b, 3c (e)-m-5a, 5b-n-. in this study, we discovered that a single mutation occurred at the corresponding position of the start codon of ck/ch/lln/98i due to which this virus lacked orf 3a. on the other hand, a putative transcription regulatory sequence (trs), ctgaa-caa, upstream the corresponding position of the 3a gene was detected. therefore, we considered that the absence of orf 3a in ck/ch/lln/98i resulted due to a point mutation at the corresponding position in natural conditions, suggesting that orf 3a was not necessary for the replication of the virus. this point mutation brought on a novel genomic organization at the 3′-encoding regions of ck/ch/lln/98i: -s-3b, 3c-m-5a, 5b-n-. recently, mardani et al. submitted 3 australian ibv strains, v6-92 and v18-91 lacking orfs 3a, 3b, and gene 5 and q3-88 lacking orfs 3a, 3b, and 5a in the genbank database (with accession numbers dq490219, dq490220, and dq490212, respectively). these results and ours in the present study suggest that although the general gene order of ibv is 5′-s-3a, 3b, 3c-m-5a, 5b-n-3′ in the 3′-encoding regions, the absence of nonstructural protein genes was tolerated by the virus, as in other coronaviruses (de haan et al., 2002) . this implies that the native order is not functionally essential, as proven by engineered mutants (casais et al., 2005; hodgson et al., 2006) . it was believed that the s1 domain of ibv is the most divergent region of the molecule and is responsible for determining its serotype. on the contrary, apart from the s1 gene, the other parts in the 3′-encoding region were conserved among ibv serotypes. similar to the other ibvs (boursnell et al., 1987; cavanagh, 2007) , genes 5, n, and m of our 27 viruses were conserved both in nucleotide sequence and in length. however, the sequence length of other genes varied. the 9-bp deletion at the 3′-end of the s2 part of ck/ch/lsc/99i, the 30bp insertion at the 5′-end of the 3b gene of ck/ch/lgd/04iii, and the 21-bp deletion at the 3′-end of the 3c gene of ck/ch/ ldl/97i and ck/ch/ldl/98i may indicate that the deletions or insertion of these sequences are not essential for ibv replication. recombination is undoubtedly a feature of the replication and evolution of ibv (bochkov et al., 2006; dolz et al., 2006; kottier et al., 1995; kusters et al., 1990; ladman et al., 2006; shieh et al., 2004; wang et al., 1993) and other coronaviruses. it is believed that the conditions for recombination amongst the ibv strains in the field are as follows: an extremely large number of chickens, most maintained at high density; ease of spread of the virus; and serotype co-circulation, including proof of co-infection with more than one serotype in a given flock (cavanagh, 2007) . this is true in the case of ck/ch/lln/98i and ck/ch/lsd/03i. in china, intensive chicken farms are concentrated in the shandong and liaonin provinces, where these 2 viruses have been isolated, respectively (liu et al., 2006b) . since most of the chickens in these districts are maintained at a high density and intensively exposed to live vaccines, including mass-type and j9, during the breeding period, the vaccines must certainly be implicated as a source for mass-and j9-like sequences. the lx4-or ck/ch/ltj/95i-like sources of genetic material were probably a strain that naturally infects these affected flocks (liu and kong, 2004; liu et al., 2006b) . the natural recombination event demonstrated here could suggest that the continuous use of live vaccines may actually contribute to natural recombination and ibv-associated disease (wang et al., 1993) . a potential recombination junction site was identified in the 5′-portion of the s1 gene upstream the hvr (binns et al., 1986; wang et al., 1993) ; however, ck/ch/ lln/98i and ck/ch/lsd/03i sites in this study were predicted to be located at the 3c and m genes, respectively. this might be due to the sequence conservation in those regions because the polymerase may attach to heterologous templates through base pairing of similar sequences on the newly synthesized strand when recombination occurred. the predicted proteins of orf 3a, 3b, 5a, and 5b were small and the functions of these gene products are not known. recently, it was reported that these proteins were accessory proteins (casais et al., 2005; hodgson et al., 2006; shen et al., 2003) . hodgson et al. (2006) modified gene 3 of ibv by using a reverse genetics system and discovered that the recombinant virus titers without the 3a protein produced in ck cells, embryos, and tracheal organ cultures (tocs) were similar to those of the wildtype virus, although they declined earlier in tocs, probably due to the absence of the 3a protein. thus, these authors concluded that neither the tricistronic arrangement of gene 3 nor the 3a protein is essential for replication per se, suggesting that this protein may have roles in vivo (hodgson et al., 2006) . our results indicate that a 43-bp nucleotide sequence was deleted at the 3′end of the 3a gene in the ibv strain ck/ch/lsd/03i, resulting in a frameshift event and a c-terminally truncated protein, with 47 deduced amino acids. however, this strain had growth ability in embryos similar to that of other ibv strains. moreover, the pathogenicity and replication in chicks were also identical to ibvs with the normal 3a gene in our study, suggesting that the deleted sequence was not essential for virus replication and pathogenesis either in vitro or in vivo. however, it is intriguing to note that the ck/ch/lln/98i lacking orf 3a had a visibly lower titer in embryos at 72 h post-inoculation compared with the other ibv strains carrying normal orf 3a. the virus adapted ability in embryos varied among ibv strains, and we could not conclude that such a change may be due to the defect of orf 3a. however, the other genes in the 3′-encoding region of ck/ch/ lln/98i had highly conserved sequences and structures with other strains. although the relationship between the change in the virus titer and orf 3a defect in this virus required confirmation by using the reverse genetics system, ck/ch/lln/98i lacking orf 3a as a wild-type ibv strain could be a more useful study tool as compared to the mutant engineered using the reverse genetic system. it is hypothesized that gene 3 may play important roles in the coronavirus pathogenesis to avian species. the replication and pathogenicity of ck/ch/lln/98i was compared to those of the other ibv strains with normal orf 3a by infecting chickens at a similar dose (eid 50 ) of virus. all 5 ibv strains could infect chickens, induce specific antibody, and secrete offspring viruses from the respiratory tract. although we did not qualify the secreted viruses and evaluate the precise differences of replication ability from these viruses in chickens, our observation suggested that ck/ch/lln/98i lacking orf 3a could effectively replicate in vivo and infect chickens. comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6/82 with that of ibv beaudette molecular epizootiology of avian infectious bronchitis in russia the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3′ untranslated region identifies the virus as a close relative of infectious bronchitis virus gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys infectious bronchitis coronavirus associated with an enteric syndrome on a quail farm the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens antigenic and molecular characterization of isolates of the italy 02 infectious bronchitis virus genotype a comparative sequence ananlysis to revise the current taxonomy of the family coronaviridae turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review neither the rna nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) experimental evidence of recombination in coronavirus infectious bronchitis virus sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus infectious bronchitis virus s1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization existence of gene 5 indicates close genomic relationship of turkey coronavirus to infectious bronchitis virus association of the infectious bronchitis virus 3c protein with the virion envelope a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china a polycistronic mrna specified by the coronavirus infectious bronchitis virus isolation of avian infectious bronchitis coronavirus from domestic peafowl (pavo cristatus) and teal (anas) infectious bronchitis virus: s1 gene characteristics of vaccines used in china and efficacy of vaccination against heterologous strains from china genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between s1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ ldl/97i strain of infectious bronchitis coronavirus. the vet the molecular biology of coronaviruses emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication complete nucleotide sequences of s1 and n genes of infectious bronchitis virus isolated in japan and taiwan identification of a new membrane associated polypeptide specified by the coronavirus infectious bronchitis virus evidence of natural recombination within the s1 gene of infectious bronchitis virus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol growth kinetics of embryo-and organ-culture adapted beaudette strain of infectious bronchitis virus in embryonated chicken eggs key: cord-003334-ion97n4b authors: de silva senapathi, upasama; abdul-cader, mohamed sarjoon; amarasinghe, aruna; van marle, guido; czub, markus; gomis, susantha; abdul-careem, mohamed faizal title: the in ovo delivery of cpg oligonucleotides protects against infectious bronchitis with the recruitment of immune cells into the respiratory tract of chickens date: 2018-11-15 journal: viruses doi: 10.3390/v10110635 sha: doc_id: 3334 cord_uid: ion97n4b the in ovo delivery of cytosine-guanosine (cpg) oligodeoxynucleotides (odns) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (tlr)21 signaling pathway. although the delivery of cpg odns in ovo at embryo day (ed) 18 has been shown to reduce infectious bronchitis virus (ibv) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered cpg odns are capable of protecting chickens against a post-hatch challenge is unknown. thus, our objectives were to determine the protective effect of the in ovo delivery of cpg odns at ed 18 against ibv infection encountered post-hatch and, then, to investigate the mechanisms of protection. we found significantly higher survival rates and reduced ibv infection in the chickens following the pre-treatment of the ed 18 eggs with cpg odns. at 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (cd)8α+ and cd4+ t lymphocytes, and an up-regulation of interferon (ifn)-γ mrna in the respiratory tract of the chickens. overall, it may be inferred that cpg odns, when delivered in ovo, provide protection against ibv infection induced morbidity and mortality with an enhanced immune response. infectious bronchitis (ib) is mainly an acute and severe disease of the respiratory system of chickens [1] . the causative agent, infectious bronchitis virus (ibv), belongs to the family coronaviridae [2] . there is increasing evidence of ibv infection being reported in birds other than chickens [3, 4] . although ibv induced changes are observed primarily in the mucosal surfaces of the respiratory tract, the virus is also known to cause pathology in the female reproductive tract and kidneys, with a varying degree of severity dependent upon the type of strain that infects and replicates in the aforementioned tissues [5] [6] [7] . ever since the first record of ib in the early 1930s [8] , periodic ib outbreaks associated with the isolation of heterogeneous strains of ibv have been reported globally [1, 9] . major losses to the broiler meat industry are due to carcass condemnation at processing, a poor feed conversion ratio resulting in poor weight gain, and mortality. ibv is considered a highly infectious agent with near 100% morbidity, and with mortality reaching 10-25% [10, 11] . in breeder and layer flocks, the major losses are due to reduced egg production during and after infection with ibv. the egg drop during ibv infection has been estimated to be between 3-50% [11] . furthermore, the downgrading of eggs because of a poor internal egg quality and egg shell quality also account for considerable production losses [12] . the standard preventive measures, such as strict quarantine and biosecurity measures [13] , do not seem to sufficiently control the disease. thus far, the most efficient method for controlling ibv is by vaccination [14] . the immunization of chickens against ibv is mainly by live attenuated and killed vaccines [15] . however, the emergence of variant ibv strains/serotypes arising from vaccinated flocks, among other factors, has led to vaccine failure and ib outbreaks. thus, the development of novel approaches as an alternative or adjunct in order to control the current measures against ibv is becoming increasingly important. toll-like receptor (tlr)s are a family of germ line encoded pattern recognition receptors (prrs) expressed on the surface or within the endosomal compartments of cells [16] . these receptors are crucial for recognizing whole or segments of microbial pathogens, and they initiate key host immune defenses against inciting agents. among the tlrs, tlr9 (in mammals)/tlr21 (in birds) are the only receptors capable of distinguishing bacterial, parasitic, and viral dna containing cytosine-guanosine (cpg) motifs [17] . several studies have demonstrated the immunostimulatory and therapeutic success of cpg oligodeoxynucleotides (odns) application in various host-pathogen interaction models [18] [19] [20] . the protection provided by cpg odns against lethal challenges of extracellular bacteria, such as escherichia coli [18] and salmonella typhimurium [19] , and viruses, such as low pathogenic avian influenza virus [20] and infectious laryngotracheitis virus (iltv) [21, 22] , in chickens have been well documented. cpg odns are known to induce an array of cytokines; chemokines; and effecter molecules, such as interferon (ifn) α, β and γ, interleukin (il)-1β, il-6, il-12, il-8, tumor necrosis factor (tnf)-α, and nitric oxide (no) [23] [24] [25] . these effecter molecules are believed to play a pivotal role in protecting the host against intra and extra cellular pathogens. whilst activating a variety of immune cells, it plays an integral role in bridging the innate immune system with the adaptive immune system directing immune responses toward t helper (th)1 response [26] . a study that pre-treated chicken embryos with class b cpg odns at embryo day (ed) 18 in ovo, and then challenged them with ibv ark99 strain the day after (ed 19) , showed an increased up-regulation of ifn-γ, il-1β, il-6, il-8, and oligoadenylate synthetase (oas) a in the embryonic spleen [23] . additionally, the authors saw a significant reduction in the ibv nuclear (n) gene mrna expression in various embryonic tissues pre-treated with cpg odns, compared with the control, highlighting the value of cpg odn treatment in ibv control. however, they did not demonstrate whether the in ovo cpg odn delivery is effective against the ibv challenge encountered post-hatch. in this study, we determined whether the cpg odns delivered in ovo could provide protection against a post-hatch ibv challenge. furthermore, we looked into several cytokines and immune cells that may be activated with such protection. we found that in ovo delivery was protective against ibv challenge post hatch, suggesting a potential lasting protective effect of cpg odns towards ibv infection, which could be exploited for developing control measures. the specific pathogen free (spf) eggs from white leghorn layer hens were obtained from the canadian food inspection agency (cfia), ottawa, and were incubated according to the manufacturer's instructions in digital egg incubators (kingsuromax 20 and rcom maru deluxe max, autoelex co., ltd., gimhae, gyeongnam, korea), located at the health research innovation centre (hric) 53, university of calgary. all of the animal care protocols as well as the use of live chickens, embryos, and spf eggs in our experiments, have been reviewed and approved by the health science animal care committee (hsacc, ac14-0013, 20 november 2014). at ed 11, the incubated eggs were candled in order to select viable eggs for further incubation, and the hatched birds were transported and housed in high containment poultry isolators at the prion/virology animal facility, hric, university of calgary, with access to ad libitum food, water, and necessary veterinary care. the ibv massachusetts (m)41 strain was purchased from the american type culture collection (atcc, manassas, va, usa) and was used in all of the experiments. nine day old spf viable eggs were used to propagate m41 strain of ibv, and the allantoic fluid was harvested at 3 dpi by careful aspiration. the end point dilution assay was employed to assess the viral titers using ed 9 spf eggs, and was expressed as a 50% embryo infectious dose (eid 50 ) [27] . the synthetic cpg odns, class b cpg motifs recognized by chicken tlr21 . the control odns were diluted in pbs to the same concentration (50 µg in 200 µl per egg), and were delivered via the same route (n = 6). the in ovo tlr ligand delivery was carried out as described previously [20] . on day 1 post-hatch, the birds in both groups were infected with ibv m41 strain intra-trachealy, at a dose rate of 2.75 × 10 4 eid 50 per bird, and were monitored for 11 days post-infection (dpi) for disease progression and outcome. the humane end point of the birds was determined based on the clinical score of each bird (ruffled feathers and huddling together = 1, droopy wings = 1, depression = 1, mild increase in respiratory rate = 1, increased respiratory rate with constant beak opening =2, severe increased respiratory rate marked by gasping = 3, and body weight loss = 1). the clinical score of 5 was considered the humane endpoint. the eggs were then incubated for 3 days until hatching. on the day of hatching, a subset of cpg odn-treated chickens (n = 12) were infected with ibv m41 strain intra-trachealy at a dose rate of 2.75 × 10 4 eid 50 per bird, while maintaining the rest of the birds in that treatment group as uninfected controls (n = 9). similarly, a subset of the control odn-treated chickens was infected with ibv m41 strain with the same dose (n = 18), with the remaining birds being the control odn treated birds (n = 11), and all the pbs treated birds (n = 3) were kept as controls. the birds were weighed, wing tagged and after infection, were placed in separate isolators until the subsets of the animals were euthanized at 3 (n = 5-9 per group) and 7 (n = 3-9 per group) dpi. the clinical signs were observed and recorded daily as described, and the oro-pharyngeal and cloacal swab samples obtained using puritan ® unitranz-rt ® media transport systems (vwr, edmonton, ab, canada) at 3 and 7 dpi, and the ibv genome load were quantified following the rna extraction. simultaneously, the lung tissue was collected at 3 and 7 dpi in rna save ® (biological industries, froggabio, toronto on, canada), in order to determine the viral genome loads in lungs. to evaluate the ibv n antigen in the tracheal mucosal epithelium, the tracheal tissues from the 3 dpi birds were collected and preserved in an optimum cutting temperature (oct) compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), and were snap frozen in dry ice until use in immunofluorescent assay. to observe the histopathology, the tracheal tissues of the 3 dpi birds were fixed in 10% neutral buffered formalin (vwr international, west chester, pa, usa) and sent to the histopathology diagnostic services unit at the university of calgary, faculty of veterinary medicine, for hematoxylin and eosin (h and e) staining. additionally, the trachea and lung tissues were collected in an oct compound (tissue-tek ® , sakura finetek usa inc, torrance, ca, usa), snap frozen, and subjected to immunofluorescent assay so as to quantify the key innate and adaptive immune cells. another portion of the tissues were collected iusing rna save ® (biological industries, froggabio, toronto on, canada) for the cytokine mrna expression analysis. the animal numbers represent the total number of animals in two independent experiments. the total rna from the lungs collected at 3 and 7 dpi was extracted using a trizol reagent (invitrogen, canada inc., burlington, on, canada), according to the manufacturer's guidelines. for the rna extraction of oro-pharyngeal and cloacal swabs, the e.z.n.a. ® viral rna kit (omega bio-tek inc., norcross, ga, usa) protocol was adopted as per manufacturer's guidelines. the concentration of extracted rna was measured using nanodrop1000 spectrophotometer (thermoscientific, wilmington, de, usa), with the absorbance at a 260/280 nm wavelength. two µg of total rna from the tissue samples and 200 ng of total rna from the swab samples were used to synthesize the cdna with the use of the high capacity cdna reverse transcription kit (invitrogen life technologies, carlsbad, ca, usa), as per manufacturer's guidelines. a rt-pcr assay was carried out using fast sybr ® green master mix (invitrogen, burlington, on, canada) in order to quantify the ibv n gene and cytokine mrna expressions. rt-pcr assays were conducted in a 96 well un-skirted, low profile pcr plate (vwr, edmonton, ab, canada), where the final reaction volume of the qpcr was maintained at 20 µl. each qpcr run consisted of samples of interest, a positive control/s (gene specific plasmid), negative reverse transcriptase (nrt) control (cdna construct without the multiscribe reverse transcriptase enzyme), and negative template (ntc) control. all of the cdna samples originating from the tissues, along with the plasmid dilution series used to generate the standard curves, were run in triplicate. the target genes were quantified in relation to the β actin housekeeping gene. the target gene and the housekeeping gene for each sample was run on the same plate. five picomolar (pm) of different gene specific primers (forward and reverse primers) were used in each reaction (supplementary table s1 ). the change in the mrna expression of the cytokines was assessed using the pfaffl method [28] . the optimum parameters used in the thermal cycler (cfx96-c1000) (bio-rad laboratories, mississauga, on, canada) were 95 • c for 20 seconds (s) of pre-incubation, 95 • c for 3 s, and 60 • c for 30 s for 40 amplification cycles. a melting curve analysis was performed between 95 • c and 65 • c, with a 0.5 • c raise in temperature every 5 s. the acquisition of fluorescent signals was performed at 60 • c for 30 s. for the cluster of differentiation (cd)8α+ cell and macrophage (kul01+) of the lung and trachea, 5 µm thick sections were cut from the oct preserved tissues and were fixed using cold acetone for 5 minutes (min). the tissues were then blocked by adding 5% goat serum diluted in a trizma buffered saline (tbs) buffer (trizma base: 2.42 g; nacl: 8 g in 1 l of distilled water; ph 7.6) at room temperature for 30 min. after tipping off the excess blocking buffer, as the primary antibodies, the mouse monoclonal antibody specific for chicken macrophages/monocytes, kul0+ (southern biotech, birmingham, alabama, usa), cd8α (ct-8, southern biotech, birmingham, alabama, usa), was used in a 1:200 dilution in a 5% goat serum for 30 min. the secondary antibody, goat anti-mouse igg (h+l) conjugated with dylight ® 550 (red fluorescence) (bethyl laboratories inc., montgomery, tx, usa) was then used in a 1:500 in 5% goat serum for 1 hour (h), followed by adding vectashield ® mounting medium with 4 , 6-diamidine-2 -phenylindole dihydrochloride (dapi, vector laboratories inc., burlingame, ca, usa) (blue fluorescence), placing cover slips and edges sealed with lacquer as the final step. for the cd4+ t cell staining, before blocking the tissues with 5% goat serum, sections were blocked with avidin followed by biotin (vector laboratories, inc., burlingame, ca, usa), each with 15 min incubation periods, in between washing with tbs-t for 3 min twice and with pbs for 3 min once. after blocking with 5% goat serum for 30 min, a primary antibody, cd4 (ct-4, southern biotech, birmingham, alabama, usa) was added in a 1:200 dilution in 5% goat serum for 30 min. next, biotinylated goat anti-mouse igg (h+l) (southern biotech, birmingham, alabama, usa) was used as a secondary antibody in a 1:250 dilution in a 5% blocking buffer, and was incubated for 30 min. then, dylight ® 488 (green fluorescence) streptavidin in a 15:1000 dilution was added for 30 min, followed by a final step of mounting the slides with a vectashield ® mounting medium with dapi (vector laboratories inc., burlingame, ca, usa). all of the incubations were performed in a humidifying chamber at room temperature. each incubation with an antibody was followed by washing the slides in a tbs-t buffer for 3 min twice and in pbs for 3 min once. for the quantification of the tissue kul01+, cd4+ cells, and cd8α+ cells, five areas with maximum positive fluorescent signals of kul01+, cd4+ cells, and cd8α+ cells per tissue section were captured at x 20 magnification, along with the corresponding nuclear stained (dapi) areas. the images were then subjected to fluorescent intensity quantification using image j software (national institute of health, bethesda, md, usa). the fluorescent intensities for the dylight ® 550 (kul01+, cd8α+ cells) and dylight ® 488 (cd4+ cells) positive signals were expressed relative to the total area (as estimated by nuclear staining with dapi), and were given as a percentage. a log-rank test was used to identify the differences in survival percentage. the kruskal-wallis test followed by the mann-whitney u test were used to identify the group differences in the clinical score data for each time point. the differences among the two groups were identified using othe student's t test. one-way analysis of variance (anova) followed by the students-newman-keuls test were used to identify the group differences in all of the other experiments. the grubbs' outlier test was performed in order to identify the outliers before the data was analyzed. the data in the graphs are shown in the original scale of the measurements. however, because of the non-normality and inability to satisfy the model assumptions of data belonging to the cell counts and cytokine mrna expression, a natural log transformation was applied to these data sets prior to analysis. model statistics were performed using graphpad prism software 5, la jolla, ca, usa. a normality test, generation of histograms, box plots, and q-q plots were performed in r statistical software, r studio version 1.0.153, boston, ma, usa. * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 *** = significant at p ≤ 0.001. we observed a significant increase in the survival rate of the cpg odn-treated chickens (p < 0.05) when compared with the control odn-treated chickens, as seen in figure 1a . also, the clinical signs in the cpg odn-treated ibv-infected group were significantly milder compared with the control odn-treated and ibv-infected group at 10 dpi (p < 0.05, figure 1b) . , and the rest were kept as in ovo cpg pre-treated uninfected controls (n = 9). similarly, a subset of birds in the in ovo control odn-treated birds was infected with ibv (n = 18), and the remaining birds were kept as in ovo control odn-treated uninfected controls (n = 11). the in ovo pbs treated birds were kept as uninfected controls (n = 11). a subset of birds from each group was sacrificed at 3 dpi (n = 5-9 per group), and the remaining birds were sacrificed at 7 dpi (n = 3-9) in order to obtain lung tissue. (c) ibv genome loads in oro-pharyngeal swabs at 3 and 7 dpi, (d) ibv genome loads in cloacal swabs at 3 and 7 dpi, and (e) ibv genome loads in 3 and 7 dpi lung. (f-g) the quantitative data and representative figures from the immunofluorescent assay of the trachea for ibv n antigen is presented. scale bar = 200 µm (h) representative images of histological observations of trachea are given. control odns -ibv: severe epithelial metaplasia with severe cellular infiltration, germinal center formation is seen (a), superficial epithelial layer has become squamous with complete loss of cilia (b) and mucus glands not detected. cpg odns-ibv: pseudostratified simple columnar epithelium and intact ciliated epithelia (arrow) is evident where some have become rounded, and a few mucus secreting glands have been distorted and elongated (arrow head). cpg odns-control, control odns-control, and pbs-control: no lesions, normal pseudostratified ciliated columnar epithelium (c) with mucus secreting glands. log-rank test was used to identify the differences in the survival rate, and the kruskal-wallis test followed by the mann-whitney u test were used to identify the differences in the clinical scores at selected time points. the student's t test was performed to identify group differences in the oropharyngeal and cloacal genome loads, and one-way analysis of variance (anova) followed by the students-newman-keuls post hoc test was used to identify the differences in the lung ibv genome loads and ibv n antigen amount in the trachea. the differences were considered significant at * = significant at p ≤0.05, ** = significant at p ≤0.01 *** = significant at p ≤0.001. c-h: the animal numbers and results represent the pooled data of the two independent experiments. in order to assess the ibv genome loads in the lungs, and to determine the degree of virus shedding through the feco-oral route, the ibv n gene was quantified at 3 and 7 dpi in the birds that were pre-treated with in ovo cpg odns, control odns, and pbs. we observed a significant reduction in the viral genome loads in the oro-pharyngeal swabs collected 3 and 7 dpi (p < 0.05; figure 1c ), but did not observe a difference in the ibv genome loads in the cloacal swabs (3 and 7 dpi) between the treatment groups (p > 0.05; figure 1d ). however, significantly lower levels of lung viral genome load in the in ovo cpg odn pre-treated ibv infected group compared to in ovo control odn pre-treated ibv-infected group were observed at 3 dpi (p < 0.0001; figure 1e ). at 7 dpi, although the control odn pretreated ibv infected lung had a significantly higher ibv genome load when compared with the uninfected controls (p < 0.0001; figure 1e) , the difference of the ibv genome load in the lungs between two ibv infected groups was not significant (p > 0.05; figure 1e ). we observed a significant reduction of the ibv-n antigen in the tracheal mucosal epithelium of the in ovo cpg odn pre-treated-ibv infected birds compared with the in ovo control odn pre-treated-ibv infected birds (p < 0.0001; figure 1f-g) . this was also seen in the histology of the trachea for the degree of tracheal damage following ibv infection. the mucosal epithelium of the in ovo control odn treated-ibv infected group showed severe metaplasia with severe mononuclear cell infiltration, where the superficial epithelial layer had been replaced by squamous cells. a complete erosion/loss of the entire mucosae was evident in several areas of the trachea. also, mucus secreting glands were absent from the remaining mucosae. in contrast, in the in ovo cpg odn pre-treated-ibv infected birds, the epithelium was a pseudostratified simple columnar epithelium mostly with intact cilia on the surface. mucus glands were present with some distortion and elongation (figure 1h ). in general, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd4+ and cd8α+ t numbers in the trachea compared with the uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure 2a-c) . similarly, the in ovo cpg odn pre-treated ibv infected group recorded higher macrophage and cd4+ and cd8α+ t numbers in lungs compared to uninfected and control odn pre-treated groups, although not all of the specific comparisons reached statistical significance (figure 2d-e) . in the trachea and lungs, the macrophage and cd8α+ t recruitment patterns, respectively, indicated that the in ovo delivered cpg odns are capable of increasing the recruitment of these cells in both the ibv infected and uninfected chickens (figure 2a the quantitative data following immunofluorescent assays done for the trachea (a) macrophages, (b) cluster of differentiation (cd)4+ t cells, and (c) cd8α+ t cells are given. the quantitative data following the immunofluorescent assays done for lung (d) macrophages, (e) cd4+ t cells, and (f) cd8α+ t cells are given. one-way anova followed by the students-newman-keuls post hoc test were used to identify the group differences. the differences were considered significant at * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 *** = significant at p ≤ 0.001. the results represent the pooled data of two independent experiments. considering that we observed a significant reduction in the ibv induced morbidity and mortality of in ovo cpg odn pre-treated birds correlating with varying degrees of increased macrophages, cd4+, and cd8α+ t cells in the tracheal and lung tissues, we needed to further elucidate the mechanisms by which these immune cells were efficiently recruited. several cytokine mrna expression levels in the lungs were analyzed at 3 dpi, and our data showed a significant increase in the up-regulation of only the ifn-γ mrna expression in the in ovo cpg odn pre-treated lungs compared with the in ovo control odn pre-treated lungs, in both the ibv infected (p < 0.01) and uninfected (p < 0.05) groups (figure 3a-c) . in ovo delivery of poultry vaccines has been performed routinely for decades by the poultry industry [29] . in ovo delivery targets the deposition of cpg odns in the amniotic cavity. subsequently, the ingestion of cpg odns containing amniotic fluid by the developing embryo distributes cpg odns in the respiratory and gastrointestinal tracts, leading to immune cell recruitment in these two body systems [22] . we have shown in this study that cpg odns when delivered in ovo are capable of protecting young chickens against a post-hatch ibv infection induced ib. in ovo cpg odn-treated birds displayed reduced ibv viral loads in the lungs and a decreased ibv replication and pathology in the trachea, which is associated with high survival rates and low morbidity. we found that the macrophages in the trachea and cd4+ and cd8α+ t cells in the lungs play important roles in this process, as increases in these cells were observed in the in ovo cpg odns treated group. lastly, we saw an up-regulation of ifn-γ mrna in the in ovo cpg odn pre-treated lungs, suggesting the critical role of this cytokine in the in ovo cpg odn-induced clearance of ibv infection. the host survival after day 1 post-hatch ibv infection was seen as significant in the presence of cpg odn administration in ovo compared with the controls, and a similar protective effect of in ovo delivered cpg odns has been recorded against the post-hatch iltv infection [21, 22] , and e. coli and salmonella thypimurium septicemia [18, 19] . in the current study, the protection-mediated by the in ovo administered cpg odns was associated with significantly lower ibv replication in the trachea and ibv genome loads in the lungs at 3 and 7 dpi. consequently, the ibv genome loads in the oro-pharyngeal swabs were significantly reduced at 3 and 7 dpi. however, we did not observe a significant reduction in the ibv genome loads in the cloacal swabs at 3 and 7 dpi, because of the high variability of the ibv genome loads within the control odn pre-treated group. it is difficult to explain why we observed a discrepancy in the ibv genome loads between the oro-pharyngeal and cloacal swabs, as the in ovo delivered cpg odns have been shown to recruite immune cells into the gastrointestinal mucosa [22] . our data confirm that, when delivered in ovo, the cpg odns are able to recruit macrophages into the trachea at 3 dpi (four days of age), when compared to the in ovo delivered control odns in both the ibv infected and uninfected groups. previously, we saw that the in ovo delivered cpg odns increased the macrophages in the trachea at one day of age [21, 22] . this macrophage recruitment to the trachea is associated with a lower ibv replication in the tissue, and it is possible that the cpg odn-mediated increase of the macrophages seen in the trachea in this study, played a central role in limiting the viral replication by three possible mechanisms. first, these cells may have efficiently and rapidly phagocytized the virus-infected cells and aided in virus elimination. second, they may have alerted the adaptive immune system to the invasion via active antigen presentation to the t cells. third, it may have contributed to the t and b cell activation and proliferation through the release of cytokines [30, 31] . our observation of the increased recruitment of cd4+ and cd8α+ t cells in the lungs following in ovo cpg odns delivery indicated that cpg odns could act as a mitogen, as has been shown previously [32] . this cpg odn-mediated increased cd4+ and cd8α+ t cell recruitment also could be due to the increased survival of these t cells in the lungs [32] . interestingly, we saw an expansion of the cd8α+ t cell population, but not the cd4+ t cell population in the in ovo cpg odn pre-treated-ibv infected lungs. although a portion of this increase of cd8α+ cell recruitment could be potentially attributable to the ibv specific cd8+ t cells, we did not determine whether these cd8α+ t cells are in deed ibv specific. we are at a loss as to why we did not see a similar cd4+ and cd8α+ t cell response in the trachea, but it is possible that the in ovo delivered cpg odn-mediated cd8α+ t cell recruitment is tissue specific [22] . it is also important to note that our data is limited to 3 dpi, and we do not know whether the in ovo delivered cpg odn-mediated cd4+ and cd8α+ t cell recruitments in the trachea are occurring in other time points. of the examined immune mediators, ifn-γ, a dominant product of the t helper (th)1 type cells, was upregulated in the cpg odn pre-treated ibv infected and uninfected groups, when compared with the control odn pre-treated ibv infected and uninfected groups. the source of the lung ifn-γ mrna could be the cd4+ t cell lymphocytes and cd8α+ cytotoxic lymphocytes [33, 34] , and we observed an increased recruitment of the cd4+ and cd8α+ t cells in the lungs in our experiment. two other immune mediators that were induced by the cpg odns and originated from the innate immune cells, such as the macrophages in the lungs, are the chemoattractant, il-1β, and the no production inducer, inos [21] . in the current study, we did not observe that the cpg odns or ibv induced the mrna expression of il-1β or inos. this discrepancy in the cpg odn-mediated lack of il-1β and inos expression can be explained by the difference in the time points observed. thapa et al. [21] observed an increase il-1β mrna expression in the lungs pre-hatch, and we observed a lack of il-1β mrna expression post-hatch following in ovo cpg odns delivery. the significance of the observations described in our study are two-fold. first, we found that the in ovo administration of cpg odns is capable of limiting ibv replication in the lungs and trachea, leading to an increased survival and reduced morbidity in the early post-hatch birds. second, in our study, the early recruitment and maintenance of key immune cells, such as cd8α+ and cd4+ t cells and macrophages, and the up-regulated ifn-γ mrna, exhibited not only an initiation of the early innate response, but also an effective and early adaptive host response mediated by the cpg odns, which would facilitate protection against the ibv infections encountered in birds in their immediate post-hatch life. further experiments elucidating the mechanisms of the cpg odn-mediated adoptive response, such as cell-and antibody-mediated immune responses in chickens and the duration of protection provided by this ligand against ibv, would be greatly beneficial in order to better understand the protective effects of cpg odns, and may aid in the development of more effective ibv control measures. to conclude, we show that the cpg odn-mediated protective response against post-hatch encountered ibv infection is associated with the up-regulation of ifn-γ mrna expression (in the lungs) and the enhanced recruitment of macrophages (in trachea) and cd4+ and cd8α+ t cells (in the lungs). our findings, although preliminary, may provide a basis for developing novel control strategies in the long term against ibv infection in chickens. infectious bronchitis virus types: incidence in the united states induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens pathogenicity of australian strains of avian infectious bronchitis virus isolation of avian infectious bronchitis coronavirus from domestic peafowl (pavo cristatus) and teal (anas) histopathology and immunohistochemistry of renal lesions due to infectious bronchitis virus in chicks cytopathology of chick renal epithelial cells experimentally infected with avian infectious bronchitis virus infectious bronchitis virus: immunopathogenesis of infection in the chicken the early history of infectious bronchitis global distributions and strain diversity of avian infectious bronchitis virus: a review association of the chicken mhc b haplotypes with resistance to avian coronavirus avian infectious bronchitis virus ultrastructural study of infectious bronchitis virus infection in infundibulum and magnum of commercial laying hens identification and epizootiology of infectious bronchitis in a closed flock adjuvants designed for veterinary and human vaccines severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus cpg oligodeoxynucleotide and double-stranded rna synergize to enhance nitric oxide production and mrna expression of inducible nitric oxide synthase, pro-inflammatory cytokines and chemokines in chicken monocytes a novel toll-like receptor that recognizes bacterial dna protection of chickens against escherichia coli infections by dna containing cpg motifs protection of neonatal broiler chicks against salmonella typhimurium septicemia by dna containing cpg motifs toll-like receptor (tlr) 21 signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production in ovo delivery of cpg dna reduces avian infectious laryngotracheitis virus induced mortality and morbidity in ovo cpg dna delivery increases innate and adaptive immune cells in respiratory, gastrointestinal and immune systems post-hatch correlating with lower infectious laryngotracheitis virus infection cpg oligodeoxynucleotides activate innate immune response that suppresses infectious bronchitis virus replication in chicken embryos cpg-odns induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs bacterial dna-induced nk cell ifn-γ production is dependent on macrophage secretion of il-12 interactions between bacterial cpg-dna and tlr9 bridge innate and adaptive immunity a simple method of estimating fifty per cent endpoints the use of real-time quantitative pcr for the analysis of cytokine mrna levels hatchery vaccination against poultry viral diseases: potential mechanisms and limitations immunostimulatory cpg-modified plasmid dna enhances il-12, tnf-α, and no production by bovine macrophages interleukin-1-induced promotion of t-cell differentiation in mice immunized with killed listeria monocytogenes creating space: an antigen-independent, cpg-induced peripheral expansion of naive and memory t lymphocytes in a full t-cell compartment distinct effects of t-bet in th1 lineage commitment and ifn-γ production in cd4 and cd8 t cells il-12 up-regulates il-18 receptor expression on t cells, th1 cells, and b cells: synergism with il-18 for ifn-γ production this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to acknowledge the staff of the prion/virology animal facility at foothill campus, university of calgary, for the experimental animal management. the authors declare no conflicts of interest. key: cord-273846-l0elcfe8 authors: ganapathy, kannan; cargill, peter walker; jones, richard charles title: effects of cold storage on detection of avian infectious bronchitis virus in chicken carcasses and local antibodies in tracheal washes date: 2005-02-24 journal: j virol methods doi: 10.1016/j.jviromet.2005.01.024 sha: doc_id: 273846 cord_uid: l0elcfe8 in order to test the survivability of infectious bronchitis virus (ibv) in dead chicken carcasses during 24 h of cold storage, 7 week-old specific-pathogen-free chickens were infected with virulent ibv massachusetts strain m41, and were killed humanely 10 days later. carcasses were stored in a cold room at 4 °c. after 1, 3, 6, 9, 12 or 24 h of storage, necropsies were carried out. trachea, lung, kidney and rectum were collected for virus isolation by tracheal organ culture (toc) or embryonated chicken eggs (ece), and detection by nested reverse-transcriptase polymerase chain reaction (rt-pcr). ibv was detected by rt-pcr at all sampling times, except for 1 and 6 h of storage in kidney and 9 h of storage in kidney and rectum. for ece, isolation was obtained at all sampling points, except at 1 and 24 h of storage in lungs. isolation by tracheal organ cultures was less successful, except from rectum. in addition to sampling for virus, tracheal washes were collected from each carcass to measure the ability to detect local antibodies after storage. levels of iga in tracheal washes remained high for up to 9 h of storage, suggesting that accurate sampling for research purposes when required must be carried out within this time. infectious bronchitis caused by a coronavirus is an important disease in chickens, and it mainly affects respiratory and urogenital systems (cavanagh and naqi, 2003; dhinakar raj and jones, 1997) . diagnosis of infectious bronchitis virus (ibv) is confirmed by isolation of the virus using either chicken embryonated eggs (ece) or tracheal organ culture (toc) and detection by reverse-transcriptase polymerase chain reaction (rt-pcr) (cavanagh and naqi, 2003; gelb and jackwood, 1998) . tracheal swabs, oropharyngeal swabs and tissues such as trachea, lungs, kidney, oviduct and caecal tonsils are normally used for isolation (cavanagh and naqi, 2003; gelb and jackwood, 1998) . it is recommended that carcasses should be submitted to the laboratory as soon as possible but no reports are available to indicate an appropriate time limit, beyond which virus detection is impossible. this paper provides information on the probability of ibv recovery from target tissues in carcasses stored at 4 • c for up to 24 h post-killing. three different methods of demonstrating the presence of ibv, namely isolation by toc or ece, and detection by nested rt-pcr were used. the trachea is recognised as a main target organ for ibv infection, hence an important site for research into study local immune responses (dhinakar raj and jones, 1997; gillette, 1981; gomez and raggi, 1974) . in such investigation, tracheal washes are collected for detection of local antibodies (dhinakar raj and jones, 1996; hawkes et al., 1983) and this is normally done soon after killing. however, no details on the optimal time intervals between killing and collection of tracheal washes have been established. this experiment therefore provided the opportunity to measure the levels of iga and igg in tracheal washes of chicken carcasses stored at 4 • c and sampled at the same intervals. white leghorn specific-pathogen-free chicken eggs (lohmann animal health, cuxhaven, germany) were incubated and hatched at our laboratory. chicks were housed in isolation rooms in an experimental house. food and water were provided ad libitum. the massachusetts strain m41 was used after numerous passages in ece. the titre was 6.9 log 10 median egg infective dose 50 per ml . prior to this, the virus had undergone 10 passages in toc and 2 passages in ece. chickens were inoculated when seven weeks old, with 100 l of ibv by the oculo-nasal route. the birds were monitored for clinical signs and were humanely killed at 10 days post-infection. carcasses were stored at 4 • c. at 1, 3, 6, 9, 12 and 24 h of storage, four carcasses were randomly chosen for tracheal wash collection and virus detection. tracheal washes were collected as described by dhinakar raj and jones (1996) and stored at −70 • c until further use. they were assayed for ibv-specific iga and igg by indirect elisa (below). pieces of trachea, lung, kidney and rectum were aseptically collected for isolation or rt-pcr. a similar group of uninfected chickens kept in a separate isolation pen were used as a control. each trachea was scraped with a sterile surgical blade and the mucus and epithelium were vortexed in 0.9 ml of virus isolation medium [eagles serum-free mem with glutamine, streptomycin (50 g/ml) and penicillin (50 iu/ml)]. pieces of lung, kidney or rectum (after squeezing out faecal contents) were homogenised using a sterile pestle and mortar with sterile sand and 0.2 ml of the medium. subsequently, more medium was added to make a final 1:10 (w/v) dilution of the sample. prior to centrifugation, a sterile cotton swab was dipped into each of the tissue homogenates for rt-pcr detection of ibv. swabs were left to dry at room temperature then kept in a cupboard at room temperature until used. the tissue homogenates were centrifuged at 1500 × g for 5 min and the supernatants were collected and stored at −70 • c until processed for virus isolation. three different methods were used to detect presence of ibv in the homogenised tissues. isolation of the virus was carried out in chicken tocs as described by cook et al. (1976) . three replicates of each homogenate underwent three passages and those tocs with complete ciliostasis at the third passage were subjected to rt-pcr, to confirm presence of the virus. virus isolation was carried as described by gelb and jackwood (1998) using specific-pathogen free chicken eggs of 9-11 days of incubation. this was done for up to three passages. after the third passage, eggs were incubated for 7 days and chilled overnight. chorioallantoic fluid was collected aseptically and stored at −70 • c for until used. the embryos were examined for typical lesions of ibv. the chorioallantoic fluid from embryos with lesions consistent with ibv after up to three passages was considered positive for virus. those where embryo lesions were inconclusive after the third passage was subjected to rt-pcr to confirm the presence of ibv. the swabs were pooled according to tissues and sampling intervals. rna was extracted as described previously . briefly, each was dipped into 1 ml of guanidine isothiocynate (solution d) containing 2-mercaptoethanol and being left at −20 • c for several hours. then, 500 l of the sample was transferred to a new microcentrifuge tube and 50 l of sodium acetate (ph 4.1) and 650 l phenol chloroform were added. the mixture was vortex mixed and centrifuged for 5 min. the top layer was transferred to 500 l isopropanol, mixed and precipitated overnight at −20 • c. rna was precipitated using 100% ethanol and the resulting precipitate was suspended in a solution containing sterile tissue culture water, dithiothreitol and ribonuclease inhibitor. rt-pcr was conducted according to method of cavanagh et al. (1999) . ibv-specific iga and igg in tracheal washes were assayed using an indirect elisa (dhinakar raj and jones, 1996) except that the plates were coated with partially purified ibv m41 antigen. this was done by ultracentrifugation of previously clarified allantoic fluid at 20,000 × g for 90 min and the resulting pellet was washed and resuspended in phosphate buffered saline (ph 7.2). the virus suspension was overlaid onto 25% sucrose and ultracentrifuged at 20,000 × g signs of sneezing, head-shaking and watery eyes were observed from day 2 until day 7 post-infection. at necropsy on 10 days post-infection, no gross lesions were found. for trachea, virus was isolated at 3 and 9 h of storage only and in lungs between 2 and 12 h but from never more than two out of four chickens (table 1) . at 1, 3 and 12 h of storage virus was isolated in the kidney, and was consistently detected in rectum. virus was consistently detected at all sampling points except at 1 and 24 h of storage in lungs. in total, rectum provided 100% recovery, followed by trachea and kidney (63%), and lungs (58%). ibv was detected at all sampling intervals in all four organs, except at 1, 6 and 9 h of storage for kidney and at 9 h of storage for rectum (table 1) . all control tissues remained negative for virus by isolation and rt-pcr. fig. 1 shows levels of iga and igg in tw at different sampling intervals. levels of iga at 1, 3, 6 and 9 h of storage were similar and were significantly higher than 12 and 24 h of storage. however, no significant differences were found between the sampling intervals, except levels of iga at 9 h of storage were significantly higher than 12 h of storage. for igg, only trace amounts were detected. the main aim of this paper was to provide some information on the survivability of ibv in chicken carcasses during 24 h of cold storage. this, in turn, should influence the ability to confirm a diagnosis on materials that is not fresh. the chickens were deliberately killed at 10 days post-infection, as it is well recognised that it is more difficult to recover virus at this stage than at less than 7 days post-infection (cavanagh and naqi, 2003; cook, 2001) . isolation was attempted in toc or ece, which have been reported to be equally sensitive for ibv recovery (cook et al., 1976; darbyshire et al., 1975; de wit, 2000) and also detected by nested rt-pcr cavanagh and naqi, 2003; elhafi et al., 2004) . in this study, it appears that irrespective of the ibv detection method, the virus was found in all tissues examined for up to 24 h of storage. this suggests that in difficult circumstances, necropsy examination and tissue collection of carcasses kept at 4 • c could be safely delayed up to 24 h post-killing. interestingly, irrespective of the method of isolation, consistently higher numbers of recoveries were obtained from the rectum. it is known that clearance of ibv infection first occurs from the respiratory tissues followed by other tissues, with later clearance from kidney and digestive tract (lucio and fabricant, 1990 ). in addition, ambali and jones (1990) reported ibv isolation from rectal tissue and replication in the epithelium, suggesting that apart from excretion of the virus via this route, the tissue may play an important role in generating new virus when respiratory replication has declined. thus, the rectum could be an important but additional tissue for diagnosis of ibv. as for the kidneys, since we have used a non-nephrotropic virus, the frequency of virus detection was low and to be expected. the host, agent and environmental factors may have influenced the results of this experiment (dhinakar raj and jones, 1997) and a change in these factors, particularly the strain of virus used may have produced a different outcome. the nested rt-pcr employed in this study, is regularly used in our laboratory for detection of ibv in pooled swabs. for diagnosis of the disease, use of pooled swabs is a routine practice as it is convenient, and represents a flock rather than individual birds. in our study, we also used pooled swabs taken from homogenised tissues where rna was extracted for rt-pcr. this appears to be the first report where such an extraction has been performed. other workers have extracted rna from allantoic fluid (cavanagh and naqi, 2003; gelb and jackwood, 1998) , directly from tracheal or oropharyngeal swabs (capua et al., 1999; cavanagh et al., 1999; jackwood et al., 1997; elhafi et al., 2004) or directly from non-processed tissues (falcone et al., 1997) . in this study, positive pcr reactions were found on all occasions, except after 1, 6 and 9 h of storage for lungs and 9 h of storage for rectum. this could have been due to low amount of viral genomes in the tissues or other non-specific inhibitory factors (kwon et al., 1993) , as positive isolations at these times were obtained by ece. generally, it appears that rt-pcr has detected positive samples at similar rate as ece, and was better than toc. this may demonstrate that direct swabs from homogenised tissues could be used for detection of ibv by nested rt-pcr, which may take no more than 48 h. in researching into local immune responses induced by ibv, tracheal washes are collected frequently and assayed for iga and igg (hawkes et al., 1983; dhinakar raj and jones, 1997) . this study shows that levels of iga in the tracheal wash remain high for up to 6 h of storage with slight decline at 9 h of storage, and sharply drops thereafter. thus, where complex experiments on local immune responses are to be undertaken, and especially if multiple sampling needs to be done, it should be noted that for optimal detection of local immunoglobulins, tracheal washes should be collected within 9 h of storage. early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus co-circulation of four types of infectious bronchitis virus (793/b 624/1, b1648 and massachusetts) infectious bronchitis 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infectious bronchitis: virus-neutralizing antibody in tracheobronchial secretions local immunity to avian infectious bronchitis in tracheal organ culture presence of viral antigens and antibody in the trachea of chickens infected with avian infectious bronchitis virus further development and use of a molecular serotype identification test for infectious bronchitis virus polymerase chain reaction a biotin-labelled dna probe for detection of infectious bronchitis virus in chickens tissue tropism of three cloacal isolates and massachusetts strain of infectious bronchitis virus key: cord-269150-d1sgnxc0 authors: tan, yong wah; hong, wanjin; liu, ding xiang title: binding of the 5′-untranslated region of coronavirus rna to zinc finger cchc-type and rna-binding motif 1 enhances viral replication and transcription date: 2012-02-22 journal: nucleic acids res doi: 10.1093/nar/gks165 sha: doc_id: 269150 cord_uid: d1sgnxc0 coronaviruses rna synthesis occurs in the cytoplasm and is regulated by host cell proteins. in a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-utr) of sars coronavirus (sars-cov) rna as bait against a human cdna library derived from hela cells, we found a positive candidate cellular protein, zinc finger cchc-type and rna-binding motif 1 (madp1), to be able to interact with this region of the sars-cov genome. this interaction was subsequently confirmed in coronavirus infectious bronchitis virus (ibv). the specificity of the interaction between madp1 and the 5′-utr of ibv was investigated and confirmed by using an rna pull-down assay. the rna-binding domain was mapped to the n-terminal region of madp1 and the protein binding sequence to stem–loop i of ibv 5′-utr. madp1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (rtcs) in ibv-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. using small interfering rna (sirna) against madp1, defective viral rna synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral rna synthesis. during the replication of mammalian viruses, it is inevitable for host proteins to be involved in the viral life cycles. in fact, coronaviruses require host proteins to aid in the stages from virus entry to progeny release. entry of the virus particle into a host cell requires the recognition of specific cell surface proteins, which act as receptors for the virus spike (s) protein (1) (2) (3) (4) (5) (6) . upon entry into host cells, the ribonucleocapsid uncoats and releases the 5 0 -capped viral genome, a single-stranded positive-sense rna. the genomic rna ranges from 27 to 32 kb in length, is the largest known of its kind and is structurally similar to host mrna (7) . the replicase gene, which spans the 5 0 two-thirds of the genome, is translated by host ribosomes into two large polyproteins, pp1a and pp1ab via a frameshift event (8) (9) (10) . the polyproteins are autoproteolytically processed into a maximum of 16 nonstructural proteins (8, (11) (12) (13) (14) (15) (16) , which are assembled into replicationtranscription complexes, including the main enzyme rna-dependent rna polymerase (nsp12) (17, 18) . this complex is required for generating new full-length virus rna in replication as well as subgenome-length rnas to be used for translation of virus structural and accessory proteins. in addition to their role in rna synthesis, these nonstructural proteins may have multiple functions, such as the suppression of host mrna translation as well as mrna degradation by nsp1 of sars coronavirus (sars-cov; [19] [20] [21] , which may play a role in the suppression of immune response mounted by the host upon infection. the replication-transcription complex (rtc), which is located on membrane bound vesicles in the cytoplasm (22) , is required for genome replication through continuous transcription and subgenomic rna synthesis via discontinuous transcription (18, 23, 24) . apart from the replicase gene products, a viral structural protein, the nucleocapsid (n), is also required for efficient viral rna synthesis (25, 26) . the resulting genome-size transcripts are destined to be packaged into progeny virions while the subgenomic, positive-sense transcripts are being translated into four structural proteins, spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins, as well as other accessory proteins. in virus rna synthesis, the replicase complex is indispensable but not an exclusive participant. several host proteins have been identified to be able to interact with regulatory signals within the untranslated regions in the viral genome of betacoronavirus mhv. these include the polypyrimidine tract-binding protein (ptb) (27, 28) with the leader sequence, hnrnp a1 (27, 29, 30) and hnrnp q (31) with the 3 0 -utr. more recently, poly(a)-binding protein (pabp), hnrnp q and glutamyl-prolyl-trna synthetase (eprs) were found to play a role in coronavirus rna synthesis through their interaction with the 3 0 -utr of alphacoronavirus tgev (32) . in addition, interaction of viral proteins with host proteins, such as the recently identified interaction between coronavirus nsp14 and ddx1 (33) , may also play important enhancement functions in coronavirus replication and infection cycles. in this study, we describe the interaction of a cellular protein, madp1 (zinc finger cchc-type and rna binding motif 1) with the 5 0 -utr of ibv and sars-cov, using yeast-based three hybrid screen (34) and rna-binding assays. subsequently, the rna-binding domain of madp1 and the rna secondary structure responsible for the interaction were mapped and defined. using indirect immunofluorescence, we confirmed that madp1, despite being reported as a nuclear protein (35) , was detected in the cytoplasm of virus-infected cells and partially co-localized with the rtcs. upon silencing of madp1 using sirna, viral rna synthesis on general has been affected, resulting in a lower replication efficiency and infectivity. all wild-type and mutant madp1 expressing constructs were based on the vector pxj40flag which contains both the cmv and t7 promoter and all expressed proteins were n-terminally tagged with the flag epitope. for the over-expression of the wild-type and mutant madp1 proteins, h1299 cells grown to 100% confluency were infected with recombinant vaccinia-t7 virus for 2 h (h), and the constructs were transfected into the infected cells using effectene transfection reagent (qiagen). cells were lysed with lysis buffer [140 mm nacl, 10 mm tris (ph 8.0), 1% np-40] 22 h post-transfection. template dna was amplified from plasmid dna encoding the 5 0 end of ibv genome with various sets of primers targeting different regions of the 5 0 -utr (tables 1 and 2) , with the sense primers containing the t7 promoter sequence (36) . biotinylated rnas were in vitro transcribed with t7 rna polymerase (roche applied science) in the presence of biotin rna labeling mix (roche applied science) at 37 c for 2 h. template dnas were removed by digestion with rnase-free dnase i (roche applied science) and the labeled rnas purified with ultrapure phenol:chloroform:isoamyl alcohol (invitrogen) then solubilized in nuclease-free water. biotinylated rna at 0.1 mm was incubated with cell lysates over-expressing egfp, flag-tagged madp1 or its mutant proteins, respectively, in the presence of 10 mm dithiothreitol (dtt), 100 mg/ml yeast trna (ambion) and 1 u/ml protector rnase inhibitor (roche applied science) in a final volume of 200 ml at room temperature for 30 min. the mixtures were incubated with 40 ml (50% slurry) of streptavidin agarose beads (sigma aldrich) at room temperature for 30 min. the beads were collected by centrifugation and washed three times with rnase p (rp) buffer (50 mm kcl, 1 mm mgcl 2 , 10 mm hepes, ph 8.0), suspended in 25 ml of sodium dodecyl sulfate (sds) sample buffer with 100 mm dtt. bound proteins were resolved by sds-polyacrylamide gel electrophoresis (sds-page) and detected with appropriate antibodies. african green monkey kidney cells (vero) grown to 50% confluency in four-chamber glass slides were transfected to over-express flag-tagged madp1 or vector control using effectene for 16 h. transfected cells were infected with wild-type ibv or mock-infected with vero cell lysate (vero cells with serum-free medium subjected to three freeze-thaw cycles at minus 80 c and room temperature, respectively) for 1 h. infection was allowed to progress for 2 h after virus removal and the cells were treated with actinomycin d at 15 mg/ml (sigma aldrich) for 4 h; 1 mm of brutp (sigma aldrich) was transfected into the cells with superfect (qiagen) for 3 h. cells were fixed at 10 h post-infection with 4% paraformaldehyde for 15 min and permeabilized with 0.2% triton-x 100 for 10 min. treated cells were blocked in 10% goat serum, stained with primary antibodies mouse anti-brdu and rabbit anti-flag (sigma aldrich) and subsequently probed with alexafluor 488 anti-rabbit and 594 anti-mouse (invitrogen) antibodies. images were captured with olympus fluoview upright confocal microscope using a sequential laser scanning protocol. h1299 cells grown to 30% confluency were transfected with 100 nm of either siegfp (5 0 -gcaacgugaccc ugaaguucdtdt-3 0 ) or simadp1 (5 0 -caaugacuu guaccggauadtdt-3 0 ) using dharmafect 2 sirna transfection reagent (dharmacon) for 72 h. cells were infected with recombinant ibv-luc at a multiplicity of infectivity of $1 (moi & 1) and incubated for 2 h at 37 c, 5% co 2 . the virus-containing medium was replaced with fresh serum-free medium and the cells were either harvested immediately (0 h) or continued to be incubated at 37 c until specific time points post infection (4, 8, 12, 16, 20 or 24 h) . infected cells were subjected to lysis, either through three freeze-thaw cycles (at à80 c and room temperature, respectively) without removal of media, or using lysis buffer after removal of media. firefly luciferase activity which was used as an indication of viral activity for the recombinant virus was measured using luciferase assay system (promega) according to manufacturer's instructions using the cell lysates. an end-point dilution assay, the 50% tissue culture infectious dose (tcid 50 ), the amount of virus that will produce pathological change in 50% of inoculated cell culture, of the infected cells was used as a measurement of virus titer. the tcid 50 of the infected cells at each time point was determined by using the freeze-thawed infected cells. for each sample, a 10-fold serial dilution was performed and five wells of vero cells on 96-well plates were infected with each dilution. the numbers of infected wells were collated and tcid 50 of each sample was calculated using the reed-muench method (5) . reverse transcription-polymerase chain reaction determination of the replication and sub-genomic transcription efficiency of ibv total rnas were prepared from the infected cells at their specified time points using trizol reagent (invitrogen) reverse transcription (rt) was performed with expand reverse transcriptase (roche) according to the manufacturer's instructions using the sense primer ibv leader (5 0 -26ctattacactagccttgcgct46-3 0 ) for the detection of negative-stranded subgenomic rna (sgrna) and the antisense primer ibv24803-r (5 0 -24803ctctgg atccaataacctac24784-3 0 ) for the detection of positive-stranded sgrna. both primers were then used for pcr. if transcription of subgenomic mrnas did occur, a 415-bp pcr product corresponding to the 5 0 -terminal region of subgenomic mrna 5 and a 648-bp fragment corresponding to the 5 0 -terminal region of subgenomic mrna 4 would be expected. similarly, rt was carried out with the sense primer ibv14931-f (5 0 -14931gcttatccactagtacatc14949-3 0 ) for the detection of negative-stranded genomic rna. sense primer ibv14931-f and the antisense primer ibv15600-r (5 0 -15600cttctcgcacttctgcactagca15578-3 0 ) were used for pcr. if replication of viral rna occurred, a 670-bp pcr fragment would be expected. oligonucleotides were designed based on simadp1 sequence and cloned into psilencer 2.1 neo (ambion) according to manufacturer's instructions. negative control silencer construct was supplied with the cloning kit. constructs psilencer-nc (negative control) and psilencer-madp1 were transfected into h1299 cells with effectene transfection reagent. transfected cells were selected with 500 mg/ml g418 (sigma) and the selected clones were subjected to screening for madp1 knockdown efficiency. selected h1299-shnc and h1299-shmadp1 stable cell lines were maintained in media containing 500 mg/ml of g418. in order to find candidate host proteins that may be involved in the replication and transcription of coronavirus rna, a yeast-based three-hybrid (34) screen against a human cdna library using the 5 0 -utr of sars-cov rna as bait was performed. screens were also performed using the negative sense 5 0 -utr and 3 0 -utr as bait. each screen yielded about six to eight colonies which were sequenced and non-sense sequences of the candidates were eliminated. in total, the screen identified three candidates, madp1, hax1 and ribosomal protein l27a as binding partners to sars-cov positive sense 5 0 -utr, negative sense 5 0 -utr and negative sense 3 0 -utr, respectively. although it was interesting to find ribosomal protein l27a interacting with the table 1 . nucleotide sequences of primers used to amplify dna templates for in vitro transcription primer name sequence t7_egfp_510-528r pt_egfp_f anti-sense 3 0 -utr, which was not required for viral protein translation, subsequent functional studies of the protein would prove to be complicated as the virus itself relies heavily on the host ribosome to translate viral proteins, necessary for the infection to proceed. therefore, it was not chosen for further studies. hax1 was reported to function as an anti-apoptotic protein, which was not the focus of our screen and was therefore not chosen for further studies as well. madp1 was reported as a member of the alternative splicing pathway, which implied a possible role in facilitating distal rna sequences to be brought into close proximity, corresponded well with current evidence on the mechanism of discontinuous transcription. therefore, it was chosen as the sole target for this study. the 5 0 -utr of coronavirus genomic rna interacts specifically with madp1 the interaction between madp1 and the coronavirus 5 0 -utr was confirmed by using over-expressed flag-tagged madp1 in a biotin-rna pull-down assay. based on the efficiency of flag-tagged madp1 co-purification with the biotinylated rna, the full-length, mammalian-expressed madp1 was found to be able to interact with the 5 0 -utr of ibv and sars-cov rna ( figure 1a ). over-expressed flag-tagged protein was used to facilitate detection, as there was no commercially available antibody to the protein at that time. it was noted that ibv 5 0 -utr showed higher binding affinity to the flag-tagged madp1 than did sars-cov 5 0 -utr ( figure 1a ). the specific interaction between ibv 5 0 -utr and madp1 and its functional implication in coronavirus replication were therefore chosen for subsequent characterization. to check the specificity of the interaction, a competition assay based on the biotin-rna pull-down assay was performed. total cell lysates containing flag-tagged madp1 were incubated with 0.1 mm biotinylated ibv 5 0 -utr in the presence of increasing concentrations of either unlabeled specific competitor rna probe (ibv 5 0 -utr) or unlabeled non-specific probe (egfp rna) composed of nucleotides 1-528 of the egfp coding sequence, from 0 to 0.2 mm. western blot analysis of the co-purified flag-tagged protein showed that increasing concentrations of unlabeled specific competitor rna led to the decreasing co-purification of madp1 with the biotinylated rna probe ( figure 1b) . however, increasing concentrations of unlabeled non-specific competitor rna did not result in detectable change to the efficiency of madp1 co-purification ( figure 1b) . simultaneously, a protein exhibiting a non-specific rna-binding activity, the flag-tagged ibv-n, was used as a control. total cell lysates containing the flag-tagged ibv n protein was incubated with 0.1 mm of the biotinylated ibv 3 0 -utr, in the presence of increasing concentrations of either the unlabeled specific probe or an unlabeled non-specific probe, egfp rna, of an equal length. western blot detection of the co-purified flag-tagged n protein revealed that increasing concentrations of both unlabeled rna probes increasingly reduced the efficiency of n protein co-purification with the biotinylated rna probes ( figure 1b ). these results confirmed that madp1 could interact specifically with the 5 0 -utr of ibv rna. over-expressed flag-tagged madp1 translocates from the nucleus to the cytoplasm madp1 was identified as a component of the 18 s u11/12 snrnp (37) and its subcellular localization was determined to be in the nucleoplasm (35) . ibv replication and transcription, on the other hand, take place in the cytoplasm of the infected cells. therefore, to validate the likelihood of madp1 interacting with the viral 5 0 -utr, immunofluorescence was used to track the subcellular localization of both flag-tagged madp1 and de novo synthesized viral rna in both mock-infected and ibv-infected cells. flag-tagged madp1 was overexpressed in cultured vero cells, which were then infected with ibv and treated with actinomycin d to inhibit host transcription. the newly synthesized viral rna, a marker for the rtcs, was labeled with brutp. the cells were fixed at 10 h post-infection to allow sufficient labeling of the newly synthesized viral rna and to minimize the formation of large syncytial cells. in uninfected cells, flag-tagged madp1 was localized in the nucleus exclusively ( figure 2 ). upon infection by ibv, flag-tagged madp1 appeared to be present in the cytoplasm as well ( figure 2) . interestingly, the cytoplasmic localization pattern of flag-tagged madp1 appears to be partially overlapped with that for the rtcs, although further studies would be required to ascertain if madp1 would be a part of the rtcs ( figure 2 ). as a negative control for the over-expressed protein, vector transfected cells probed with flag antibody showed negative staining for the over-expressed protein ( figure 2 ). similar colocalization patterns were also observed in ibv-infected h1299 cells (figure 2 ). to define the segment and structural elements of ibv 5 0 -utr required for its interaction with madp1, four truncated mutant rna fragments were synthesized, as shown in figure 3a , by in vitro transcription. 5 0 -utrá1 contains stem-loops i-iv (38), 5 0 -utrá2 and 5 0 -utrá3 spans stem-loops i to iii and ii to iv, respectively, whereas 5 0 -utrá4 spans the rest of the 388 nucleotides. the biotin-labeled rna transcripts were used in the biotin-rna pull-down assay ( figure 3b ) to check the efficiency of flag-tagged madp1 co-purification with rna. results showed that madp1 was co-purified only with transcripts which contain stem-loops i-iii of the 5 0 -utr (5 0 -utrá1 and 5 0 -utrá2). in addition, stem-loop i appeared to be essential for interacting with madp1 as its absence in 5 0 -utrá3 abolished the interaction with madp1 ( figure 3b ). the rest region of the 5 0 -utr (5 0 -utrá4) did not appear to interact with madp1 ( figure 3b ). to confirm further the role of stem-loop i in the interaction between madp1 and ibv 5 0 -utr, two mutants were constructed, based on 5 0 -utrá2. 5 0 -utrá2m1 carried two-point mutations at nucleotide residues 11 and 12 from ga to cu, which would disrupt the structure of stem-loop i ( figure 3c ), and 5 0 -utrá2m2 carried additional mutations at residues 25 and 26 from uc to ag ( figure 3c ), which would restore the secondary structure of stem-loop i. the mutant rnas spanning stemloops i-iii were assessed for its ability to bind madp1. the result indicated that the integrity of stem-loop i may be essential for the interaction between the 5 0 -utr with madp1 ( figure 3d ), as the stem-loop disrupting mutation (5 0 -utrá2m1) failed to interact with madp1. the stem-loop restoring mutation at nucleotide residues 25 and 26 from uc to ag was able to restore partially the interaction (5 0 -utrá2m2) ( figure 3d ). this result affirmed the conclusion that the secondary structure of stem-loop i of ibv 5 0 -utr is indispensable for its interaction with madp1. the rna recognition motif (rrm) of madp1 is responsible for its interaction with ibv 5 0 -utr madp1 contains two nucleic acid binding domains, the rna recognition motif (rrm) in the n-terminal region and the universal minicircle sequence binding protein (umsbp) in the central region. in order to identify the domain involved in the interaction between madp1 and ibv 5 0 -utr, a series of truncation mutants of the protein were created ( figure 4a ). the first three mutants, madp1n which contains the rrm domain, madp1m spans the zinc finger domain and madp1c contains mostly phosphorylation sites, were assessed for their ability to interact with ibv 5 0 -utr. only madp1n retained a low level of the rna-binding activity ( figure 4b , 1n) and negligible activity was detected for the other two truncated proteins ( figure 4b, 1m, 1c) . as the rna-binding activity for madp1n fragment was much lower compared to the full-length protein, three more mutants were created to extend the madp1n fragment ( figure 4a ). an extension of 14 or 31 amino acid residues was made for mutants madp1x and madp1z, respectively. a truncation at the n-terminus by 40 residues as well as an extension by 14 amino acid residues was made for madp1y. it was observed that both madp1x and madp1z bound to ibv 5 0 -utr more strongly than did the full-length protein as well as madp1n mutant protein ( figure 4b, 1x, 1z) . madp1y, on the other hand, bound weakly to the rna fragment ( figure 4b, 1y) . hence, the 14 amino acid extension beyond the rrm (madp1x) may have been required to preserve the integrity of the protein structure and that the 40 amino acid residues at the n-terminus of madp1 are required for efficient rna binding. as the rrm domain was determined to be responsible for the interaction, information available on this domain indicated three amino acids at its active site, which interact with nucleic acid residues via their aromatic and hydrophobic side chains. for madp1, the identified active site was composed of phenylalanine 55 and valine 53, respectively, while tyrosine 13 may have acted as an anchor for the phosphate backbone via electrostatic interactions. hence, three mutants with either a single alanine substitution for tyrosine 13 (y13a), a double alanine substitution for valine 53 and phenylalanine 55 (v53f55a) or triple alanine substitutions for all three residues (yvf), were constructed ( figure 4a ). these three mutants were over-expressed in h1299 cells as flag-tagged proteins, and the lysates were assessed for their respective rna-binding affinities for full-length ibv 5 0 -utr ( figure 4c ). interaction of deletion mutants of madp1 with ibv 5 0 -utr. cell lysates prepared from h1299 cells over-expressing flag-tagged wild-type madp1 or its truncation mutants were used for biotin-rna pull-down assay using the full-length ibv 5 0 -utr. both the crude lysates (labeled c) and protein bound on the streptavidin beads (labeled e) were resolved by sds-page and detected by western blot with anti-flag antibody. egfp over-expressed cell lysate was included as a negative control. (c) interaction of three madp1 mutant constructs, y13a, v53f55a and yvf, with ibv 5 0 -utr. the three full-length madp1 constructs with amino acid mutations at the predicted rna-binding sites were transfected into h1299 cells and used in a biotin-rna pull-down assay with the full-length ibv 5 0 -utr. all mutants resulted in a reduction in rna-binding affinity for the biotinylated rna molecule and the reduction was most dramatic for triple residue mutant yvf ( figure 4c ), implying cooperative binding demonstrated by the three residues. this finding confirms that the madp1 rrm is involved in the interaction with ibv 5 0 -utr. to demonstrate the significance of the interaction between madp1 and ibv 5 0 -utr, an sirna duplex designed to silence madp1 expression (simadp1) and a negative control sirna targeting egfp protein (siegfp) were figure 5a ). densitometric analyses identified a reduction between 40% and 80% of madp1 mrna was achieved by this sirna which resulted in a reduction between 70% and 90% of negative stranded genomic viral rna, 40-80% of negative stranded subgenomic viral rna and 50-90% of positive stranded subgenomic viral rna. western blot analysis also noted a reduction in the expression of viral structural genes, between 50% and 90% reduction for s and n proteins, with a reduction between 40% and 80% of madp1 protein ( figure 5b ). virus titers as represented by the tissue culture infectious dose (log10 tcid50) at each infection time point was reduced by a minimum of 3-fold and up to 10-fold compared to siegfp-transfected cells beyond 4 h of infection ( figure 5c ). firefly luciferase activity of cell lysates harvested at different time points showed a minimum of 50% reduction upon the silencing of madp1, which supports further the observation that the total viral protein production was much reduced ( figure 5d ). to eliminate the possibility that the phenotype observed in madp1-silenced cells during ibv infection was due to an off-target effect of the sirna duplex used, four additional sirna duplexes targeting different regions of madp1 were used in various combinations with simadp1 ( figure 6 ) to check their effect on ibv infection, as illustrated by the expression of the luciferase gene ( figure 6b ). all six combinations of five different sirna duplexes resulted in a reduction in the luciferase activity of the infected cells by either 70% (sicombi 3 and 4), without simadp1 or more than 90% (sicombi 1, 2, 5 and 6) with simadp1, compared to negative control, siegfp-transfected cells ( figure 6b ). this implies that, in general, knocking down madp1 with any sirna results in a reduction of virus infection. a stable cell clone expressing short hairpin rna to madp1 (shmadp1) was selected from h1299 cells and the madp1 mrna level was confirmed using northern blot ( figure 7a ). the expression of madp1 and the effect of madp1-knockdown on ibv infection were tested by comparing with a g418-selected cell line without expression of shmadp1 (non-targeting control, shnc). the results showed that, in general, silencing of madp1 with shrna reduced the amount of viral mrna production before 16 h post-infection ( figure 7b ). the amount of virus mrna is higher in shmadp1 cells compared to shnc cells beyond 16 h of infection as infection in shnc cells progressed much faster and most cells died and detached ( figure 7b ). the shmadp1 cell line was then transfected with constructs expressing flag-tagged wild type madp1 (fm), triple residue mutant (fm(yvf)), two mrna mutants resistant to silencing by simadp1 based on wild-type madp1 (fmmut) and the triple residue mutant (fmmut(yvf)), negative vector control (f) and egfp (e), respectively. the two sirna-resistant mutants were constructed by mutating the sirna-targeting sequence with degenerate codons, so that the protein sequence of madp1 was maintained. these transfected cells were subsequently infected with ibv-luc and harvested at 19 h post-infection. western blotting results showed an obvious increase in the amount of ibv n expression in cells over-expressing silencing-resistant wild-type madp1 (fmmut) as well as a slight increase in cells over-expressing both normal triple residue mutant (fm(yvf)) and silencing-resistant triple residue mutant (fmmut(yvf)) ( figure 7c ). an assessment of the luciferase activity of total cell lysates showed that over-expression of triple residue mutants fm(yvf) and fmmut(yvf) resulted in a slight increase of the luciferase activity in shmadp1 cells, whereas over-expression of silencing-resistant wild-type madp1 (fmmut) resulted in a more drastic increase of the luciferase activity in shmadp1 cells ( figure 7d ). it was noted that although madp1 interacted with both sars-cov and ibv 5 0 -utr, the interaction was rather weak for the former. a comparison of the predicted stemloop i structures from both coronaviruses indicated a marked difference in their primary sequence as well as the secondary structures. hence, a third coronavirus, hcov-oc43, whose stem-loop i which deviated further from ibv than sars-cov, was assessed for its binding to madp1 ( figure 8a ). it was found that the binding of madp1 to the 5 0 utr of hcov-oc43 was as weak, if not weaker than sars-cov. it was also noted that the predicted stem-loop i structure of hcov-oc43 contained a bulge which encompassed a larger area of the stem compared to sars-cov ( figure 8b ). bulges were conspicuously absent from the ibv stem-loop i ( figure 8b ). in addition to the differences in the secondary structures between the coronaviruses, there was a lack of sequence similarity as well ( figure 8b ). previous studies on the involvement of host proteins in viral rna synthesis have revealed a number of proteins which are able to interact with the utrs of viral genomes (29, 30, 32, (40) (41) (42) (43) . some of these proteins may also interact with other viral proteins as well (30, 40) . our attempts to identify host proteins involved in this early process of the coronavirus life cycle yielded madp1. this protein was shown to be localized to the nucleoplasm but excluded from the nucleolus, but its role in rna splicing remains to be determined (37) . madp1 contains two conserved rna-binding domains, the rna recognition motif (rrm) 1 and universal minicircle sequence binding protein (umsbp) domains (a zinc finger cchc-type) (35) . the former was determined to be the domain responsible for the interaction between madp1 and ibv 5 0 -utr. the madp1 rrm 1 domain interacts with nucleic acid residues via aromatic and hydrophobic side chains at its active site, which in the case supplied by phenylalanine 55 and valine 53, respectively. tyrosine 13 may have acted as an anchor for the phosphate backbone via electrostatic interactions. in this study, interaction between madp1 and the sars-cov and ibv 5 0 -utr was initially identified by a yeast-based three hybrid screen and subsequently confirmed using an in vitro rna pull-down assay with ibv 5 0 -utr. a deeper look at the details of this interaction revealed that the rna recognition motif, but not the zinc finger motif, of madp1, is responsible for the interaction. this interaction is also shown to be specific and stem-loop i of ibv 5 0 -utr is essential for the interaction to occur. although madp1 was reported to be a nuclear protein (35) , it could be detected in the cytoplasm of ibv-infected cells and partially overlaps with the de novo synthesized viral rna, which marks the location of the rtcs in infected cells in the presence of actinomycin d. silencing of madp1 resulted in a marked reduction in syncytium formation upon ibv infection. a closer examination revealed that the synthesis of both genome-(grna) and subgenome-length rnas (sgrna) was compromised, resulting in a drastic reduction of viral structural protein expression and release of viral progeny (titers), hence the overall reduction of viral infectivity in the cells. across different coronaviruses, the leader sequence situated in the extreme 5 0 end of the genome, is composed of stem-loops i and ii. mutations introduced into either stem-loop i or ii resulted in non-viable viruses, impaired (sense and anti-sense) sgrna synthesis, but not the full-length grna synthesis (38, 44) . it was, however, observed in this study that silencing of madp1 did render an impact on grna synthesis, although to a lesser extent compared to sgrna synthesis. this might have been due to a secondary effect of decreased sgrna synthesis, as proteins encoded by sgrnas may enhance viral rna synthesis (45) . the predicted structure of stem-loop ii indicated a strong secondary interaction, which is highly conserved across different groups of coronaviruses. the predicted stem-loop i structure, on the other hand, appears to fold into a hairpin of low thermodynamic stability, shows a wider sequence variation and is characterized by the presence of bulges, non-canonical base pairing as well as a prevalence of a-u base pairing (46) . it has been shown in mhv that the structural liability of stem-loop i is a critical driving force in the 5 0 -and 3 0 -utr interaction (44) . comparing the predicted stemloop i structures of ibv to sars-cov and hcov-oc43 ( figure 8b) , it was noted that there exists a difference in the loop sequence. in addition, ibv stem-loop i has a shorter stem and the absence of bulges, although the structure may be as unstable thermodynamically as that of sars-cov and hcov-oc43, due to the extremely high prevalence of weak base pairing between a and u as well as the presence of a non-canonical base pair at the base of the stem (46) . hence, sequence and structural differences may be one of the possible explanations for the observation of a weaker binding between madp1 and sars-cov or hcov-oc43 5 0 -utr than with ibv 5 0 -utr. in fact, the relatively weaker binding of madp1 to the stem-loop i restoring mutant (5 0 -utrá2m2) demonstrated in this study supports that primary sequences in the 5 0 -utr may play a certain role in this interaction. most studies on host involvement in coronaviral rna synthesis were so far performed using mhv (29) (30) (31) 42, 43) . identification of the interaction between madp1 and 5 0 -utr as well as its functional involvement in coronavirus replication, in this study, therefore may represent the first host protein identified to play a role in viral rna synthesis by interacting with the 5 0 -utr of the viral rna in a gammacoronavirus. the functional implication of the interaction between madp1 and ibv 5 0 -utr may be extended to the rest of the members of the coronavirus family. in the case of hnrnp a1, it was initially reported to be functionally important for viral rna synthesis for group ii virus mhv (29, 30) . subsequently, its involvement in viral rna synthesis was also confirmed in tgev, a group i coronavirus (32) . in this study, we have shown that betacoronaviruses hcov-oc43, sars-cov and gammacoronavirus ibv can bind to madp1, albeit with different affinities. due to the lack of a high containment facility, the functional implication of the relatively weaker interaction between sars-cov 5 0 -utr and madp1 was not further studied. it is, therefore, yet to be demonstrated if this weaker binding dictates less dependency on madp1 in sars-cov rna replication and infectivity. current evidence indicates that madp1 is compartmentalized in the nuclei of cultured cells (35) , markedly differing from the cytoplasmic, perinuclear localization of the coronavirus rtcs (47) (48) (49) . as there was no report on the possibility of madp1 shuttling between the nucleus and cytoplasm, our observation using indirect immunofluorescence that over-expressed madp1 upon ibv infection became partially localized in the cytoplasm may represent a first report that madp1 could be localized outside the nucleus. this could have been achieved with either an existing shuttling mechanism used by a nuclear protein or the assistance of viral factors. for example, ibv n protein is known to enter the nucleus while maintaining a predominantly cytoplasmic localization (50, 51) . alternatively, binding of viral rna may partially retain the newly synthesized madp1 in the cytoplasm, as observed in this study. it was observed that over-expression of flag-tagged madp1 was unable to fully restore ibv infection in madp1-knockdown cells, even though the expression level of the introduced madp1 construct far surpassed the endogenous level, as observed by western blot analysis. considering the fact that only 30% of cells were transfected and over-expressed madp1 protein despite the presence of a higher level of the protein in the transfected cells, it is understandable that the expression of viral proteins could not be restored after combining both transfected and untransfected cells. interestingly, over-expression of silencing-sensitive madp1 was unable to cause an increase in virus infection, comparing to that was observed for silencingresistant madp1 (fmmut) in shmadp1 cells, even though their expression levels were comparable. this lends further support to the conclusion that madp1 is actively involved in the replication and infectivity of ibv. although the functional studies involving ibv, a chicken coronavirus, and a human protein, madp1, were conducted using human and african green monkey cells, which were non-native. it is noteworthy that madp1 (homologene 12095) is conserved in humans (homo sapiens), chimpanzees (pan troglodytes), wolves (canis lupus), cattle (bos taurus), mice (mus musculus), rats (rattus norvegicus) and chickens (gallus gallus). the african green monkey genome is not available at ncbi, but an alignment search using basic local alignment search tool (blast) of the madp1 amino acid sequence against the rhesus macaque (macaca mulatta) refseq protein library yields a 99% sequence similarity between the two species. the chicken homolog, on the other hand, bears 85% amino acid sequence similarity, but with an almost identical match in the n-terminal 120 amino acids, to the human madp1 protein. as the predicted interaction domain lies in the n-terminus, it is highly likely that the homologs from other species could replace human madp1 in the interaction studies. in conclusion, the involvement of madp1 in coronavirus rna synthesis and its significance are demonstrated in this study in the tissue culture system. further studies with an madp1 knock-out animal system, which is currently not available, would be required to confirm further the involvement of madp1 in coronavirus rna synthesis. human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells coronavirus genome structure and replication the role of programmed-1 ribosomal frameshifting in coronavirus propagation identification of heptaand octo-uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of sars-cov orf 3a variants a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting virus-encoded proteinases and proteolytic processing in the nidovirales an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of coronavirus infectious bronchitis virus is dispensable for viral replication in cultured cells functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3c-like proteinase functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products characterization of viral proteins encoded by the sars-coronavirus genome the molecular biology of coronaviruses a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rna-dependent rna polymerase nidovirus transcription: how to make sense coronavirus transcription: a perspective the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription viral and cellular proteins involved in coronavirus replication polypyrimidine-tractbinding protein affects transcription but not translation of mouse hepatitis virus rna heterogeneous nuclear ribonucleoprotein a1 binds to the 3 0 -untranslated region and mediates potential 5'-3 0 -end cross talks of mouse hepatitis virus rna heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus syncrip, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus rna synthesis host cell proteins interacting with the 3 0 end of tgev coronavirus genome influence virus replication the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication a three-hybrid screen identifies mrnas controlled by a regulatory protein isolation, expression, and characterization of the human zcrb1 gene mapped to 12q12 amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells the human 18s u11/u12 snrnp contains a set of novel proteins not found in the u2-dependent spliceosome a u-turn motif-containing stem-loop in the coronavirus 5' untranslated region plays a functional role in replication towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 host protein interactions with the 3 0 end of bovine coronavirus rna and the requirement of the poly(a) tail for coronavirus defective genome replication mitochondrial hsp70, hsp40, and hsp60 bind to the 3 0 untranslated region of the murine hepatitis virus genome mitochondrial aconitase binds to the 3 0 untranslated region of the mouse hepatitis virus genome structural lability in stem-loop 1 drives a 5 0 utr-3' utr interaction in coronavirus replication selective replication of coronavirus genomes that express nucleocapsid protein group-specific structural features of the 5 0 -proximal sequences of coronavirus genomic rnas membrane association and dimerization of a cysteine-rich, 16-kilodalton polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus conflict of interest statement. none declared. key: cord-265258-2rmtsyns authors: domanska‐blicharz, k.; lisowska, a.; pikuła, a.; sajewicz‐krukowska, j. title: specific detection of gii‐1 lineage of infectious bronchitis virus date: 2017-07-03 journal: lett appl microbiol doi: 10.1111/lam.12753 sha: doc_id: 265258 cord_uid: 2rmtsyns infectious bronchitis virus (ibv) is a worldwide prevalent rna virus that causes highly contagious and economically devastating disease in chicken. the virus exists in many different genetic forms which made the disease control very difficult. the present study describes the development and validation of taqman probe‐based real‐time reverse transcription‐polymerase chain reaction (real‐time rt‐pcr) targeting the s1 coding region of s gene characteristic for the gii‐1 lineage (formerly the d1466‐like variant) of ibv. these strains are quite different from other european ibv belonging to different lineages of the gi genotype. the developed method was 30‐fold more sensitive than used so far for standard nested rt‐pcr with detection limit of 56 rna copies per reaction. the specificity of the assay was also evaluated with a panel of different poultry pathogens. repeatability and reproducibility of the method was very high with coefficients of variation lower than 4%. one hundred and twenty‐seven ibv‐positive samples were tested by this method and gii‐1 strains were detected in four of them (3·15%) which indicate a decrease in the gii‐1 ibv prevalence in poland. the assay was proven to be a valuable tool for rapid diagnosis of gii‐1 lineage of ibv strains and moreover it enabled the monitoring of viral loads which can be used to assess disease progression. significance and impact of the study: this study reports a taqman probe‐based real‐time reverse transcription‐polymerase chain reaction (real‐time rt‐pcr) for rapid and accurate identification of gii‐1 lineage (formerly d1466‐like variant) of infectious bronchitis virus (ibv). the assay revealed to be more sensitive than standard nested rt‐pcr assay, previously used for this purpose. the developed assay has been tested on numerous field samples and revealed 3·15% prevalence of this lineage of ibv in polish chicken population. moreover, this new assay enables the assessment of viral load measurement which might be useful for epidemiology and pathogenesis studies. infectious bronchitis virus (ibv) infects chickens of all ages causing respiratory, urogenital, reproductive and/or digestive systems diseases. infectious bronchitis virus belongs to the gammacoronavirus genus (order nidovirales, family coronaviridae, subfamily coronavirinae) (carstens 2010; dong et al. 2007 ) and its genome consists of an approximately 30-kb-long single-stranded, positivesense rna. the genome structure and the biology of the virus predispose for changes in ibv genome (ovchinnikova et al. 2011; woo et al. 2009 ). the most important are changes in the spike protein gene (s), especially in its s1 part which encodes extracellular protein subdomain since they may imply a change in the antigenicity or pathogenicity of the virus. so far, many genotypes, serotypes or protectotypes have been identified, depending on the method used for differentiation. however, even the most commonly used genotyping results were sometimes ambiguous because there were no standard rules that should be followed. that's why new classification based on s1 coding region sequence which distinguished and named 32 lineages arranged in six genotypes has been recently proposed (valastro et al. 2016) . the gii-1 lineage (named also d1466 or d212 variant) of ibv was detected for the first time in the netherlands in the late 1970s as an aetiological factor of problems connected with egg losses (davelaar et al. 1984; de wit et al. 2011) . interestingly, the s1 coding region of this variant was different from other european ibv strains which resulted in its classification to the gii genotype together with dutch v1397 strain (valastro et al. 2016) . for many years the d1466 strains has been only occasionally recognized but results of a molecular survey conducted between 2005 and 2006 indicated increasing problems connected with this variant in most countries of western europe (worthington et al. 2008) . in poland this variant emerged in 2011 and subsequently has been identified in different parts of the country (domanskablicharz et al. 2012) . for ibv detection different molecular assays have been described including conventional reverse transcriptionpolymerase chain reaction (rt-pcr) (adzhar et al. 1996; farsang et al. 2002) , labelled taqman probe-or sybr green-based real-time rt-pcr (real-time rt-pcr) assays or reverse transcription loop-mediated isothermal amplification (rt-lamp) (callison et al. 2006; chen et al. 2010; fellahi et al. 2016; jones et al. 2011; meir et al. 2010) . the next step is identification of detected ibv strain, which relays on amplification, sequencing and blast (basic local alignment research tool) or phylogenetic analysis of the sequence of s1 coding region. recently proposed ibv classification is based on the sequence of the entire s1 part with a length of 1620 nt but this is only possible for highly specialized molecular laboratories (valastro et al. 2016) . the main obstacle is the lack of universal primers and sometimes many primers should be tested before final strain identification which could be a very time and labour consuming. on the other hand, information about ibv circulating in the field should be quickly obtained as it is important for selecting suitable protective vaccine. the real-time rt-pcr assays seem to be effective tools for ibv-type identification and although s1 coding region is prone to mutations, methods aimed in this gene detection has been recently developed and used for gi-1 (mass, connecticut), gi-9 (arkansas), gi-11 (sai), g1-16 (asaii) and giv-1 (de072/ga98) lineages (acevedo et al. 2013; marandino et al. 2016; roh et al. 2014 roh et al. , 2013 . for detection of gii-1 (d1466-like) ibv lineage standard nested rt-pcr assay is commonly applied. however, this method is a time-consuming and labour intensive as it needs multiple manipulation of genetic material. moreover, these multiple steps of rna/dna handling might result in cross-contamination and as consequences in false positive results. the real-time rt-pcr assay helps to avoid these disadvantages. here, we describe the development of a taqman probe-based real-time rt-pcr for the detection of gii-1 (d1466-like) ibv lineage. we also describe the conclusions from the routine use of this method, which was implemented in our laboratory in 2014. optimizing of gii-1 real-time rt-pcr in the present work we have successfully developed a highly sensitive taqman probe-based real-time rt-pcr assay for detection of gii-1 lineage of ibv. so far, standard nested rt-pcr assay was commonly used for this purpose (cavanagh et al. 1999; worthington et al. 2008) . both methods target similar fragment of s1 coding fragment which is crucial for ibv-type determination. however, the primers and probe designed for real-time rt-pcr met single mismatches in nucleotide sequences of d1466-like ibv strains previously detected in poland and they include some degenerative ones (two in the reverse primer and two in the probe) to avoid failure in this genotype detection. the standard curve and linear regression analysis were performed using serial dilutions of known quantity of gii-1 ibv rna (fig. 1) . c t values ranged from 17á70 to 37á03 cycles (table 1) with a coefficient of determination (r 2 ) of 0á996. the slope of à3á168 reveals a very high rt-pcr efficiency (106%). the limit of detection of the gii-1 ibv real-time rt-pcrs and universal for all gammacoronavirus were similar at the level of an approximately 56 copies of viral rna. the sensitivity of the standard nested rt-pcr was almost 30-fold lower and detected the presence about 1500 copies of viral rna (table 1) . moreover, the assay developed in the present study showed to be highly specific as no fluorescent signals were detected with other tested ibv lineages or chicken rna viral pathogens. the intra and interassay precision of the new method were assessed using serial dilutions of d1466 ibv rna ranging from 8á2 9 10 6 down to 5á6 9 10 1 copies. the repeatability cvs of d1466 real-time rt-pcr ranged from 0á17 to 0á69%, while the reproducibility cvs ranged from 0á99 to 3á55% (table 2) . the assay developed in this study has been tested on numerous field tissues and swabs samples. the total of 190 field samples from chicken (broilers, commercial layers and broiler breeders) were tested by gammacoronavirus universal real-time rt-pcr assay in the period between 2014 and 2016. one hundred and twenty-seven (66á8%) were positive for ibv. when these samples were tested by the gii-1 real-time rt-pcr, 4 of 127 (3á15%) were positive. interestingly, no d1466-positive sample was detected in 2014 but three samples in 2015 and one in 2016 were identified. molecular studies carried out between 2005 and 2006 in western european countries showed growing problems associated with d1466 genotype (worthington et al. 2008) . in 2005, the average frequency of the d1466-like virus in belgium, the netherlands and germany was about 3-5%, and in 2006 the presence of this variant increased to 7, 10 and 16% respectively. in poland, in the period from november 2011, when the first case of d1466 ibv was detected, to december 2013, the standard nested rt-pcr was used and this assay enabled detection of 26 positive samples indicating the presence of this variant at 11á7% of studied chicken flocks. our study clearly shows a gradual decrease in the gii-1 ibv prevalence in poland. the assay here developed enables the estimation of the amount of viral rna copies, which could be a useful indicator of the infection phase and correlate with viraemia or clinical disease (mackay et al. 2002) . the real-time rt-pcr is able to detect as few as 56 rna molecules of this ibv lineage. when testing field samples, the c t values obtained were relatively high (mean c t values of 32á4 ae 3á8) which address the mean amount of virus rna about 1500 copies per reaction. the low number of gii-1 ibv rna copies in most cases did not parallel the c t values obtained in universal real-time rt-pcr which were much lower. this was due to the presence of other ibv lineages in these samples. subsequent applied assays aimed at the identification of other ibv lineages revealed the presence gi-1 (mass-like), gi-13 (793b-like), gi-19 (qx-like) or gi-23 (var2-like) in almost all of them. there was only one case where the c t values in both assays were equal indicating a low number of rna copies (c t about 32). it seems that this variant replicates poorly in the tissues of chickens and its excretion with the faeces is also low. the poorly replicating viruses are unlikely to cause disease symptoms. in fact some of the d1466-positive chicken flocks were described as healthy by the maintenance staff. therefore, pathogenicity and virulence of the gii-1 strains seem to be insignificant. we also revealed the usefulness the rt-ddpcr for rna concentration determination. usually, standard curves are synthetically generated with plasmids or transcript molecules with the same sequence as the gene of interest. however, its concentration depends not only on the dilution but also on the quantification methods which themselves are prone to uncertainties. the rt-ddpcr is a method that greatly facilitates rna measurement and overcomes these limitations. the primers and probe constructed in this study for real-time rt-pcr were successfully used in rt-ddpcr. however, due to the fact that only few laboratories use this technology routinely, our efforts have focused on real-time rt-pcr. in conclusion, the taqman probe-based real-time rt-pcr assay described here is a time-saving, specific, sensitive and reliable method of detection of gii-1 lineage (d1466-like) of ibv which could successfully replace standard nested rt-pcr. moreover, this new assay enables the s1 coding fragment sequences of d1466-like ibv strains were retrieved from the genbank database and aligned using mega6 (http://www.megasoftware.net, molecular evolutionary genetics analysis ver. 6.0) software package. primers and probe were designed manually based on a conserved region within the aligned s1 part of s gene sequences of 12 strains of gii-1 ibv lineage. forward primer d5-f (5 0 -ttacagcctggcaatgtctt-3 0 ) located at nucleotide position 458-477 according to the sequence of d1466 strain (genbank accession no. m21971); reverse primer d6-r (5 0 -caacatcctcmrtaaagttagaac-3 0 ) located at nucleotide position 521-544, and taqman d-probe (fam-cyagtgtgtttctaaayggcaacctt-bhq1) located at nucleotide position 488-514 amplified and detected 87-bp fragment. in silico specificity of the primers and probe was assessed by blastn search function (http://www.ncbi.nlm.nih.gov/blast). the primers and probe were synthesized by genomed sp. z o.o. (warsaw, poland). tissue samples (kidney, oviduct, respiratory and intestinal tracts) and oropharyngeal/cloacal swabs from various broiler chickens, commercial layers and broiler breeder farms were submitted for laboratory diagnosis. in the period between 2014 and 2016 a total of 190 flocks from different regions of poland were obtained. for molecular assays optimization the live attenuated ib vaccine (nobilis ib d1466; msd animal health, boxmeer, the netherlands) was used. tissue samples were homogenized in 10-20% (w/v) suspension and vaccine and swabs hydrated in phosphate-buffered saline (pbs) with addition of antibiotics (100 l penicillin and 100 lg streptomycin per ml), centrifuged at 3000g for 15 min. total rna was extracted from 250 ll of tissues homogenates, vaccine and swabs (five pooled swabs) supernatants into 50 ll rnase-free water using commercial kits (rneasy mini kit; qiagen, hilden, germany) according to the manufacturer's instruction. standard nested rt-pcr amplification was performed using the primers described by cavanagh et al. (1999) . the first reaction was carried out with onestep rt-pcr kit (qiagen) in a 25-ll final reaction mixture volume containing 5 ll 59 onestep rt-pcr buffer, 5 ll 5xq solution, primers to a final concentration of 600 nmol l à1 , 400 nmol l à1 of each dntp, 2á5 ll rna template and nuclease-free water. the nested reaction was carried out with taq dna polymerase kit (eurx, gdansk, poland) in a 25-ll volume containing 12á5 ll 29 taq pcr master mix, nested primers to a final concentration of 600 nmol l à1 , 2á5 ll cdna from the first reaction and nuclease-free water. thermocycler running conditions were: 94°c for 30 s, 49°c for 30 s and 72°c for 30 s, repeated 35 times and thereafter a final extension step at 72°c for 5 min. additionally the first reaction was preceded by a reverse transcription at 50°c for 30 min and by polymerase activation at 95°c for 15 min. pcr products were visualized and documented by electrophoresis in a 1á8% agarose gel stained with ethidium bromide under uv light using a minibis pro system (dnr bio-imaging systems ltd., neve yamin, israel). the real-time rt-pcr assays were performed using 7500 fast real-time pcr system (applied biosystems) in a 96-well optical plate format. amplification of d1466like variant s1 cdna fragment was carried out in a 25-ll mixture volume containing 12á5 ll 29 quantitect probe rt-pcr master mix (quantitect probe rt-pcr kit, qiagen), 0á25 ll quantitect rt mix, primers to a final concentration of 400 nmol l à1 , probe to a final concentration of 200 nmol l à1 , 2á5 ll rna template and nuclease free water. thermal cycling conditions included one cycle at 50°c for 30 min for reverse transcription, one cycle at 95°c for 15 min for taq polymerase activation and 40 cycles of 95°c for 10 s, and 60°c for 60 s for cdna amplification. fluorescence was acquired during each extension step. negative controls contained pcrgrade water. universal real-time rt-pcr assay for all gammacoronaviruses detection was run with the same chemistry and condition, but with the primers and probe according to callison et al. (2006) . the reverse transcription droplet digital pcr (rt-ddpcr) with the same primers and probe as in d1466 real=time rt-pcr was used to quantify the rna concentration. the rna isolated from 10-fold dilutions of d1466 ib vaccine were analysed using a droplet digital pcr qx100 system (bio-rad laboratories, inc., hercules, ca, usa) courtesy of the department of microbiology, national veterinary institute (uppsala, sweden). the 25 ll rt-ddpcr reaction mixture contained 1 9 supermix, 2 u ll à1 reverse transcriptase, 300 nmol l à1 dtt (bio-rad laboratories, inc.), d1466 primers (400 nmol l à1 )/probes (200 nmol l à1 ) and 8á2 ll nucleic acid. reverse transcription was carried out at 50°c for 30 min followed by denaturation at 95°c for 10 min, and dna was amplified with 40 pcr cycles at 95°c (30 s) and 60°c (1 min). read-out of positive vs negative droplets was performed with the droplet reader and the absolute quantification of pcr target was executed using quantasoft v1.6 software (biorad laboratories, inc.). each dilution was tested in duplicate and the amount of rna were medium value from the measurements obtained. then, the same dilutions of viral rna containing a known number of molecules were tested in real-time rt-pcr. additionally, the amplification efficiency and coefficient of determination were calculated for the gii-1 real-time rt-pcr. to assess repeatability and reproducibility, each dilution was tested on different dates and by two different laboratory technicians (in triplicate in each combination). obtained fluorescence threshold crossing point values (c t values) from each run was then used for calculation of the mean, standard deviation and coefficient of variation. to evaluate the specificity of the real-time rt-pcr, the rna preparations from other common poultry viral pathogens (gi-1, gi-12, gi-13 and gi-19 lineages of ibv, infectious bursal disease virus, newcastle disease virus, avian influenza virus, avian reovirus, astrovirus and rotavirus) were used. a duplex sybr green i-based real-time rt-pcr assay for the simultaneous detection and differentiation of massachusetts and non-massachusetts serotypes of infectious bronchitis virus universal oligonucleotides for detection of infectious bronchitis virus by polymerase chain reaction development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens ratification vote on taxonomic proposals to the international committee on taxonomy of viruses longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions reverse transcription loop-mediated isothermal amplification for the rapid detection of infectious bronchitis virus in infected chicken tissues occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands d1466-like genotype of infectious bronchitis virus responsible for a new epidemic in chickens in poland detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern china molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain comparison of sybr green i real-time rt-pcr with conventional agarose gel-based rt-pcr for the diagnosis of infectious bronchitis virus infection in chickens in morocco development and validation of rt-pcr tests for the detection and s1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples real-time pcr in virology development of rt-qpcr assays for the specific identification of two major genotypes of avian infectious bronchitis virus development of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus in chickens, and comparison of rt-pcr and virus isolation molecular characterization of infectious bronchitis virus isolates from russia and neighbouring countries: identification of intertypic recombination in the s1 gene evaluation of infectious bronchitis virus arkansastype vaccine failure in commercial broilers detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-pcr and correlation with virus detection in embryonated eggs s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification infectious bronchitis virus variants: a review of the history, current situation and control measures coronavirus diversity, phylogeny and interspecies jumping a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from we thank the staff of the department of microbiology of the national veterinary institute (uppsala, sweden) for allowing analyses on droplet digital pcr qx100 system (bio-rad laboratories, inc.) for rna concentration determination. this work was partially funded by know (leading national research centre) scientific consortium "healthy animal -safe food", decision of ministry of science and higher education no. 05-1/know2/2015. no conflict of interest declared. key: cord-291174-rym84kni authors: yang, yazhi; yang, dawei; shao, yingge; li, yi; chen, xifeng; xu, yuanyuan; miao, jinfeng title: a label-free electrochemical assay for coronavirus ibv h120 strain quantification based on equivalent substitution effect and aunps-assisted signal amplification date: 2020-10-23 journal: mikrochim acta doi: 10.1007/s00604-020-04582-3 sha: doc_id: 291174 cord_uid: rym84kni a label-free electrochemical strategy is proposed combining equivalent substitution effect with aunps-assisted signal amplification. according to the differences of s1 protein in various infectious bronchitis virus (ibv) strains, a target dna sequence that can specifically recognize h120 rna forming a dna-rna hybridized double-strand structure has been designed. then, the residual single-stranded target dna is hydrolyzed by s1 nuclease. therefore, the content of target dna becomes equal to the content of virus rna. after equivalent coronavirus, the target dna is separated from dna-rna hybridized double strand by heating, which can partly hybridize with probe 2 modified on the electrode surface and probe 1 on aunps’ surface. thus, aunps are pulled to the surface of the electrode and the abundant dna on aunps’ surface could adsorb a large amount of hexaammineruthenium (iii) chloride (ruhex) molecules, which produce a remarkably amplified electrochemical response. the voltammetric signal of ruhex with a peak near − 0.28 v vs. ag/agcl is used as the signal output. the proposed method shows a detection range of 1.56e(−9) to 1.56e(−6) μm with the detection limit of 2.96e(−10) μm for ibv h120 strain selective quantification detection, exhibiting good accuracy, stability, and simplicity, which shows a great potential for ibv detection in vaccine research and avian infectious bronchitis diagnosis. [figure: see text] electronic supplementary material: the online version of this article (10.1007/s00604-020-04582-3) contains supplementary material, which is available to authorized users. in the last few decades, viruses are a real menace to safety. the pandemic dimension spread of coronavirus disease poses a severe threat to the health and lives of seven billion people worldwide [1] . rapid identification of viruses should be one of the best ways to prevent disease outbreaks and is of great significance to medical healthcare [1] . ibv, one kind of coronaviruses, is a positive-sense single-stranded enveloped rna virus with a length of 27-32 kb. one ibv strain, h120 strain, usually needs to be identified from other strains for immunoprophylaxis and vaccine production, for example, nna strain. both of them are composed of structural and nonstructural proteins [2, 3] . the spike (s) glycoprotein is one of the major structural proteins which can be posttranslationally cleaved into s1 and s2 subunits [4] . s1 subunit determines the genotype, serotype, and phenotype of ibv, which is the most significant protein for virus identification [5] . it is known that gene sequences of h120 and nna strains yazhi yang and dawei yang contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/10.1007/s00604-020-04582-3) contains supplementary material, which is available to authorized users. are highly similar [6] [7] [8] ; as a result, an obstacle in immunoprophylaxis presents because of the lack of accurate identification method. therefore, developing a rapid and sensitive method for identification and quantification of different ibv strains based on hypervariable region of s1 gene can effectively solve the problem, which plays important roles in ib early diagnosis and control, especially for vaccine production. up to now, a number of methods have been fabricated to diagnose acute ibv infections based on ibv virus rna detection and antibody response. these common methods include immunofluorescence assay (ifa) [9] , agar gel precipitation test (agpt) [10] , virus isolation (vi) [11] , immunoperoxidase assay (ipa) [12] , reverse transcriptase polymerase chain reaction (rt-pcr) [13] , and enzyme-linked immunosorbent assay (elisa) [4] . among the above methods, the sensitivity is generally unsatisfactory and those methods are relatively expensive and laborious. meanwhile, one of present challenges in the virus detection field is the need for further non cross-reactive, stable, and sensitive biosensors [14] . thus, the development of ultrasensitive and fast methods to detect ibv is still a great challenge and absolutely necessary. to improve the sensitivity, the signal amplification strategy should be utilized [15, 16] . recently, some studies have shown that nanomaterials play an essential role in nanotechnology and biomedical applications [17, 18] . among different nanomaterials, gold nanoparticles (aunps) have attracted tremendous interests [19, 20] , due to its characteristics including easy synthesis manipulation, precise control over the physicochemical properties, strong binding affinity for thiols, and distinct electronic properties [21] . aunps have been used as efficient sensors for the detection of dna and rna based on different sensing strategies [22, 23] . among the electrochemical methods, it has attracted great attention due to its properties [24, 25] , including convenient operability, simple instrumentation, low cost, and on-site detection [26, 27] . thus, the development of labelfree electrochemical biosensor for the assay of ibv detection based on the aunps amplification is very promising. materials and apparatus al2000 dna marker and 1 kb dna ladder marker were obtained from nanjing zhongding biotechnology co. ltd. prime script ii 1st strand cdna synthesis kit, sybr premix ex taq ii (tli rnaseh plus) bulk, pmd19-t vector cloning kit, and ecori were purchased from dalian bao biological engineering co. ltd. the axyprep dna gel extraction kit was obtained from axygen (usa). chloroauric acid, sodium citrate, and tris(2-carboxyethyl)-phosphine (tcep) were purchased from sigma-aldrich chemical co. ltd. (usa). other reagents in the method were of analytical grade. all solutions in the study were prepared with ultrapure water, which was obtained from milli q water purification system (usa). the dna immobilization buffers are 10 mm tris-hcl, 1 mm edta, 0.1 m nacl, and 10 μm tcep (ph 7.4). the reaction buffer is phosphate-buffered saline (10 mm na 2 hpo 4 , 2 mm nah 2 po 4 , ph 7.4) with 140 mm nacl and 5 mm mgcl 2 . dna oligonucleotides (hplc purified) were synthesized by sangon biotechnology co. ltd. (shanghai, china). all the electrochemical measurements were performed on chi 660d electrochemical workstation (shanghai, china). dna sequences used in this electrochemical assay were shown in table 1 . the axyprep body fluid viral dna/rna miniprep kit was used to extract viral rna. the prime script ii 1st strand cdna synthesis kit and roche's pcr enzyme faststart universal sybr green master (rox) were for rna inversion and s1 gene amplification, respectively. the s1 gene amplification reaction system (total volume 25 μl) contained 12.5 μl of 2 × pcr master mix, 1 μl of cdna products, 9.5 μl of ddh 2 o, and 1 μl of 10 pmol of s1 forward and s1 reverse primer (table s1 ). amplification procedures were as follows: 95°c for 5 min, then 33 cycles of 95°c for 30 s, 52°c for 30 s, 72°c for 1 min, and finally 10 min at 72°c. the products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. after purification, pcr product was connected with pmd19-t. then, it was transformed into susceptible e. coli by conventional method and coated on plates with ampicillin and lb. white colonies were selected and inoculated in lb medium containing ampicillin. finally, the plasmids were extracted for sequencing and identification by enzyme digestion. the concentration of standard plasmids was measured by ultraviolet spectrophotometer and copy number was calculated through the equation: copies/ml = plasmid concentration × 6.02 × 10 2 3 × 10 − 9 /(plasmid length × 660). after table 1 list of dna sequences used in this experiment name sequence (5′-3′) sh-h120-probe1 ttt ttt tca ggt gag tta h120-probe 2-sh gat cat aat ata tat ata t h120-target att atg atc taa ctc acc tga determination of copy number, a series of standard plasmids at different copies were prepared for real-time fluorescence quantitative pcr to obtain the standard linear curve, which was used to calculate the concentration of real virus samples. the synthesis of aunps was according to our previous report [28] . then, 100 μl of aunps colloid was incubated with 40 μl of 10 μm probe 1 and 360 μl of pbs buffer. the mixed solution was oscillated slowly in 37°c for 12 h. after that, 2 m nacl was added slowly at room temperature for 4 times. the final concentration of nacl was 0.3 m. the salt was used to block the negative charges on dna strands to allow high density loading and rearrangement of dna on aunps' surface. after incubation at room temperature for another 8 h, the solution was centrifuged at 12,000 rpm for 20 min. then, the solution was purified through three times of centrifugation at 12,000 rpm for 20 min. finally, the aunps were resuspended in 20 mm tris-hcl (ph 8.0) solution and stored at 4°c. the characterizations of probe 1 dnamodified aunps were investigated by ultraviolet visible absorption spectroscopy (fig. s1 ). pretreatment of gold electrode was according to our previous report [29] . the electrode was electrochemically activated in 0.5 m h 2 so 4 . probe 2 of 0.5 μm was incubated with gold electrode for 12 h at room temperature. then, the modified electrode was immersed into an aqueous solution of 1 mm mercaptohexanol (mch) for 60 min to inhibit nonspecific dna adsorption [30] . the target dna was incubated with viral rna at 90°c for 5 min and dropped to room temperature slowly. after that, the reaction mixture was incubated with 1 μl of 80 u/μl nuclease at 37°c for 30 min. then, the reaction mixture was placed at 90°c for 15 min and annealed at 4°c. then, the final reaction mixture was incubated with probe 2-modified electrode. finally, probe 1-functionalized aunps was incubated with the modified electrode for 2 h. in this work, a threeelectrode cell was used with an ag/agcl as the reference electrode, a platinum wire as the counter electrode, and a gold electrode as the working electrode. two electrochemical techniques, linear sweep voltammetry and chronocoulometry, were conducted in the experiment. a tris-hcl solution (ph 7.4) of 10 mm containing 50 μm ruhex and 10 mm pbs buffer was used. the peak current at − 0.28 v was recorded and used to quantify the concentration of ibv h120 strain. the detailed working principle is illustrated in scheme 1. herein, a label-free electrochemical assay based on equivalent substitution effect and aunps-assisted signal amplification is developed for identification and quantification detection of ibv h120 strain. we firstly design the h120 target dna which can recognize rna of h120 strain specifically, and the residual single-stranded h120 target dna can be hydrolyzed by s1 nuclease. then, the h120 target dna is separated from the dna-rna hybridized double strand by heating. thus, the concentration of virus rna of h120 strain is equivalently substituted by the target dna. the target dna can partly hybridize with the probe 2 modified on the electrode surface and probe 1 on the surface of aunps. due to an electrostatic interaction, positively charged signal molecules, hexaammineruthenium (iii) chloride (ruhex), can be adsorbed onto the probe 1 modified on aunps which is pulled on the electrode surface through the target dna, causing an intense electrochemical response. by analyzing the electrochemical response, a sensitive identification and quantification assay for ibv h120 strain is thus established and the method proposed in this work has a great potential for ibv detection in medical research and early ib diagnosis. as shown in fig. 1a and b, electrophoresis results indicate pcr products of s1 gene are successfully amplified. electrophoresis results of standard plasmids are shown in fig. 1c . after being completely cleaved by ecori enzyme, two bands can be seen in the electrophoretic diagram that demonstrate the successful construction of plasmid. after construction of the standard plasmid containing characteristic sequence in s1 gene of h120 strain, virus copy numbers in plasmids are measured by real-time fluorescence quantitative pcr. amplification and melting curve of plasmid in fig. 2a and b indicate that there is no nonspecific amplification. a linear relationship between ct value and the logarithm of the initial copy number of the standard plasmid (fig. 2c) is obtained, and the linear regression equation is y = − 3.3191x + 33.0203 (r 2 = 0.99951). thus, a series of h120 standard samples at different concentrations is successfully prepared for the following electrochemical detection. meanwhile, according to calculation, the concentration of h120 virus sample is 1.56e −6 μm (rsd = 0.67%, n = 3). in this work, the experimental conditions have been optimized to achieve the best signal-to-noise level. first, the annealing condition of target dna and ibv h120 strain has been investigated. in this assay, we have adopted two kinds of annealing (fig. s2a) . one is 90°c water bath for 5 min and then natural cooling to room temperature, and the other is metal bath cooling by means of gradient temperature (90°c for 5 min, 70°c for 10 min, 50°c for 10 min, 30°c for 10 min, 10°c for 25 min). as shown in fig. 3a , the former performance is better. thus, 90°c water bath for 5 min with natural cooling to room temperature is chosen for the following experiments. second, the concentration of ruhex is investigated (fig. s2b) . in this assay, we have applied 5 μm and 50 μm ruhex for the detection of ibv h120 strain. as shown in fig. 3b , the electrochemical signal with 50 μm ruhex is better. thus, 50 μm is chosen as the optimized concentration. finally, the electrolyte of this assay is studied (fig. s2c) . we have respectively used 10 mm tris-hcl (ph 7.0) and 10 mm pbs (ph 7.0) as the electrolytes. as shown in fig. 3c , the tris-hcl buffer is better than pbs buffer. therefore, the tris-hcl buffer is chosen as the electrolyte. the sensitivity of the sensing system is evaluated under the optimized experimental conditions. firstly, we detect different concentrations of ibv h120 strain; a gradual increase of ruhex peak current corresponding with the elevated concentration of h120 strain from 1.56e −9 to 1.56e −6 μm is obtained ( fig. 4a and b) . and the peak current is linear with the logarithm of ibv h120 strain. the linear range of ibv h120 strain from 1.56e −9 to 1.56e −6 μm with the detection limit at 2.96e −10 μm is obtained (s/n = 3, rsd = 1.65%, n = 3). the linear regression equation is y = 7.9821 + 0.6406x (r 2 = 0.99837), where y is the peak current and x is the logarithm of ibv h120 strain concentration. to further evaluate the specificity of our proposed method, the chronocoulometry is used. we have detected h120 and nna strain using h120 target dna. as shown in fig. 4c , nna strain does not produce significant electrochemical signal and h120 strain could scheme 1 schematic illustration of the electrochemical method for ibv h120 strain detection fig. 1 agarose gel electrophoresis diagrams. a lane m: 2000 bp dna marker, lane 1 negative control, lane 2: h120 s1 gene sample. b lane m: 2000 bp dna marker, lane 1: negative control, lane 2: nna sample. c lane m: 1 kb dna ladder marker, lane 1: h120 plasmid, lane 2: h120 plasmid is cleaved by ecori enzyme produce significant electrochemical signal, which signifies the excellent specificity of this method. in order to evaluate the performance of the present sensor system, a comparable table for ibv analysis methods has been listed in the electronic supplementary material (table s2) . though the construction of aunps is time-consuming and the introduction of the signal amplification makes the detection system a little bit complicated, it still can be seen from the table that our analysis method is more excellent than others due to the following attractive advantages. first, the ibv h120 strain rna detection process is transformed into dna detection which can effectively avoid rna degradation. second, the use of gold nanoparticles greatly increases the electron transfer efficiency of electrode surface. third, aunps-assisted electrochemical signal amplification highly enhances the sensitivity of ibv detection. in this work, we designed the sequence of the target dna based on the hypervariable region in the s1 gene between different ibv strains, then, constructed the standard plasmid containing characteristic sequence of s1 gene in h120 strain, fig. 2 real-time fluorescence quantitative pcr plot. a amplification plots. b melting curves of h120 standard samples at 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , and 10 9 copy numbers. c the linear relationship between ct value and the logarithm of the initial copy numbers fig. 3 chronocoulometric-int numerical bar graph. a the annealing condition of target dna and virus rna. b the chronocoulometric response of ruhex. c the ph 7.0 electrolyte and at last, proposed a label-free ultrasensitive electrochemical assay applicable for the detection of ibv h120 strain in real sample, which can distinguish between h120 and nna. in this electrochemical method, h120 strain detection is based on equivalent substitution effect and aunps-assisted signal amplification with a detection range from 1.56e −9 to 1.56e −6 μm. compared with previously reported ibv detection methods that may focus on the portable and simplified analysis, our electrochemical assay still has some advantages. for example, converting rna detection to dna detection effectively avoid rna degradation. the use of gold nanoparticles and aunpsassisted electrochemical signal amplification greatly increases the electron transfer efficiency and the sensitivity of ibv detection. furthermore, by analyzing the hypervariable region of s1 gene and replacing the target and probe dna sequence by other customized sequences, the developed sensing strategy can be easily used to detect other virus. therefore, the method might hold a great potential for further applications in virus bioanalysis, early clinical diagnosis, and biomedical research. ultrasensitive detection of pathogenic viruses with electrochemical biosensor: state of the art pathogenicity of a qx-like avian infectious bronchitis virus isolated in china pathogenesis and diagnostic approaches of avian infectious bronchitis a novel immunochromatographic strip for antigen detection of avian infectious bronchitis virus the first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in algeria the effect of diatomaceous earth in live, attenuated infectious bronchitis vaccine, immune responses, and protection against challenge genetic and biological characteristics of four novel recombinant avian infectious bronchitis viruses isolated in china the establishment and characteristics of cell-adapted ibv strain h120 recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification diagnosis of infectious bronchitis in chickens by polymerase chain reaction and fluorescent antibody technique development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and mycoplasma gallisepticum in chickens detection ofinfectious bronchitis virus with the use of real-time quantitative reverse transcriptase-pcr and correlation with virus detection in embryonated eggs electrochemical biosensors for pathogen detection dna-functionalized porous fe 3 o 4 nanoparticles for the electrochemical detection of h120 rna: a linear sweep voltammetry corresponding to different concentration (1.56e −9 μm, 1.56e −8 μm, 1.56e −7 μm, 1.56e −6 μm, n = 3). b the linear relationship between the peak current and the concentration of h120 rna. c chronocoulometric response of (a) 10 μl h120 virus, (b) 10 μl nna virus, (c) 0 μl h120 virus construction of self-powered mirna biosensor with target recycling amplification electrochemical detection of mirna combining t7 exonuclease-assisted cascade signal amplification and dna-templated copper nanoparticles exonuclease and nicking endonuclease-assisted amplified electrochemical detection of coralyne polydopamine nanosphere@silver nanoclusters for fluorescence detection of multiplex tumor markers electrochemical immunosensor based on chitosan-gold nanoparticle/carbon nanotube as a platform and lactate oxidase as a label for detection of ca125 oncomarker nuclease assisted target recycling and spherical nucleic acids gold nanoparticles recruitment for ultrasensitive detection of microrna triple signal amplification strategy for ultrasensitive determination of mirna based on duplex specific nuclease and bridge dna-gold nanoparticles direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification a highly sensitive gold nanoparticle-based electrochemical aptasensor for theophylline detection a new electrochemical method for the detection of cancer cells based on small molecule-linked dna integration of fluorescence imaging and electrochemical biosensing for both qualitative location and quantitative detection of cancer cells electrochemical detection of tobramycin based on enzymesassisted dual signal amplification by using a novel truncated aptamer with high affinity switchable dna tweezer and g-quadruplex nanostructures for ultrasensitive voltammetric determination of the k-ras gene fragment a colorimetric aptasensor for the antibiotics oxytetracycline and kanamycin based on the use of magnetic beads and gold nanoparticles stable and reusable electrochemical biosensor for poly (adp-ribose) polymerase and its inhibitor based on enzyme-initiated auto-parylation star trigon structure-aided dna walker for amplified electrochemical detection of dna immunosensor based on antibodyfunctionalized mos 2 for rapid detection of avian coronavirus on cotton thread chiral zirconium quantum dots: a new class of nanocrystals for optical detection of coronavirus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors thank merial co. ltd. (nanjing, china) for providing ibv h120 and nna strains. conflict of interest the authors declare that they have no conflict of interest. key: cord-265508-t1nfyzf5 authors: boursnell, m.e.g.; brown, t.d.k. title: sequencing of coronavirus ibv genomic rna: a 195-base open reading frame encoded by mrna b date: 1984-08-31 journal: gene doi: 10.1016/0378-1119(84)90169-0 sha: doc_id: 265508 cord_uid: t1nfyzf5 abstract dna sequencing of genomic cdna clones of avian infectious bronchitis virus (ibv) has been carried out. 770 bases have been determined which include genomic sequences spanning the 5' termini of the two smallest mrnas of the 3'-coterminal “nested” set: mrna a and mrna b. this region contains the complete coding sequences for mrna b which are additional to those present in mrna a. two open reading frames are present, predicting proteins of m rs 7500 and 9500. avian ibv, in common with other coronaviruses, has a single-stranded, polyadenylated, infectious rna genome approx. 20 kb in length (stern and kennedy, 1980a) . in infected cells multiple subgenomic positive-stranded rnas are produced . for ibv and mhv these have been shown to consist of a 3'-coterminal "nested" set (stem and kennedy, 1980a; lai et al., 1981; leibowitz et al., 1981) . for both ibv and mhv the messenger function of these subgenomic rnas has been demonstrated (rottier et al., 1981; stem et al., 1982; siddell, 1983) . however, certain differences of genome organisation have become apparent between (stern and kennedy, 1980b; lai et al., 1981) . second, the coding function of the various messenger rnas is different. both ibv and mhv contain three main structural polypeptides: the nucleocapsid, the membrane (el), and the spike or peplomer (e2) polypeptides (cavanagh, 1981; siddell et al., 1983) . in both systems the smallest rna codes for the nucleocapsid protein but in mhv the next smallest rna codes for the membrane polypeptide, whereas in ibv the membrane polypeptide is coded for by the third smallest rna (siddell et al., 1980; siddell, 1983; rottier et al., 1981; stern et al., 1982; stem and sefton, 1984) . these differences are summarised in fig. 1 . the organisation of the messenger rnas and in vitro translation studies have led to the hypothesis that the 5'-most sequences of each mrna, which are not present in the next smallest mrna, contain the complete coding sequences for the major protein product produced by that messenger species (stern and kennedy, 1980b; lai et al., 1981) . however, since the only coronavirus sequences published are those of the smallest rna species skinner and siddell, 1983) it has not been possible to examine this hypothesis at the rna sequence level. rna sequence data from this region might also enable us to predict the properties and thus aid the identification of possible polypeptides coded for by mrna b. in this paper we report the nucleotide sequence of a cloned cdna copy of ibv genomic rna in the region corresponding to the 5' end of mrna b. the sequence shows that the 5'-most sequences of mrna b could code for a hydrophobic 7.5-kda1 protein. the preparation of cdna clones has been previously described (brown and boursnell, 1984) . briefly, virion rna was isolated from ibv strain beaudette grown in embryonated eggs. cdna was produced by oligo(dt)-primed reverse transcription of the rna, followed by self-primed reverse transcription to generate the second strand. s 1 nucleasetreated cdna was dc-tailed using terminal transferase, annealed to dg-tailed pst i-cleaved pat 153 (twigg and sherratt, 1980) and transformed into escherichia co/i hb 10 1. ampicillin-sensitive colonies were selected for further characterisation. viral clones were identified by hybridisation with a probe prepared by polynucleotide kinase labelling of alkali-treated, full-length ibv genomic rna. restriction sites were mapped on a series of clones and this enabled construction of a continuous map, sequences at 3'-terminus of the viral genome was confirmed by hybridisation with a kinase-labelled poly(u) probe. (c) formaldehyde-agarose gel analysis of ibv mrnas 1 s yo formaldehyde-agarose gels were run essentially as described by maniatis et al. (1982) . total rna samples from ibv-infected chick kidney cell cultures were run overnight at 60 v on 16 cm vertical gels. ibv mrnas were detected by blotting onto nitrocellulose and probing with nick-translated cloned ibv sequences (maniatis et al., 1982 kinase, were sequenced essentially as described by maxam and gilbert (1980) . the depurination reaction was carried out in 66"; formic acid for 10 min at 20' c, after which the samples were treated in the same way as the pyrimidine reaction. for sequencing some regions of the dna, restriction digests of the viral insert were recloned into the plasmid puc9 allowing sequencing from adjacent vector restriction sites (messing and vieira, 1982) . sequence data were stored and analysed on an apple iie microcomputer using the programs of larson and messing (1983) and on a vax 1 l/780 minicomputer using the programs of staden (1984) . results 770 bp of dna sequence from one ibv genomic clone, c5.136 (brown and boursnell, 1984) have been determined. this sequence corresponds to the genomic rna sequence stretching from 2.40 kb to 1.63 kb from the 3' end of the viral genome. in 95% of the sequence has been determined on both strands, and on each strand most regions have been sequenced more than once from different restriction sites. fig. 2c shows the positions of the initiation and termination codons in the three reading frames and the positions of the 5' ends of mrnas a and b as determined by sl nuclease mapping (brown and boursnell, 1984) . it should be noted that s 1 nuclease mapping will determine the 5' end of the "body" of the mrna (see discussion) . the dna sequence of 770 nucleotides, with a translation of the three main orfs, is shown in fig. 3 (brown and boursnell, 1984) . in skinner and siddell, 1983 ) reveals no significant homology at the rna or protein level. although some homology might be expected it should be noted that no serological crossreaction between ibv and any of the mammalian coronaviruses has been reported . the region of the ibv sequence presented in this paper contains the 5' ends, on the viral genome, of mrnas a and b. the messenger rnas of coronavirus ibv are probably transcribed from non-contiguous regions of the viral genome. work on the murine coronavirus mhv has shown that a common 5' leader sequence, originating from the extreme 5' end of the viral genome, is fused to the body of each messenger rna (lai et al., 1982; 1983; spaan et al.. 1983; baric et al., 1983) . it is likely that the ibv messenger rnas have the same structure. however, whether or not leader sequences are present, sl nuclease mapping experiments have shown that the ends ofthe bodies ofmrnas a and b lie at positions 139 and 443 respectively (brown and boursnell. 1984 ) as shown in fig. 3 . of the two orfs which are present at the 5' end of mrna b, the m, 7500 orf seems to be the most likely candidate for translation in vivo. the location of the coding sequence for the putative m, 7500 polypeptide fits in well with the hypothesis that the major polypeptide product of each mrna is translated from those 5' sequences not present in the next smallest rna. if the a4, 9500 polypeptide were translated then this would no longer be true, since its coding region stretches well into mrna a. furthermore, the rna sequences flanking the initiation codons for the putative a4r 7500 and nucleocapsid genes correspond well to those preferred for functional eukaryotic initiation codons (kozak, 1983) . however, the sequence gnnauga around the aug codon at the start of the m, 9500 orf is rare in this context. in addition the aug codon at the start of the mr 7500 orf is the first initiation codon to occur in the body of mrna b ; since initiation of translation at anything other than the first aug codon is known to be rare (kozak, 1983) this suggests that this orf codes for the major product of mrna b. the amino acid sequence of the putative m, 7500 polypeptide shows it to be hydrophobic in nature and to have an unusual composition in that 26"" (17 out of 65) of its residues are leucine. of the six possible triplets coding for leucine, one (uua) is used 8 times out of 17. this unusual composition and ccdon bias, which would not be expected from a chance orf, suggests that this polypeptide is translated in vivo. a computer analysis of the sequences presented here has been carried out using the program analy seq (staden, 1984) . this program uses certain criteria to select one of the three reading frames as being the most likely protein coding frame. although better suited to analysing large orfs, it is interesting to note that a search based on looking for codon biases above those expected from the base composition selects 857; of the m, 7500 orf as the most likely coding frame. the 15"; of codons not selected are not in a single block, which might have suggested a sequencing error leading to an artificial frameshift. all of the nucleocapsid which has so far been sequenced was selected as the most likely coding frame, but none of the a4, 9500 orf. we have carried out in vitro translation of total and poly(a) + rna populations from ibv-infected chick kidney cell cultures using a rabbit reticulocyte lysate system. however, analysis of the products on lo-18% polyacrylamide gradient gels containing urea could not resolve any small polypeptides due to high background in the relevant low-m, range. a similar problem has been found by stem and sefton (1984; stern, d.f., personal communication) who have carried out in vitro translation studies of gelpurified and gradient-fractionated mrnas and have identified no major specific product from mrnas b or d. at present, therefore, it is not possible to say definitively whether either of these polypeptides is produced. in a search for small structural polypeptides, a [ 3h]leucine-labelled preparation of virus has been analysed on a 12.5% polyacrylamide tube gel (cavanagh, d., personal communication) , using the phosphate buffer system of swank and munkres (197 1) . this showed that there were three detectable polypeptides of apparent m,s: 16000, 12000, and 10000. the percentage of the total counts in the gel accounted for by these polypeptides was < 1%. thus, if either of these orfs codes for a structural polypeptide, it must only be present in very small quantities. it is also possible that they could code for non-structural polypeptides. these would be difficult to identify in ibv-infected cells by pulse-labelling techniques without using immunoprecipitation to lower the background of host-cell incorporation which is poorly shut off by ibv infection. the availability of sequence data for these putative polypeptides, however, opens up the possibility of using immunoprecipitation with antisera prepared against synthetic oligopeptides to search for the presence of these polypeptides in ibv-infected cells. sequence of the nucleocapsid gene from murine coronavirus mhv-a59 characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader sequences on nascent chains avian infectious bronchitis virus genomic rna contains sequence homologies at the intergenic boundaries structural polypeptides of coronavirus ibv a rapid boiling method for the preparation of bacterial plasmids comparison of initiation of protein synthesis in prokaryotes. eukaryotes and organelles mouse hepatitis virus a59: mrna structure and genetic localization of the sequence divergence from hepatotropic strain mhv-3 further characterisation of mrnas of mouse hepatitis virus: presence of common 5'-end nucleotides presence of leader sequences in the mrna of mouse hepatitis virus apple i1 computer software for dna and protein sequence data the virusspecific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm molecular cloning -a laboratory manual sequencing end-labelled dna with base-specific chemical cleavages the puc plasmids, an m13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers translation ofthree mouse hepatitis virus strain a59 subgenomic rnas in xerropus heris oocytes coronavirus jhm: coding assignments of subgenomic mrnas coronavirus jhm: cell-free synthesis of structural protein ~60 the biology of coronaviruses coronavirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein coronavirus mrna synthesis involves fusion of non-contiguous sequences graphic methods to determine the function ofnucleic acid sequences coronavirus multiplication strategy, 1. identification and characterisation of virus-speci coronavirus multiplication strategy, ii. mapping the avian infectious bronchitis virus intracellular rna species to the genome structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus coronavirus multiplication: locations of virion proteins on the avian infectious bronchitis virus genome molecular weight analysis of sulfate trans-complementable copy-number mutants of plasmid colei we are grateful to penny gatter, anne foulds and ian foulds for excellent technical assistance. this research was carried out under research contract no. gbi-2-01 l-uk of the biomolecular engineering programme of the commission of the european communities. key: cord-265499-pbf11zy1 authors: dove, brian k.; you, jae‐hwan; reed, mark l.; emmett, stevan r.; brooks, gavin; hiscox, julian a. title: changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus date: 2006-03-03 journal: cell microbiol doi: 10.1111/j.1462-5822.2006.00698.x sha: doc_id: 265499 cord_uid: pbf11zy1 the nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. while many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. in this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. we found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm. the nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, rna processing, cell cycle regulation, cell growth and as a sensor of cell stress (carmo-fonseca et al ., 2000; rubbi and milner, 2003; andersen et al ., 2005; lam et al ., 2005) . given that the nucleolus is crucial to the successful functioning of a cell it is no surprise that both animal and plant viruses target this structure, whether to recruit nucle-olar proteins to facilitate virus infection or usurp host cell function (hiscox, 2002; 2003; rowland and yoo, 2003; weidman et al ., 2003; kim et al ., 2004) . proteomic analysis revealed that the nucleolus is composed of over 400 proteins which can be grouped into discrete functional classes related to the activity of the nucleolus (andersen et al ., 2002; . predominant among these is nucleolin, which can account for approximately 10% of the protein content within the nucleolus. nucleolin is a multi-domain and multifunctional protein that has been shown to be involved in ribosome assembly, rrna maturation, nucleo-cytoplasmic transport and cell proliferation (ginisty et al ., 1998; srivastava and pollard, 1999) . nucleolin is highly phosphorylated with the potential to bind to multiple rna targets and this may reflect its variety of functions (ginisty et al ., 1999) . while mammalian nucleolin has a predicted molecular mass of approximately 77 kda (depending on the species), the apparent molecular mass is between 100 and 110 kda, and has been attributed to the amino acid composition of the n-terminal domain, which is highly phosphorylated. cell culture studies have shown that nucleolin is stable in proliferating cells, but undergoes self-cleavage in quiescent cells (chen et al ., 1991) and has been shown in primary cells to be regulated at both the level of transcription and translation (bicknell et al ., 2005) . morphologically the nucleolus can be divided into a fibrillar centre (fc), a dense fibrillar component (dfc) and an outer granular component (gc), each with defined functions (thiry and lafontaine, 2005) . as befits the nature of the nucleolus, many nucleolar proteins undergo complex multi-protein : protein and protein : rna interactions. for example, nucleolin associates with a variety of proteins including b23 (li et al ., 1996; liu and yung, 1999) , the tumour suppressor protein p53 (daniely et al ., 2002) , human la antigen (intine et al ., 2004) , telomerase (khurts et al ., 2004) and also rna (serin et al ., 1997; allain et al ., 2000; ginisty et al ., 2001) . any perturbations to a nucleolar protein, either in localization to the nucleolus or abundance, whether induced by infection, cellular disease or cell state (e.g. the cell cycle), is likely to have down stream consequences for host cell metabolism (hiscox, 2002; zimber et al ., 2004; andersen et al ., 2005) . we have been studying the interaction of viruses with the nucleolus using the avian coronavirus, infectious bronchitis virus (ibv). although the principal site of repli-cation of this virus is the cytoplasm (lai and cavanagh, 1997) , nevertheless one of the most abundant viral proteins produced during infection, nucleocapsid (n) protein, can localize to the cytoplasm and nucleolus in infected cells and cells expressing this protein from an expression vector wurm et al ., 2001) . n protein can also associate with nucleolin, a nucleolar protein (chen et al ., 2002) . while nucleolar localization of n protein has also been described for the closely related arteriviruses (rowland et al ., 1999; tijms et al ., 2002; rowland and yoo, 2003; yoo et al ., 2003) and may thus be a common property of nidoviruses, in the case of the severe acute respiratory syndrome (sars) coronavirus n protein, nucleolar localization has not been observed in infected cells using antibodies to detect n protein (rowland et al ., 2005; you et al ., 2005) . although it has been described in cells expressing n protein (li et al ., 2005) , albeit with low frequency (you et al ., 2005) . ibv n protein (beaudette strain) is a 409-amino-acid basic protein with a predicted molecular weight of 45 kda. analysis of the protein by non-reducing sds-page revealed a monomeric form with an apparent molecular weight of approximately 55 kda and a timeric form of approximately 165 kda, the latter form not being present under reducing conditions (chen et al ., 2003) . these data suggested several post-translational modifications including disulphide bridge formation and phosphorylation (chen et al ., 2003) . although coronavirus n proteins have the potential to be phosphorylated at multiple residues, mass spectroscopic analysis of ibv and also the porcine coronavirus, transmissible gastroenteritis virus (tgev) (calvo et al ., 2005) n proteins, indicated that relatively few sites were occupied. analysis demonstrated that ibv n protein was phosphorylated at four sites, ser190 and ser192 and thr378 and ser379 . the majority of species were either mono-or biphosphorylated and mapped to predicted protein kinase a (pka) in the case of ser190 and ribosomal s6 kinases (rsk) in the case of ser192 and casein kinase ii (ckii) phosphorylation sites for both thr378 and ser379. n protein has several functions during virus infection, with both roles in modulating virus replication and altering host cell processes. n protein is involved in the control of virus rna synthesis (almazan et al ., 2004; schelle et al ., 2005) and surface plasmon resonance studies demonstrated that phosphorylated n protein has a high affinity for viral rna . ibv n protein has been shown to induce aberrant cytokinesis and delay cell growth chen et al ., 2002) . sarscoronavirus n protein can activate the ap-1 transcription pathway (he et al ., 2003) , re-organise actin and induce apoptosis (surjit et al ., 2004) . the murine coronavirus n protein can stimulate transcription of prothrombinase fgl2 (ning et al ., 1999; 2003) . using ibv we tested the hypothesis that virus infection leads to perturbations in the morphology and proteins of the nucleolus which may have downstream consequences for host cell function. a number of viruses have been shown to either target nucleolar proteins for use in virus infection or viral proteins have been shown to localize to the nucleolus (hiscox, 2002; 2003; rowland and yoo, 2003; weidman et al ., 2003) . given that the nucleolus is a multi-protein complex, we hypothesized that viral interactions with the nucleolus would disrupt its morphology, which we defined as changes in the distribution of nucleolar proteins that alter the shape of the nucleolus as revealed by microscopy. nucleolar morphology was examined during infection with ibv using confocal microscopy with fluorescently tagged nucleolar proteins as well transmission phase contrast microscopy (you et al ., 2005) . two marker proteins were selected, b23 tagged c-terminal to dsred (dsred-b23) and nucleolin tagged c-terminal to egfp (egfp-nucleolin), and as described previously, using these techniques different regions of the nucleolus could be identified (you et al ., 2005) (fig. 1a) . under transmission phase contrast (and bright field analysis), the nucleolus is highly refractive due to the high protein content . the only area which is not highly refractive is the fc, which is enriched in nucleic acid; this appears as a less refractive area in the nucleolus (fig. 1a) . the fc and dfc are also apparent in cells expressing dsred-b23 and egfpnucleolin (you et al ., 2005) . there was no change in the distribution of either egfp ( to further investigate the change in nucleolar morphology, the subcellular localization of egfp-nucleolin was examined at 24 h pi. three principal nucleolar morphologies were observed in virus-infected cells. in approximately 64% of infected cells the nucleolus had the same apparent morphology to that of mock-infected cells. second, in approximately 23% of infected cells, there was a punctate distribution of egfp-nucleolin ( fig. 3a) , which could be more closely observed through z-sectioning through the plane of the nucleolus (fig. 3b ). third, in approximately 13% of infected cells, an increase in the apparent size of the fc (fig. 3c ) compared with mockinfected cells (fig. 3d ). the diameter of the fcs between infected and mock-infected cells were compared by fluorescent intensity (an example of this in infected cells is shown in fig. 3e ) and revealed that by examining 15 fields of view from three independent experiments, the average diameter of the fc in mock-infected cells was 1.3 ± 0.1 µ m. in contrast, the average diameter of the fc in infected cells exhibiting the larger phenotype was 2.6 ± 0.4 µ m. we investigated whether the nucleolar protein profile was altered in coronavirus-infected cells, specifically focusing on nucleolin. vero cells were infected with ibv and nucleolar extracts purified at 24 h pi. the purity of nucleolar extracts was confirmed by visual microscopy (data not shown) and western blot analysis of nucleolar material using marker proteins for the nucleolus (nucleolin) and the nucleus/nucleoplasm (lamin b) (fig. 4a ). the results indicated that nucleolin was enriched in the purified nucleolar material compared with prior purification stages that contained increased levels of nuclei and nucleoplasm as shown by the increased detection of lamin b in these samples. thus both bright field microscopic analysis (data not shown) and western blot indicated that the nucleolar preparations were enriched in nucleoli. the protein content from equivalent numbers of nucleoli (as determined by counting the numbers of nucleoli using bright field microscopy) was separated using denaturing sds-page on a 4-12% gradient gel (invitrogen) (fig. 4b ). comparison of the two proteomes indicated that novel protein species were present in nucleolar proteomes from virusinfected cells (indicated by the arrows labelled with the numeral 1), and that the abundance of some cellular nucleolar proteins was altered between virus-infected and mock-infected cells (indicated by the arrows labelled with the numeral 2). western blot analysis using either antibody to n protein or nucleolin confirmed that n protein was present in the nucleolar extracts from infected cells but not mock-infected cells and that there was a slight increase in the amount of nucleolin between infected or mock-infected cells (fig. 4b) . however, although proteins extracted from equivalent number of nucleoli were analysed, quantification of the relative amounts of protein in these structures between mock and infected cells is problematic as variations in nucleolar size and density are difficult to control. a change in the morphology of the nucleolus was observed in ibv-infected cells using egfpnucleolin as a marker protein (e.g. fig. 3b ), leading to the prediction that the levels of nucleolin might increase in virus-infected cells, which was observed in fig. 4b . the ability of n protein to localize to the nucleolus was confirmed using live cell imaging of cells expressing egfp fused to the n-terminus of ibv n protein (egfp-ibv n protein) ( fig. 4c ) (you et al ., 2005) . previous reports have indicated that nucleic acid binding proteins can localize to cell nuclei as an artefact of fixation conditions, a specific example being herpes simplex virus vp22 protein (lundberg and johansson, 2001; 2002) . however, both live cell imaging of egfp-ibv n protein in transfected cells and the presence of ibv n protein in nucleoli purified from infected cells would indicate that localization of ibv n protein to the nucleolus is not an artefact of fixation conditions. to test the hypothesis that interaction of a virus with the nucleolus can lead to the redistribution of nucleolar proteins we examined the localization of the tumour suppressor protein p53. the reasons for choosing this protein is that coronavirus infection can result in changes to the cell cycle and cell growth chen et al ., 2002; chen and makino, 2004) and that the coronavirus n protein (among others) can mediate these effects timani et al ., 2005) . in addition, disruption of the nucleolus has been reported to stabilize p53 as part of a response to cell stress, with the prediction that viruses may disrupt p53 to prevent this response from occurring (rubbi and milner, 2003; yuan et al ., 2005) . in mock-infected cells, p53 was located in the nucleolus/ nucleus and cytoplasm (fig. 5a) ; however, in virusinfected cells p53 was redistributed from the nucleolus/ nucleus to the cytoplasm and was punctate in distribution ( fig. 5b and c) . we confirmed that the nucleolus was still intact in these cells using transmission phase contrast (fig. 5c ). western blot analysis indicated that there was no change in the amount of p53 between mock and infected cells at 24 h pi (fig. 5d) . the data indicated that an ibv protein(s) might colocalize with p53 in the peri-nuclear region, as given by the yellow signal generated through the proximity of p53 labelled with texas-red and ibv labelled with fitc. the polyclonal antibody used to detect ibv was raised in rabbits against virus and therefore, could be predicted to detect (at least) the spike, membrane and envelope surface glycoproteins as well as the n protein. given that n protein can reduce cell growth chen et al ., 2002) we hypothesized that n protein might be responsible for sequestration of p53. to test this, we expressed egfp-ibv n protein in vero cells and examined for colocalization with p53; however, none was observed (fig. 6a) . given that n protein is probably part of the virus replication complex, then n protein may interact with p53 in the context of virus-infected cells. therefore, we examined the interaction of p53 with egfp-ibv n protein in infected cells. the data showed that both p53 (red) and ibv proteins (blue) colocalized (as shown by the purple signal, arrowed), and the egfp-ibv n protein was recognized (in part) by the anti-ibv polyclonal antibody (cyan signal) (fig. 6b ). if p53 and egfp-ibv n protein colocalized then we would expect a yellow signal; however, similar to egfp-ibv n protein expressed alone, there was no obvious colocalization of egfp-ibv n protein and p53 apart from an isolated area proximal to the peri-nuclear region (arrowed yellow) (fig. 6b) . however, colocalization between egfp-ibv n protein, p53 and ibv proteins detected using the polyclonal antibody (white signal) can also be observed in the cytoplasm (fig. 6c) . the redistribution of p53 may account for the observed reduction in cell growth observed in cells expressing n protein chen et al ., 2002) . although live cell imaging in conjunction with a fluorescent tagged protein can be used to determine whether that protein localizes to the nucleolus, the resolution is not sufficient to determine if the protein has a specific localization in the nucleolus. using egfp-ibv n protein we investigated whether ibv n protein localized to a specific region of the nucleolus. ideally such experiments would be conducted in infected cells. however, while we have polyclonal antibody that can be used to detect n protein, the accessibility of a protein to antibody in the nucleolus can be problematic (sheval et al ., 2005) , especially if the protein of interest is highly concentrated in the nucleolus. vero cells were transfected with pegfp-ibv-n and fixed 24 h post transfection. nucleoli were visualized by staining with propidium iodide (pi), which we have found stains the fc, dfc and gc. as described previously two distinct populations of cells were observed, either n protein localized in the cytoplasm or n protein localized in the cytoplasm and the nucleolus chen et al ., 2002; you et al ., 2005) . this study was undertaken with a higher resolution confocal microscope and we observed a third population of cells in which n protein was predominantly localized in the cytoplasm, but also was present in low levels in the nucleus and nucleolus (an example is shown in fig. 7a) . indeed, analysis of these cells indicated that not all of the nucleolar volume was occupied by n protein (fig. 7a , right hand images). to investigate this further, cells expressing egfp-ibv n protein were fixed and stained with pi to visualize the entire nucleolar volume (examples are shown in fig. 7b , top images). the data indicated that while egfp-ibv n protein and pi colocalized in many parts of the nucleolus, there were some areas of the nucleolus not occupied by 6 . a. confocal analysis to investigate the potential colocalization of p53 (red) in cells expressing egfp-ibv n protein (green). b and c. confocal analysis of the distribution of ibv proteins (blue), egfp-ibv n protein (green) and p53 (red) in ibv-infected cells at 24 h pi. colocalization between egfp-ibv n protein plus p53, egfp-ibv n protein plus ibv proteins, p53 plus ibv proteins, and between the three complexes, appears yellow, cyan, purple and white respectively. the nucleolus is indicated (no). size bar is 10 µ m. n protein (in red). further magnification of these cells at ×63 and enhanced ×4.5 revealed that indeed n protein occupied less area of the nucleolus than that stained by pi (fig. 7b, lower images) . this finding was confirmed by orthogonal reconstruction (fig. 7c ) of the region of the cell delineated in fig. 7b , lower image, indicated by the white vertical line. on the basis of this data we hypothesized that n protein localized to the dfc. in order to test this prediction we analysed the distribution of n protein in the nucleolus in more detail using a plasmid which expressed a fluorescent protein (ecfp) fused n-terminal to a mutant of n protein (pecfp-nr1+2) which localizes to the nucleolus and nucleus but not cytoplasm and the nucleolar marker protein dsred-b23. as stated above b23 provides a marker for the dfc and is absent from the fc and gc, and therefore provided the nucleus is visible, these domains can also be visualized. cells were cotransfected with pecfp-nr1+2 and pdsred-b23 and fixed at 24 h post transfection. the confocal microscope was used to take approximately 0.41 µm sections through the nucleus/nucleolus (fig. 7d , note ecfp false coloured green). as can be observed, dsred-b23 localizes predominately to the dfc. ecfp-nr1+2 localizes predominately to the nucleus and nucleolus and more specifically to the dfc. interestingly the apparent diameter of the fc in this nucleolus was approximately 2.0 µm, which reflects the diameter of the nucleolus with the large phenotype observed in infected cells. therefore, we postulate that nucleolar localization of n protein causes a disruption to the fc. in summary, infection of cells with ibv causes disruption in the nucleolar morphology and proteins and redistribution of p53 from the nucleolus and nucleus. we hypothesize that such changes would have downstream consequences for host cell metabolism and may contribute to the cellular pathogenesis of the virus. alterations in the nucleolus are not limited to virus infection and a recent study has indicated that the nucleolar proteome can change in response to conditions of cell growth (andersen et al., 2005) . therefore, there may be an intimate relationship between nucleolar perturbations induced by virus infection and the normal nucleolar functioning in the host cell. vero cells (an african green monkey kidney-derived epithelial cell line) were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs). ibv beaudette us, an ibv strain adapted for growth in vero cells (alonso-caplen et al., 1984) , was propagated in vero cells and the virus harvested at 24 h pi. virus titre was calculated by plaque assay titration in vero cells. all cell culture experiments in this study were conducted in the absence of antibiotic or antifungal agents. cells were infected with ibv at a moi of 1 when they were 60% confluent and incubated for 1 h at 37°c after which the initial infection media were replaced with cell growth media. pegfp-ibv-n, which, when transfected into cells, would lead to the synthesis of the fluorescent fusion protein egfp-ibv n, was constructed by cloning the ibv n gene from plasmid ptriexibvn (chen et al., 2002) by polymerase chain reaction (pcr) using forward and reverse primers containing the first and last 20 nucleotides of the ibv n gene sequence (beaudette us strain, accession number: aaa46214). primers incorporated a 5′ bamhi site and a 3′ ecori site. pcr products were gel purified by recochip (takara) as per the manufacturer's instructions, and subcloned into pcr2.1 topo vector (invitrogen). dna was purified by alkaline lysis and digested using the bamhi and ecori sites before being ligated into pegfpc2 (clontech) using t4 dna ligase (invitrogen), as per the manufacturer's instructions. pecfp-nr1+2 was constructed as described for pegfp-ibv-n, except the forward primer was ggccggtcctcgagc catggcaagcggtaaagcagctgg and the reverse primer g accggtcccgcggctaatctcttgtaccctgattggatc and the insert was digested with xhoi and sacii. the clones were verified by sequencing and expression of the fusion protein by western blot (data not shown). pegfp-nucleolin and pdsred-b23 were generous gifts of dr david matthews at the university of bristol and when transfected into cells led to the expression of the fluorescent fusion protein egfp-nucleolin and dsred-b23 respectively. transfection was carried using lipofectamine (invitrogen) according to the manufacturer's instructions. live cell imaging was performed using a nikon eclipse ts100 microscope utilizing the appropriate filter for each tag (e.g. filter b-2a, excitation 450-490 nm for egfp). fluorescence images were captured using a nikon digital sight ds-l1. confocal sections of fixed samples were captured on a lsm510 meta microscope (carl zeiss) equipped with a 40×, na 1.4, oil immersion lens. pinholes were set to allow optical sections of 1 µm to be acquired. egfp was excited with the 488 nm argon laser line running at 2% and emission was collected through a lp505 filter. pi and texas-red were excited with the helium:neon 543 nm laser line in all cases and emission was collected through a lp560 filter. far red was excited at 633 nm. z-sections of cells were generated by a two-step methodology. first, serial confocal sections of egfp or ecfp were acquired with the meta detector. texas-red or pi was then collected as described using the same z-settings. z-steps were collected 0.5 µm apart to allow over sampling of the data. the two sets of z-stacks were then pseudo-coloured and merged using the 'copy' facility within the lsm510 meta software. egfp-nucleolin and egfp-ibv n protein were visualized by direct fluorescence. ibv proteins were visualized using a rabbit anti-ibv polyclonal antibody to the major structural proteins as described previously chen et al., 2002) or a chicken anti-ibv polyclonal antibody (charles river) and were visualized using appropriate secondary antibodies conjugated with either fitc or alexa fluor 633 (molecular probes) (far red, false coloured blue in this study). the tumour suppressor protein p53 was visualized by labelling with a mouse anti-p53 antibody (d0-1) (santa cruz biotechnology) and with a texas-red conjugated anti-mouse secondary antibody (molecular probes). where appropriate the cell nucleus and nucleoli were stained with fluorescence grade pi (molecular probes), omitting the rnase treatment step. all fluorescence was measured in the linear range as the detector is a photomultiplier, and the range indicator was utilized to ensure no saturated pixels were obtained on image capture. images were scanned 16 times. no cross-talk between channels was determined by switching off the appropriate excitation laser and imaging the corresponding emission. for example, potential cross-talk between red and far red was assessed by switching off the 543 (red) laser and imaging in the cy5 channel emission. nucleoli were purified according to previously published methodologies (andersen et al., 2002) with modification for vero cells (emmett et al., 2004) , the crucial stage being sonication, where the efficiency of release of nucleoli was determined after each sonication step by live cell imaging. detection kit (amersham/pharmacia) according to the manufacturer's instructions. briefly, each sample was separated on a 10% nupage bis-tris precast polyacrylamide gel (invitrogen) in mops running buffer. protein was transferred onto a polyvinylidene difluoride membrane (invitrogen) in invitrogen transfer buffer for 1 h at 20°c. the membrane was blocked for 1 h in tbs plus 0.05% tween 20 and 5% milk. to detect ibv n protein, primary rabbit anti-ibv polyclonal sera, diluted 1:10 000 in tbs plus 0.05% tween 20, and incubated with the membrane for 2 h at 20°c. the membrane was washed three times in tbs plus 0.05% tween then the membrane was incubated with secondary goat antirabbit igg conjugated to horseradish peroxidase (diluted 1:1000 in tbs plus 0.05% tween) for 1 h at 20°c. to detect nucleolin the protocol was the same apart from the primary antibody was rabbit anti-nucleolin (santa cruz). to detect the tumour suppressor protein p53 we used a mouse anti-p53 antibody (d01, santa cruz) and to detect gapdh we used a mouse anti-gapdh antibody (6c5, abcam), with appropriate secondary antibody. proteins were quantified prior to separation using a bicinchoninic acid assay (pierce). molecular basis of sequence-specific recognition of pre-ribosomal rna by nucleolin the nucleoprotein is required for efficient coronavirus genome replication replication and morphogenesis of avian coronavirus in vero cells and their inhibition by monensin directed proteomic analysis of the human nucleolus nucleolar proteome dynamics nucleolin is regulated both at the level of transcription and translation phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells to be or not to be in the nucleolus murine coronavirus replication induces cell cycle arrest in g0/g1 phase increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus mass spectroscopic characterisation of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding using surface plasmon resonance stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation the cell cycle and virus infection nucleolin functions in the first step of ribosomal rna processing structure and functions of nucleolin two different combinations of rna-binding domains determine the rna binding specificity of nucleolin activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein brief review: the nucleolus -a gateway to viral infection? the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus nonphosphorylated human la antigen interacts with nucleolin at nucleolar sites involved in rrna biogenesis nucleolin interacts with telomerase involvement of the nucleolus in plant virus systemic infection the molecular biology of coronaviruses the nucleolus the dynamics of the nucleolus bioinformatic analysis of the nucleolus c23 interacts with b23, a putative nucleolar-localization-signalbinding protein sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in vivo interaction of nucleophosmin/b23 and protein c23 during cell cycle progression in hela cells is vp22 nuclear homing an artifact? positively charged dna-binding proteins cause apparent cell membrane translocation the nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene induction of prothrombinase fgl2 by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor-4 alpha nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences the localisation of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells disruption of the nucleolus mediates stabilization of p53 in response to dna damage and other stresses selective replication of coronavirus genomes that express nucleocapsid protein two rna-binding domains determine the rna-binding specificity of nucleolin a higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies molecular dissection of nucleolin's role in growth and cell proliferation: new insights the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors birth of a nucleolus: the evolution of nucleolar compartments nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus nuclear/nucleolar localization properties of cterminal nucleocapsid protein of sars coronavirus the interaction of cytoplasmic rna viruses with the nucleus localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rnaassociated protein fibrillarin sub-cellular localisation of the severe acute respiratory syndrome coronavirus nucleocapsid protein genetic inactivation of the transcription factor tif-ia leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis nuclear bodies and compartments: functional roles and cellular signalling in health and disease this work was funded by the award of bbsrc project grant (number bbs/b/03416) to jah and gb, and bbsrc committee studentship (bbssp200310434) to jah. the confocal microscope facility in the astbury centre for structural molecular biology was funded by the wellcome trust and srif and we would like to thank gareth howell for his help in using this facility. key: cord-268454-w4qca90s authors: rim, aouini; nacira, laamiri; jihene, nsiri; said, salhi; khaled, miled; ahmed, rejab; abdeljelil, ghram title: viral interference between h9n2-low pathogenic avian influenza virus and avian infectious bronchitis virus vaccine strain h120 in vivo date: 2019-06-17 journal: comp immunol microbiol infect dis doi: 10.1016/j.cimid.2019.06.004 sha: doc_id: 268454 cord_uid: w4qca90s the interaction between a low pathogenic avian influenza virus (a/ck/tun/145/2012), a h9n2 tunisian isolate, and a vaccine strain (h120) of avian infectious bronchitis, administered simultaneously or sequentially three days apart to chicks during 20 days, was evaluated using elisa antibody levels, quantitative reverse transcription–polymerase chain reaction (qrt-pcr) analyses and histopathology examination. first, the in vivo replication interference of avian influenza virus (aiv) and infectious bronchitis virus (ibv) was evaluated using qrt-pcr to detect accurately either aiv or ibv genomes or viral copy numbers during dual infections. second, we have determined the amount of specific antibodies in sera of chick’s infected with aiv alone, ibv alone, mixed aiv + ibv, ibv then aiv or aiv ibv 3 days later using an elisa test. finally, histopathological analyses of internal organs from inoculated chicks were realized. quantitative results of aiv and ibv co-infection showed that interferences between the two viruses yielded decreased viral growth. however, in the case of super-infection, the second virus, either aiv or ibv, induced a decrease in the growth of the first inoculated virus. according to our results, vaccine application was safe and do not interfere with aiv h9n2 infection, and does not enhance such infection. in conclusion, co-infection of chicks with aiv and ibv, simultaneously or sequentially, affected the clinical signs, the virus replication dynamics as well as the internal organ integrity. the results proposed that infection with heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated. avian influenza virus (aiv) and infectious bronchitis virus (ibv) are respiratory diseases of poultry, caused by type a orthomyxovirus and gammacoronavirus, respectively, with several traits in common. they are considered the most economically important respiratory viral diseases, and they threaten the poultry industry worldwide [1] . mixed infection or co-infection with aiv and ibv has been described as a natural infection in different countries in asia and the middle-east [2, 3] . more, the ibv vaccine is used extensively in chicken farms in many countries worldwide where both ibv and low pathogenic aiv h9n2 are endemic [3, 4] . the h120 strain of ibv was one of the earliest live attenuated ibv vaccines to be developed and has continued to be use in most parts of the world. as with any live-attenuated ibv vaccine, the h120 vaccine strains must replicate in the respiratory tract if they are to stimulate protective immunity. it follows, therefore, that some damage to the epithelial layer of the respiratory tract will occur and a fine balance must be achieved between a strain that is so over-attenuated that it is unable to replicate sufficiently to stimulate immunity and one that is insufficiently attenuated so that serious damage may occur. the h120 virus meets these criteria well and is ideally suited for use in young, susceptible chicks [5] . both viruses replicate in epithelial cells of the respiratory and the intestinal tracts, where trypsin-like enzymes allow virus entry. they likely compete to infect target cells and replicate. it has been shown that many viruses require the presence of well defined cell surface proteins for cell entry. the cell surface receptor for aiv or ibv on the cell surface is the n-acetyl neuraminic acid (α2,3-sialic acid-galactose) [6] . such cell receptor also serves as a receptor protein determinant for primary attachment of group 3 corona viruses [7] , allowing fusion events and entry of both viruses. huang et al. have described as it is unknown whether co-infections with aiv and ibv exacerbate clinical signs of the disease in infected birds or produce viral interference, masking infections by either virus [8] , even though it is reported that co-infection of ibv with aiv increased not only the severity of aiv-h9n2 clinical sings and gross lesions, but also the mortality rate with extended viral shedding period of aiv [9] [10] [11] . in tunisia, aiv type a-h9n2 and ibv (h120 vaccine strain) are frequently isolated from broiler poultry farms, affecting their productions. hatchery vaccination is actually opening the door to a real control of broiler diseases. indeed, infection of animals with aiv and ibv, following simultaneous, super-infection or vaccination, may lead to severe complications having bad effect on the animal health and the farmer expenses. mixed infection of poultry with aiv and ibv induced complex clinical picture making identification and diagnosis of either one or both viruses' difficult [12, 22] . still, questions remain regarding potential interactions between these viruses in co-infected bird [8] . so, it is worthy to better understand the in vivo interactions between these viruses for an efficacious disease control by adapting the vaccination programs (kelli et al., 2010) . the objectives of this study was to evaluate the effect of multi-infections of chicks with ibv vaccine strain and field aiv isolate (a/ chicken/tun/145/12 h9n2) by inoculating them simultaneously or sequentially and determining the induced immune response (elisa test), the virus pathogenicity (clinical signs and lesions, histopathology), their body distributions and titers by quantitative reverse transcription-polymerase chain reaction (qrt-pcr). and to study consequences of vaccination on aiv challenge. the aiv (a/ck/tun/145/12-(h9n2)) strain was isolated and characterized in 2012 [13] and the ibv-h120 (massachusetts) vaccine strain was provided from pasteur institute of tunisia. both viruses were grown in 10-day-old embryonated chicken eggs (ece) at 37°c for 72 h, as described by senne [14] . allantoic fluids were collected, titrated and then the 50% egg infective dose (eid 50 ) calculated, according to the reed-muench method (1938). one-day-old conventional broiler chicks were purchased from a local hatchery. the immune status of birds contains ibv and h9n2 maternally-derived antibodies. they were then housed in three selfcontained isolation units which are ventilated under negative pressure with inlet and exhaust hepa-filtered air and maintained under continuous lighting; feed and water were given adlibitum. all experiments were conducted in accordance with ethical guidelines of the tunisian council on animal care and approved the ethics committee of the institute pasteur of tunis under the reference 2017/05/i/lemvipt/v1. a total of 150 broiler chicks was divided into one control group and five virus infected groups containing each 25 birds. the birds were inoculated via the intraocular and intranasal routes; with 0.1 ml of 10 3 eid 50 of each virus. we used low inputs of pathogenicity strains in order to follow easily viral dominance than when we used higher virus inputs of high pathogenicity because it will damage all the experiment system. the viruses were administered either alone, mixed or consecutively, 3 days apart, as previous reported and showing that the maximum of virus replication in the organism is reached three days after infection [15, 16] . clinical signs were noticed and chick body weights along with blood sample and tracheal (tc) and cloacal (cl) swabs collections from five chicks of each group, at 1, 3, 7, 11 and 20 days post inoculation (dpi), were realized. thereafter, the chicks were ethically culled and internal organs placed in 10% neutral buffered formalin for microscopic lesion examinations. the virus contents in collected swabs and organs were also determined [17, 18] . tc and cl swabs were collected, placed in 2 ml mem containing final concentrations of gentamycin (200 μg/ml), penicillin g (2000units/ml), and amphotericin b (4 μg/ml) and maintained frozen at −80°c, before being thawed and centrifuged at 3000 rpm for 15 min. viral rna was then extracted using 200 μl of virus suspensions with trizol, according to the manufacturer's instructions (invitrogen, carlsbad, ca), and precipitated with absolute ethanol. the final pellets were suspended in 20 μl rnase-free water and stored at −20°c. standard precautions to avoid contamination were taken. the number of viral copies present in each sample was computed using a newly developed one-step multiplex qrt-pcr assay in our laboratory [19] . the reaction mixture contained 2 μl of rna samples, 0.6 μl of each primer at a concentration of 10 pmol/μl and 7.5 μl of enzyme with buffer agpath (applied biosystem, usa) in a final volume of 15 μl (table 1 ). the qrt-pcr assay was done in an escospectrum 96 real time thermal cyclers with the following cycling conditions: 45°c for 10 min, initial denaturation at 95°c for 10 min, followed by 40 pcr cycles of denaturation at 95°c for 10 s, annealing and extension at 60°c for 45 s with a single fluorescence acquisition step at the end of the annealing step. for quantitative purposes, positive ct values (ct ≤ optimal cutoff point) for each analyzed sample were determined from the corresponding standard curves derived from aiv and ibv standard table 1 number of chicks positive for ibv and aiv in tracheal (tc) and cloacal (cl) swabs during single and dual infections tested by qrt-pcr. plasmid templates. more details about the cloning, extraction of recombinant plasmid and the construction of standard curves are fully provided by laamiri et al. [19] . elisa antibody tests were realized to measure specific antibody titers in the sera collected from five chicks of each experimental group at 1, 3, 7, 11 and 20dpi using id screen elisa kit, for aiv and ibv (id vet-france). antibody titers were calculated according to the kit's instructions. various internal organs (trachea, lung, liver and intestine) were collected from 5 sacrificed chicks of each group, at 1, 3, 7, 11, and 20dpi, and conserved in 10% formaldehyde solution. they were cut into small pieces and prepared for histopathological analyses using classical steps of molding in paraffin, embedding and sectioning the organs into very thin sections using a microtome. the slices were layered on a glass slide for staining with a mixture of hematoxylin and eosin (h&e), and then analyzed under a light microscope with 100x objective. data were analyzed using prism v.5.01 software (graphpad software inc. la jolla, ca, usa) and values are expressed as the mean ± standard error of the mean (sem). one-way anova with tukey post-test was used to analyze body weights. the number of birds shedding of viruses was tested for statistical significance using fishers exact test. two-way anova with bonferroni multiple comparison analysis was used to evaluate virus titers in swabs and organs. for statistical purposes, all qrt-pcr negative tc and cl swabs were given a numeric value less than 10 copies/ml. statistical significance was set at p < 0.05 unless otherwise stated, using student's test. none of the chicks infected with either aiv or ibv alone or both viruses, showed any clear clinical signs. besides, significant changes in chick body weights of virus-infected chicks were noted only at 20dpi for all infected groups except co infected ones, in comparison with the control non-infected animal group (fig. 1 ). chicks inoculated with aiv showed pneumonia, enlarged spleen, serous effusion and congested kidney and spleen. these lesions appeared 7dpi in all infected chicks. similarly, the most noticed lesions were observed at 11dpi in chicks infected with ibv, showing congested trachea and kidneys, pneumonia and enlarged spleen. simultaneously aiv and ibv infected chicks showed less pronounced lesions as compared to super-infected groups, which animals showed pneumonia, congested spleen, liver discoloration and enlarged kidneys. using qrt-pcr, the viral interference levels in various inoculated groups, as compared to the control group, was quantitatively evaluated. tc and cl viral shedding was examined and the results are shown in table 1 and figs. 2-5, representing the average values of three independent experiments with standard error bars. chicks inoculated with only ibv or aiv excreted the virus from day 3 to 20dpi, as determined by virus detection in tc and cl swabs (table 1 ). in co-infected groups, significant ibv virus excretion was noticed in both tc and cl swabs taken at 11 and 20dpi (p < 0.01). super-infected groups showed that ibv virus is detected in tc and cl swabs from 3 to 20dpi in chicks infected with aiv followed by ibv inoculated 3 days later; no aiv shedding being noticed. chicks infected with ibv then super-infected with aiv-3 days later, showed no ibv shedding either in tc or cl, during the experimental period (figs. [2] [3] [4] [5] . when comparing the kinetics of aiv or ibv virus replication at different time points after chick inoculations, the patterns of tc and cl viral titers were different depending on the virus exposure, as shown for aiv or ibv viral gene copy numbers detected by qrt-pcr (figs. [2] [3] [4] [5] . the results of single aiv infected groups showed classical viral curve with higher virus excretion, in either tc or cl swabs, at 7dpi. however, co-infected birds (aiv + ibv mixture) presented significant lower amounts of viral gene copy numbers at 7dpi, as compared to single aiv infected birds (p < 0.05) (figs. 2 and 3 ). when aiv was first inoculated and followed by ibv administration, on the contrary, when aiv was secondly inoculated 3 days after ibv infection, a clear growth of aiv was shown during the experimental period and no inhibition was noticed by the first administrated ibv (figs. 2 and 3 ). similar results were observed during single ibv infected as well as co-infected (aiv + ibv mixture) birds as demonstrated for aiv infected groups, with significant lower amounts of viral gene copy numbers at 7dpi in either tc or cl swabs (p < 0.05) (figs. 4 and 5) . significant differences of aiv and ibv shedding, using the student's t-test, was noticed in tc and cl swabs taken at 3, 7, 11 and 20dpi, from co-infected chicks (p < 0.05), in comparison with single infected groups (table 1) . the elisa test was utilized to follow antibody levels against aiv and ibv in sera from non-infected as well as infected groups. no significant aiv and ibv antibodies were observed during the experimental period, in sera from all infected groups at 1-3dpi, as compared to the control non-infected group. however, a relatively low antibody levels against aiv or ibv were observed at 7dpi in single infected groups. during mixed infection, no significant levels of specific antibodies were detected against either aiv or ibv. in super-infected birds, low levels of specific antibodies against either aiv or ibv, were detected only in birds when the super-infecting virus is either aiv or ibv (results not shown). microscopic lesions of the respiratory tract of single and dual infected groups (fig. 6, fig. 7) showed congestion of pulmonary blood vessels and perivascular hemorrhage with pale bluish mucus and heterophile accumulation in lumen of secondary bronchioles (fig. 6) . histopathological findings in the trachea demonstrated marked lesions, characterized by diffuse infiltration of sub epithelial and deep layer of mucosa with lymphocytes and histiocytes, resulting in an increase in the mucosathickness. there was also congestion of the blood vessels of the mucosa with marked lymphoid tissue hyperplasia of the lamina propria (fig. 8) . collected control organs showed no changes. the jejunum of the intestine showed desquamation of intestinal villi (fig. 7) and the liver showed congestion of portal veins with peri-portal aggregation of leukocytes, mainly lymphocytes and macrophages (fig. 7) . the aims of the study were to evaluate the biological interactions between aiv and ibv-h120 that might happen in co-infected chicks. and to study consequences of vaccination on aiv challenge. infections with more than one virus, such as aiv and ibv, seem to frequently occur in poultry production; but, the effects of such co-infections or vaccination on several host responses such as viral shedding dynamic, antibody seroconversion and clinical signs are not fully known in chicks [15, 20] . besides, reported studies did not specify the various effects of dual viral infection on immune response, virus titers and genes copy number variations and histopathological changes during replication of both viruses. for this, we have deeply evaluated more than one aspects of viral interference between aiv and ibv, as compared to other reported studies [9, 20] , in terms of body weight, humoral response, macroscopic and microscopic lesions and viral replication using qrt-pcr. the results reported by seifi et al. [20] have suggested that ibv infection increases the pathogenicity of h9n2-aiv in broiler chickens, based on serological analysis and clinical signs noticed during co-infection using high viral titers (10 6 eid 50 ) of h9n2 and ibv. however, our study showed that the majority of chicks simultaneously infected with aiv (a/ck/tun/145/12)-h9n2 and ibv-h120, did not show clear clinical signs, indicating low viral growth. it is worth noting that the virus titers used in our study were relatively lower (10 3 eid 50 ), in regards to the exacerbated clinical signs reported [20] . it was interesting to note that the shedding patterns of both h9n2 and ibv were strongly influenced by each other. in fact, during co-infection, all chicks became infected with both viruses, as shown by virus serological and molecular results, but a significant reduction in virus replication was observed during the first 3 days, as compared to singly infected controls. a significant lower amounts of viral gene copy numbers were then noted at 7dpi up to 20dpi, in either tc or cl swabs (p < 0.05), collected from simultaneously (figs. [2] [3] [4] [5] . it can be inferred that the transitory inhibition of viral replication may have an effect on the severity of the disease; the induced clinical signs were not perceptible and only microscopic lesions were noticed. these findings are in concordance with other reported results [18, 21, 16] . besides, lower specific antibody titers were produced since both viruses inhibited the growth of each other's, early after infection. our results clearly showed that viral titers in tc swabs were distinctly higher than in cl sample. they are consistent with previous study showing that aiv viral shedding happens mainly by the tc route (costa-hurtado, 2014). in addition, virus shedding was lower in all groups simultaneously or sequentially treated, as compared to single infected animals. previous in vitro or in ovo experiments have shown similar results, in which simultaneous infections with a mixture of aiv and ibv induced inhibition of the replication of both viruses during the first 3 days of infection. in fact, using cell cultures or chicken embryos, co-infections with aiv and (newcastle disease virus) ndv mixture have shown interference between both viruses, suggesting inhibition of the growth of one virus by the other [22] [23] [24] . such early inhibition may be related to competition for virus receptors on the cell surface, leading to an attachment interference phenomenon. interference could also be due to intracellular interference related to competition for replication sites or essential factors for viral replication, virus-induced interferon interference or arn or defective interfering particles [25] . both inoculated viruses may also compete for sites or essential factors of virus replication, as they are both rna viruses. it has been clearly established that the cell surface receptors for aiv are sialic acid containing glycol-conjugates [23] , whereas the cellular receptors for ibv have been proposed as α2,3-sialylated glycans [26] , both of which contain sialic acid [23] . these findings imply the existence of common receptor sites on the permissive host cells that are shared by aiv and ibv [27] . historically, the interest for viral interference phenomena has led to the discovery of interferon in the early 1940s [28, 29] ; the interferon being secreted by cells following viral infection leading to inhibition of virus replication. our results did not show an important secretion of interferon in cell cultures simultaneously infected with aiv-ibv mixture (data not shown), even though studies have reported secretion of cytokines in chickens infected with h9n2 [30] . it has also been reported that h120 and h9n2 viruses are weak interferon inducers, and the first inoculated virus could not interfere well with the second one by producing sufficient amount of interferon ( [15, 31] ; 2015 [11] ;). thus, during in vivo super-infection, our results showed an important increase of the replication of the second inoculated virus that has the ability to out-compete and dominate the first virus replication, attaining almost its initial titer obtained during single infection. this result was in concordance with some studies that have been reported (shengqiang et al., 2012), including the results in this study demonstrating the higher growth of the super-infecting virus. however, there are some reported studies that demonstrated contradictory findings in vitro, as it has been reported for the occurrence of aiv-ndv, hepatitis b virus-hepatitis c virus and aiv-ibv interferences [32] [33] [34] , showing that the pre-inoculated virus always induces a lower growth of the super-infecting virus [35] . previous studies on in vitro and in ovo interference between aiv and ibv-h120 using, respectively, cultured chicken embryo lung cells and embryonated spf eggs, have shown that the second inoculated virus slows down the process of viral replication of the first administered one [36] . at the same age, groups simultaneously infected had numerically lower antibody titers as compared to groups singly or sequentially infected. this was due to lower virus replication related to mutual inhibition during the first 3dpi and low virus titers challenging the immune system. similarly, increased histopathological lesions in the trachea of singly or sequentially infected groups were observed. viral infection has been reported to induce loss of tc cilia, hyperplasia, hyperemia, and [37, 38] . histopathological changes induced in the trachea, lungs, liver and intestine of pcr-positive chicks, during viral interference between aiv and ibv, were in accord with those previously reported [39] [40] [41] . in general, the effects of viral interference depend on their titers and their pathogenicity, the time of co-infection, the immune responses and the environmental factors leading to the adaptation of viruses to their host species. identification and assessment of factors affecting viral interference and understanding those that cause delay in virus replication will help finding the path of pathogenicity and transmission of these viruses in chicks. our results have shown an increase in virus replication in cases of super-infection, which may affect the severity of the disease. determination of factors that may influence interference during co-infection or favor a delay of one virus growth at the expense of another virus will give new insights on the pathogenesis of these viruses, allowing a better design of diagnostic tools and improved vaccine and vaccination program for better controlling the diseases. in summary, our study showed that simultaneous dual infection of one day old chick's leads to reduction of both virus growths. however, during super-infection, the second inoculated virus has a negative impact on the growth of the first virus inoculated and that the degree of interference depending upon the interval between interfering viruses. the results suggested that interference might interfere during the virus entry into the cells through a competition for shared virus sialic acid receptor that are essential for virus attachment. interference may also happen when both viruses compete for sites or essential factors of viral replication (shengqiang et al., 2012) . these findings may have a strong influence on prevention and control strategies for the spread of economically important aiv and ibv diseases in the poultry industry. according to our results, vaccine application was safe and do not interfere with aiv h9n2 infection, and does not enhance such infection. we received the approval of the ethics committee on this work. the long view: 40 years of infectious bronchitis research molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in jordan prevalence of avian respiratory viruses in broiler flocks in egypt new real time and conventional rt-pcrs for updated molecular diagnosis of infectious bronchitis virus infection (ibv) in 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infected with infectious bronchitis virus, immunol., health, and dis the authors thank the members of the local hatchery for giving the chicks, pasteur institute of tunis for realizing the experimental protocol and the national school of veterinary medicine of sidi-thabet tunisia. this work was supported by the institute pasteur of tunis and the ministry of higher education and scientific research (lr11 ip03). ra analyzed and interpreted the qrt-pcr and elisa data regarding h9n2 and h120 viruses and was a major contributor in writing the manuscript. nl performed the qrt-pcr examination, did statistical analyses of the work and contributed in writing the manuscript. ss did the experimental protocol of the chicks. ra did histopathological analysis. jn did organs examination. all authors read and approved the final manuscript. ag corrected the draft paper. key: cord-295312-68b3zio6 authors: britton, paul; casais, rosa; hodgson, teri; davis, marc; cavanagh, dave title: genes 3 and 5 of infectious bronchitis virus are accessory protein genes date: 2006 journal: the nidoviruses doi: 10.1007/978-0-387-33012-9_64 sha: doc_id: 295312 cord_uid: 68b3zio6 nan is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney, and oviduct. [1] [2] [3] interspersed amongst the ibv structural protein genes are two genes, 3 and 5 ( fig. 1 ), 4 whose role is unknown. [5] [6] [7] gene 3 is functionally tricistronic, 8 expressing three proteins, 3a, 3b, and 3c, the latter being the structural e protein of ibv. 9 expression studies have indicated that translation of the e protein is initiated as a result of ribosomes binding to a structure formed by the preceding 3a and 3b sequences. 10, 11 gene 5 is functionally bicistronic and expresses two proteins, 5a and 5b, which are expressed in ibv-infected cells. 12 to investigate the requirement for the 3a, 3b, 5a, and 5b proteins for replication, we have used our reverse genetic system [13] [14] [15] [16] [17] to produce isogenic recombinant ibvs (ribvs), after site-specific mutagenesis of the appropriate sequences, with specific modifications in genes 3 and 5. overlapping pcr mutagenesis was used to scramble the initiation codons of 3a, 3b, 5b, delete the 3ab coding sequences and introduce a kpni restriction endonuclease upstream of the gene 5 tas. the scrambled gene 5 tass and scrambled 5a initiation codon were introduced using adapters to replace the 45 bp kpni-spei fragment. the modified sequences comprising two scrambled initiation codons scaug3ab and scaug5ab were generated from sequences containing a singly scrambled atg. the modified cdnas are shown in fig. 2 . our ibv reverse genetics system is based on the use of vaccinia virus (vv) as a vector for the ibv full-length cdna. 14 recombinant vvs (rvv) containing the gene 3 and 5 modified cdna sequences were generated by transient dominant selection (tds) 18 using the eco gpt (gpt) gene as the transient selectable marker. 13, 16 the modified ibv cdnas were inserted into the beaudette sequences in vnoti/ibv fl as a result of homologous recombination and selection of rvvs expressing gpt in the presence of mycophenolic acid (mpa). mpa-sensitive vaccinia viruses, potentially containing the modified ibv cdnas, were then generated from the mpa-resistant vaccinia viruses after the spontaneous loss of the gpt gene by three rounds of plaque purification in the absence of mpa. 13, 16 two rvvs, representing each modification, identified by pcr amplification and sequence analysis, were isolated after two independent tdss. recombinant ibvs, containing each of the modified gene 3 and 5 sequences, were recovered from dna isolated from the rvvs as shown in fig. 3 and described in refs. 13-16. recombinant ibvs were characterized and used for subsequent experiments after three passages in ck cells. two independent clones of each ribv were rescued from each of the two rvv dnas, except for ribvs scaug3b and scaug3ab, for which only one ribv was recovered. the growth kinetics of the ribvs were analyzed on chick kidney (ck) cells, and the amounts of progeny virus produced, at specific time points, were determined by plaque titration in ck cells and compared with those produced from beau-r. ibv and the coronaviruses isolated from other avian species, turkey, 19 pheasant, 20 peafowl (accession no. ay641576) and partridge (accession no. ay646283), all contain a tricistronic gene 3 and a bicistronic gene 5, the latter located between the m and n genes. the conservation of the gene 3 and 5 sequences in ibv and ibv-like viruses, isolated from other avian species, indicate they may play a role in the virus replication cycle. in order to determine whether the 3a, 3b, 5a, and 5b proteins are required for the replication of ibv, we have used a number of alternative ways to oblate the expression of these gene products. we modified the ibv genome corresponding to gene 3 by scrambling the 3a and 3b initiation codons, either singly or together, and by deleting the sequence corresponding to 3a3b. we have shown that 3a is no longer produced after scrambling of the aug or deletion of the sequence (unpublished data, hodgson et al.) . we modified gene 5 by scrambling the 5a and 5b initiation codons, either singly or together, and by scrambling the sg mrna 5 tas preventing expression of the sg mrna. we have shown that sg mrna 5 is no longer produced after scrambling of the tas and that 5b is no longer produced after scrambling of the aug. 16 comparison of the growth kinetics of the ribv with beau-r, on ck cells, showed that there were no differences (fig. 4) , demonstrating that neither the ibv 3a, 3b, 5a, nor 5b proteins are essential for replication per se; they can be considered to be accessory proteins. we have rescued a ribv that lacks expression of 3a and 3b, after deletion of their sequences, and lacks expression of 5a and 5b after scrambling of the gene 5 tas indicating that both sets of gene products are dispensable in vitro. protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus a nomenclature for avian coronavirus isolates and the question of species status diseases of poultry completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus the coronaviridae the molecular biology of coronaviruses the coronaviridae a polycistronic mrna specified by the coronavirus infectious bronchitis virus identification of a new membrane associated polypeptide specified by the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus distinct structural elements and internal entry of ribosomes in mrna3 encoded by infectious bronchitis virus identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection reverse genetics system for the avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity transient dominant selection of recombinant vaccinia viruses detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys key: cord-294679-7pklrmz5 authors: wei, lei; chen, cheng; zhao, qi; li, chun; cong, le; xu, xiaoling; ma, yanlin; liao, ming; xu, yuanyuan; rao, zihe title: purification, crystallization and preliminary crystallographic analysis of avian infectious bronchitis virus nsp3 adrp domain date: 2008-08-09 journal: acta crystallographica section f structural biology and crystallization communications doi: 10.1107/s1744309108024391 sha: doc_id: 294679 cord_uid: 7pklrmz5 avian infectious bronchitis virus (ibv) encodes 15 nonstructural proteins (nsps) which play crucial roles in rna transcription and genome replication. one of them, nsp3, contains an adrp (adenosine diphosphate-ribose-1′-phosphatase) domain which was revealed in recent studies to have adp-ribose-1′-monophos­phatase (appr-1′-pase) activity. appr-1′-pase catalyzes the conversion of adp-ribose-1′-monophosphate (appr-1′-p) to adp-ribose in the trna-splicing pathway. the gene segment encoding the ibv nsp3 adrp domain has been cloned and expressed in escherichia coli. the protein has been crystallized and the crystals diffracted to 1.8 å resolution. they belonged to space group p1, with unit-cell parameters a = 41.1, b = 43.2, c = 48.9 å, α = 78.0, β = 80.0, γ = 73.6°. each asymmetric unit contains two molecules. infectious bronchitis (ib) is a contagious disease that infects avian species and causes tremendous losses in the poultry industry. its aetiological agent, avian infectious bronchitis virus (ibv), replicates not only in the epithelium of upper and lower respiratory tract tissues, but also in many tissues along the alimentary tract and elsewhere, e.g. kidney, oviduct and testes (cavanagh, 2007) . infectious bronchitis virus is a member of the genus coronavirus which can be classified into three distinct groups (spaan & cavanagh, 2004) . ibv belongs to group iii (lai & holmes, 2001) . sars-cov is also a member of the coronaviruses. in 2003, an outbreak of severe acute respiratory syndrome (sars) struck china and quickly spread to the rest of the world, causing more than 8000 fatalities. immediately, scientists initiated intensive studies on coronaviruses. considering that the genomes encoding the nonstructural proteins (nsps) are conserved among sars-cov, ibv, human-cov 229e etc., the study of ibv will facilitate the thorough understanding of coronaviruses. the ibv genome is a single-stranded positive-sense rna of 27 600 nucleotides (cavanagh, 2007) . its genome expression starts with the translation of two replicase orfs (1a and 1b). the transcriptional replicase polyproteins are proteolytically processed by virus-encoded proteinases to produce 15 nsps. these nsps are replicative enzymes required in rna transcription and genome replication (snijder et al., 2003; prentice et al., 2004) . nsp3 contains an adrp (adenosine diphosphate-ribose-1 0 -phosphatase) domain, which is also known as the macro domain. recent studies have revealed that the macro domain is an adp-ribose-binding module and it is believed to have adp-ribose-1 0 -monophosphatase (appr-1 0 -pase) activity. appr-1 0pase is involved in the trna-splicing pathway and catalyzes the conversion of adp-ribose-1 0 -monophosphate (appr-1 0 -p) to adpribose, which can be recycled in nucleotide metabolism (kumaran et al., 2004; karras et al., 2005) . the cdna encoding the ibv nsp3 adrp domain (m41 strain) was provided by professor ming liao (south china agricultural university, people's republic of china). the gene encoding the ibv nsp3 # 2008 international union of crystallography all rights reserved adrp domain (corresponding to asp1024-lys1178 of the orf1a polyprotein) was inserted between the bamhi and xhoi sites of the pgex-6p-1 plasmid. the resulting plasmid was transformed into escherichia coli bl21 (de3). the cells were cultured in lb medium containing 0.1 mg ml à1 ampicillin at 310 k. when the optical density at 600 nm (od 600 ) reached 0.6, 0.3 mm isopropyl -d-1-thiogalactopyranoside (iptg) was added and the cultures were induced at 289 k for 16 h. the cells were harvested by centrifugation and resuspended in pbs (140 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo 4 , 1.8 mm kh 2 po 4 ph 7.3) supplemented with 1 mm dtt and 0.1 mm pmsf. the cells were sonicated at 277 k and the lysate was centrifuged at 27 000g for 40 min at 277 k and the precipitate was discarded. the supernatant was loaded onto 2 ml gst-glutathione affinity columns (pharmacia). the fusion protein was then cleaved on the column (oncolumn cleavage seems to be as effective as the orthodox approach) with gst-rhinovirus 3c protease at 277 k for 18 h, resulting in five additional residues (gplgs) at the n-terminus. the cleavage buffer was pbs and the ratio of protease to substrate protein was 1:50. sizeexclusion chromatography was performed using superdex 75 (amersham bioscience, usa) in 20 mm mes, 150 mm nacl ph 6.0. the purified protein was concentrated to 25 mg ml à1 . the buffer used contained 20 mm mes, 150 mm nacl ph 6.0. hampton research crystal screen kits were used to screen crystallization conditions. crystallization trials were performed using the hanging-drop vapourdiffusion technique at 291 k. 1.0 ml protein solution was mixed with 1.0 ml reservoir solution and allowed to reach equilibrium over 400 ml reservoir solution. initial crystals were obtained from index screen condition no. 84 containing 0.2 m magnesium chloride hexahydrate, 0.1 m hepes ph 7.5, 25%(w/v) polyethylene glycol 3350. after optimization better crystals were obtained (fig. 1) . the optimized condition contained 0.12 m magnesium chloride hexahydrate, 0.1 m hepes ph 7.5, 22%(w/v) polyethylene glycol 3350. the crystal was cryoprotected in a solution containing 0.1 m magnesium chloride hexahydrate, 0.1 m hepes ph 7.5, 20%(w/v) polyethylene glycol 6000. it was mounted on a nylon loop and flashcooled in a nitrogen stream at 100 k using an oxford cryosystems cryostream. the diffraction data were collected in-house on a rigaku cu k rotating-anode x-ray generator (mm007) operated at 40 kv and 20 ma (1.5418 å ) with a rigaku r-axis iv ++ imageplate detector (fig. 2) . data were processed, integrated, scaled and merged using the hkl-2000 programs denzo and scalepack (otwinowski & minor, 1997) . the crystals diffracted to 1.8 å resolution. they belonged to space group p1, with unit-cell parameters a a typical diffraction pattern of an ibv nsp3 adrp domain crystal collected on a rigaku r-axis iv ++ image-plate detector. are shown in table 1 . semet-derivative crystals also have been obtained and diffracted to 1.8 å resolution. the structural and functional analysis of ibv nsp3 adrp domain will be published elsewhere. fields virology virus taxonomy. viiith report of the ictv avian infectious bronchitis virus nsp3 key: cord-262226-7kwkla73 authors: fang, shouguo; xu, linghui; huang, mei; qisheng li, frank; liu, d.x. title: identification of two atr-dependent phosphorylation sites on coronavirus nucleocapsid protein with nonessential functions in viral replication and infectivity in cultured cells date: 2013-07-09 journal: virology doi: 10.1016/j.virol.2013.06.014 sha: doc_id: 262226 cord_uid: 7kwkla73 coronavirus encodes an extensively phosphorylated and highly basic nucleocapsid (n) protein. previous studies have identified ser190, ser192, thr378 and ser379 as the phosphorylation sites for coronavirus infectious bronchitis virus (ibv) n protein. in this study, we show that phosphorylation at thr378 and ser379 sites is dependent on the ataxia-telangiectasia mutated (atm) and rad3-related (atr), a kinase activated during ibv replication. introduction of ala substitutions at these two sites individually, in combination of the two and together with other two sites (ser190 and ser192) into an infectious ibv clone did not affect recovery of the recombinant viruses containing the mutations. a mutant virus (ribv-nm4) carrying the four ala substitutions grew at a similar, if not better, growth rate as wild type virus. this study reveals a cellular kinase responsible for phosphorylation of a coronavirus n protein at two positions and the functional consequence of this modification on coronavirus replication. coronavirus is an enveloped virus with a single strand, positive-sense rna genome of 27-30 kb length. in cells infected with coronavirus, a 3′-coterminal nested set of 6-9 mrnas species, including the genome-length mrna (mrna1) and 5-8 subgenomic mrna species (mrna2-9), is expressed. the genomelength mrna1 encodes two overlapping replicase proteins in the form of polyproteins 1a and 1ab, which are processed by virusencoded proteinases into at least 15 putative nonstructural proteins (nsp1-nsp16) (fang et al., 2008 . the four structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), are encoded by subgenomic mrnas. in addition, several putative nonstructural proteins, such as 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, are also encoded by subgenomic mrnas thiel et al., 2003) . coronavirus n protein contains multiple functional domains. sequence comparisons and structural studies have identified three main structural domains, although their primary sequence conservation is low (lai and cavanagh, 1997; li et al., 2005) . of this the middle domain is an rna-binding domain, capable of binding both coronavirus-and non-coronavirus-derived rna sequences in vitro tan et al., 2012; zhou et al., 1996) . the motifs for ribosome binding and nucleolar localization signals have been assigned to domain iii . more recently, chang et al. (2009) proposed that all coronavirus n proteins may share a similar modular organization. in cells expressing the n protein, it localizes either to the cytoplasm alone or to the cytoplasm and nucleolus . this nucleolar localization has been shown to be a common feature of the coronavirus family . the prime function of the protein is to associate with the genomic rna to form a ribonucleoprotein complex (rnp) and viral core (davies et al., 1981; escors et al., 2001; narayana et al., 2000; riso et al., 1996) . the protein may also play an important role in the replication of the genomic rna (chang and brian, 1996) , and in the transcription and translation of subgenomic rnas (sgrna) (almazán et al., 2004; baric et al., 1988; stohlman et al., 1988; tahara et al., 1994; zúñiga et al., 2010) . in addition, n protein might inhibit host cell proliferation or delay cell growth, possibly by disrupting cytokinesis (chen et al., 2002; wurm et al., 2001) . it can also stimulate strong humoral and cellular immune response, making it a potential vaccine candidate (kim et al., 2004) . coronaviruse n protein is an extensively phosphorylated and highly basic protein. it varies from 377 to 455 amino acids in length and has high serine content (7-11%) as potential targets for phosphorylation. this protein contains several basic amino acid-rich regions functioning as nucleolar localization signals and rna-binding motifs, and a serine/arginine (s/r) rich motif (li et al., 2005 , fan et al., 2005 tan et al., 2006) . biochemical characterization and mutagenesis studies demonstrated that coronavirus n protein was also posttranslationally modified by covalent attachment to the small ubiquitin-like modifier (sumo). sumoylation may play a role in the self-association and homo-oligomerization of the n protein, and its interference of host cell division (li et al., 2005) . infectious bronchitis virus (ibv), a prototype coronavirus, is the etiological agent of infectious bronchitis, which impairs the respiratory and urogenital tracts of chickens (cavanagh, 2007) . similar to other coronaviruses, ibv n protein is heavily phosphorylated. by using mass spectroscopic analysis, two conserved amino acid clusters, ser190ser192 and thr378ser379, in ibv n protein were identified as the two regions for phosphorylation (chen et al., 2005) . subsequently, a total of six residues (ser162, ser170, thr177, ser389, ser424 and thr428) were identified as phosphorylation sites for mouse hepatitis virus (mhv) a59 n protein (white et al., 2007) , and four phosphoserines at positions 9, 156, 254 and 256 were identified in transmissible gastroenteritis virus (tgev) n protein (calvo et al., 2005) . in addition, the host cell kinase (s) responsible for phosphorylation of coronavirus n protein is beginning to emerge. one example is glycogen synthase kinase-3, which was shown to be involved in regulation of the phosphorylation of severe acute respiratory syndrome coronavirus (sars-cov) n protein . current evidence suggests that phosphorylation may play important roles in regulation of the functions of coronavirus n protein. these included regulation of its subcellular location, viral rna transcription, particle assembly, immunoreactivity and specificity (calvo et al., 2005; jayaram et al., 2005; shin et al., 2007; surjit et al., 2005; spencer et al., 2008) . however, the functional relevance of n protein phosphorylation has not been rigorously tested in the context of virusinfected cells using an infectious clone system, and host cell kinases responsible for phosphorylation of other coronavirus n proteins are not reported. ibv infection perturbs cell cycle progression and arrests cell at the s and g2/m phases (li et al., 2007; wilson and rangasamy, 2001; wurm et al., 2001) . more recently, ibv infection was shown to induce dna damage response and activation of the ataxiatelangiectasia mutated (atm) and rad3-related (atr) kinase/ checkpoint kinase-1 (chk1) pathway partly through interaction between coronavirus nsp13 and dna polymerase delta (pol δ) (xu et al., 2011) . in this study, we show that phosphorylation at the thr378 and ser379 sites is dependent on atr, a kinase activated during ibv replication. introduction of ala substitutions at these two sites individually, in combination of the two, and together with the other two sites (ser190 and ser192), into an infectious ibv clone did not affect the recovery of recombinant viruses containing the mutations. a mutant virus (ribv-nm4) carrying ala substitutions at all the four previously identified sites grew at a similar, if not better, growth rate as wild type virus. this study reveals a cellular kinase responsible for phosphorylation of a coronavirus n protein at two sites and the functional consequence of this modification on coronavirus replication in virusinfected cells. in a previous study, we showed the phosphorylation of a number of atr-specific substrates, including chk1, the best studied atr substrate on ser317 and ser345, and replication protein a2 (rpa2), a component of the heterotrimeric rpa complex, on ser4/8 in ibv-infected cells (xu et al., 2011) . the specific residues for the atm kinase activity including chk2 on thr68 and atm on ser1981, however, were not detected in the same infected cells, demonstrating the activation of the atr-chk1 pathway during ibv infection (xu et al., 2011) . during this study, we noted that, when cells were infected with ibv at high multiplicity of infection and an excess amount of samples was loaded, a batch of pchk1-specific antibodies was also able to detect clearly a band migrating slightly more rapidly than pchk1 in ibv-infected cells, but not in cells infected with the ultraviolet (uv)-inactivated ibv at 8 and 12 h post-infection (fig. 1) . it should be pointed out that this observation was not consistently made when pchk1-specific antibodies from different sources were used. it was also noted that apparently more pchk1 was detected in cells treated with uv-inactivated ibv (fig. 1) . this increased detection of pchk1 may represent cellular contamination from the inoculums, as the virus stocks used for uv irradiation were prepared by freezing/thawing the infected cells and were not purified. re-probing of the same membrane with antibodies against ibv n protein detected the n protein at the exact position ( fig. 1 ). this result suggests that the protein band detected by anti-pchk1 may be a phosphorylated form of the ibv n protein. a series of experiments was then carried out to confirm if this indeed represents a phosphorylated form of ibv n protein and if atr is responsible for its phosphorylation. first, cells infected with ibv were analyzed by western blot with antibodies against ibv n protein and atm/atr substrates. as shown in fig. 2a , the fulllength 48-kda ibv n protein and several smaller bands, representing either premature or cleavage products derived from n protein (li et al., 2005) , were detected by western blot with anti-ibv n antibodies. the 48-kda band was also detected efficiently by the anti-atm/atr substrate antibodies (fig. 2a) . in addition, a background band with unknown identity was also detected from both mock-and ibv-infected cells by this antibody (fig. 2a) . the involvement of atr in the phosphorylation of ibv n protein was then analyzed by addition of 10 and 15 μm of schisandrin b (schb), a specific atr inhibitor functioning in ibv-infected cells as demonstrated in a previous study (xu et al., 2011) cells at 4 h post-infection. to more specifically analyze the full-length ibv n with an unrelated antibody, a recombinant ibv (ribv-fn) with a flag tag added to the n-terminus of the ibv n protein was constructed and used in this study. the recombinant virus showed very similar growth kinetics as wild type ibv (fig. 2b ). the cells were harvested at 24 h post-infection and analyzed by western blot. as observed in our previous study (xu et al., 2011 ), schb exhibited a certain level of inhibitory effect on ibv replication. ibv n protein was reduced to 78% and 55% when 10 and 15 μm, respectively, of schb were added, as revealed by western blot using anti-flag antibodies and densitometry analyses (fig. 2c) . probing of the same membrane with antibodies against atm/atr substrates detected much less phosphorylated n protein in the presence of schb (fig. 2c) . quantification of the band intensities by densitometry after normalizing to the total n protein showed that the atr-dependent phosphorylation of n protein was reduced to 47% and 2% after addition of 10 and 15 μm of schb, respectively (fig. 2c ). the phosphorylation of ibv n protein by atr was then analyzed in cells overexpressing the n protein. cells transfected with ibv n construct were subjected to uv irradiation at 22 h posttransfection in the presence or absence of 10 μm of schb. western blot analysis with antibodies against ibn n and atm/atr substrates showed that significantly less phosphorylated ibv n protein was detected in the uv-irradiated, transfected cells in the presence of 10 μm of schb (fig. 2d ). in the absence of 10 μm of schb, a 2.8-fold increase of the atr-dependent phosphorylation of n protein was detected in the transfected cells after uv-irradiation, compared to that in the cells without uv-irradiation ( fig. 2d ). in the presence of 10 μm of schb, a 1.5-fold increase of the atr-dependent phosphorylation of n protein was detected by the same comparison (fig. 2d ). these results would lend more support to the conclusion that atr is one of the kinases responsible for phosphorylation of ibv n protein. to systematically map the atr-dependent phosphorylation of ibv n protein, ala substitutions at the four previously identified sites (ser190, ser192, thr378 and ser379) as well as two potential new sites (ser65 and thr109) predicted to be atr substrates (fig. 3a) were carried out. the ala substitutions were then introduced into an ibv infectious clone either individually, or in combination of two, four, five or six, and nine recombinant ibvs were recovered from these constructs (fig. 3a) . all the nine mutant viruses were passaged in cells up to eight passages and their genetic stability was determined by nucleotide sequencing of the entire n protein-coding region. the sequencing results confirmed that the ala substitutions were stably maintained in all the mutant viruses, and no additional mutations were introduced into the n protein during passaging. the growth properties of the recombinant viruses (passage 8) were then determined. as shown in fig. 3b , very similar growth kinetics was found between wild type ibv and the mutant viruses. one notable difference was that all mutant viruses reached their peak titers at 32 h post-infection, while wild type virus reached the peak titer at 24 h post-infection (fig. 3b) . wild type and seven mutant viruses (nm1-7, passage 3) were first used to infect h1299 cells. total cell lysates were prepared and analyzed by western blot with antibodies against atm/atr substrates. the results showed that the atr-dependent phosphorylation of n protein was detected only in cells infected with wild type, nm1 and nm2 mutant viruses (fig. 4a) . the same band was not detected in cells infected with other five mutant viruses (nm3-nm7) (fig. 4a) . as all these five mutant viruses contain ala substitutions at thr378 and ser379 positions, it suggests that phosphorylation of ibv n protein at these two sites is atrdependent. re-probing of the same membrane with pchk1specific antibodies detected a much reduced amount of the phosphorylated n protein in cells infected with nm4 mutant virus, and moderately reduced amounts of the phosphorylated n protein in cells infected with nm3, nm5, nm6 and nm7 mutant viruses, respectively (fig. 4a) . the additional two mutant viruses, nm8 and nm9 containing the ala substitutions at thr378 and ser379, respectively (fig. 3a) , were then used to assess the relative contribution of the two residues to the atr-dependent phosphorylation of ibv n protein. h1299 cells were infected with wild type, nm3, nm8 and nm9 mutant viruses, and total cell lysates were prepared. western blot analysis of total cell lysates with antibodies against atm/atr substrates showed, once again, detection of the atr-dependent phosphorylation of ibv n protein in cells infected with the virus (fig. 4b) . no obvious phosphorylation of ibv n protein was detected by the same antiserum in cells infected with the three mutant viruses (fig. 4b) . these results indicate that both amino acid residues are phosphorylated by atr and the neighboring sequence may affect this atr-dependent phosphorylation of ibv n protein. it was also noted that pchk1 was not readily detected by western blot using both atm/atr substrate and pchk1-specific antibodies under the conditions shown in fig. 4a and b. the reason may be due to the relatively low abundance of pchk1, compared to the phosphorylated n protein in virus-infected cells. by immunofluorescence with antibodies against ibv n protein. as shown in fig. 4a , massive syncytium formation was observed in cells infected with both wild type and mutant viruses. no distinguishable difference in syncytium formation and subcellular distribution of n protein between wild type and the mutant viruses was observed (fig. 5a) . western blot analysis of another set of the infected cells with anti-ibv n antibodies showed the presence of both full-length and other processed forms of the n protein (li et al., 2005) (fig. 5b) . interestingly, slightly more n protein was detected from cells infected with the mutant virus at this time point (fig. 5b) . the growth properties of the mutant virus were then studied by plaque formation and growth kinetics studies. as shown in fig. 5c , plaques with similar sizes were found in cells infected with both wild type and ibvnm4 mutant viruses at 24 h post-infection. analysis of the growth kinetics of wild type and the mutant viruses also showed very similar growth properties for both viruses, although the mutant virus reached its peak titers at 24 h post-infection, which was 8 h later than did wild type virus (fig. 5c) . interestingly, the mutant virus remained at peak titers until the end of the time course at 32 h postinfection (fig. 5c) . it is not clear if this may represent an altered cellular response to the infection caused by this mutant virus. the buoyant density of ibvnm4 mutant virus was then analyzed by ultracentrifugation using 10-50% sucrose gradients, after collection of the virus from culture supernatants by spinning down through a 20% sucrose cushion. eleven fractions were collected and analyzed by western blot with antibodies against ibv n protein, which was used as a marker for the presence of virus particles. both wild type and mutant viral particles were found to be present mainly in fractions 7 and 8 (50.4% for wild type and 58.4% for ibvnm4) (fig. 5d) . it was noted that more viral particles (32.0%) were found in fraction 7 for wild type ibv, but more virus particles (39.2%) were found in fraction 8 for the mutant virus (fig. 5d) . these results demonstrate that the mutant virus may have a slightly lower buoyant density than wild type virus. further study would be required to confirm if this difference may reflect the difference in the phosphorylation of the n protein. coronavirus n protein plays multiple functions during the viral replication cycle. available evidence suggests that most of its at 24 h post-infection, the cultured media were collected and cell debris was removed by centrifugation at 10,000 rpm for 10 min. the virus particles were pelleted through a 20% sucrose cushion. the pellets were resuspended in 1 ml medium and subjected to ultracentrifugation in a 10-50% linear sucrose gradient. eleven fractions were collected and the presence of virus particles in each fraction was analyzed by western blot with antibodies against ibv n protein. the percentage of virus in each fraction was calculated after determination of the amount of n protein in each fraction by densitometry. functions may be regulated by the phosphorylation of the protein (calvo et al., 2005; jayaram et al., 2005; shin et al., 2007; surjit et al., 2005; spencer et al., 2008) . in this study, two previously identified phosphorylation sites in ibv n protein, thr378 and ser379, are shown to be phosphorylated by atr, which is activated by ibv-induced dna replication stress during ibv infection of cells (xu et al., 2011) . ala substitutions of these two residues together with the other two previously identified phosphorylation sites (ser190 and ser192) as well as two predicted sites for atr individually or in combination did not affect the recovery of infectious viruses containing the mutations. in this study, we provided evidence suggesting that phosphorylation of ibv n protein at thr378 and ser379 by atr appears to play a limited, if any, regulatory function in ibv replication and infectivity in cultured cells. as ibvnm4 also covers ala substitutions at other two previously identified phosphorylation sites (ser190ser192), it would point to the possibility that phosphorylation of coronavirus n protein at these four sites may represent a by-effect of host response to virus infection. coronavirus infection may activate a diversity of host cell signal transduction pathways as well as kinases, which would in turn lead to the phosphorylation of viral n protein. this may explain why more phosphorylated n protein is incorporated into ibv virions (jayaram et al., 2005) , as extensive phosphorylation of n protein would occur at a relatively late stage of the viral infection cycle. it is currently unclear if this is specific to ibv, as some other coronaviruses, such as mhv, tend to incorporate hypophosphorylated n protein into virions (white et al., 2007) on the other hand, we cannot rule out the possibility that phosphorylation of ibv n protein may occur at other positions in cells infected with nm4 and other related mutant viruses, and phosphorylation at these alternate sites would functionally compensate for the loss of phosphorylation at these four positions. this possibility would be supported by the observation that significantly more phosphorylated n protein was detected in cells infected with nm5, nm6 and nm7 mutant viruses, than in cells infected with nm4. it appears that additional ala substitutions at ser65 and thr109 in these three mutant viruses might induce more phosphorylation of ibv n protein at a site(s) recognized by the pchk1-specific antibodies used. in this context, it would be interesting to determine which phosphorylation site(s) of ibv n protein may be recognized by this pchk1-speific antiserum. data present in this study are inconclusive, but appear to indicate that both thr378 and ser379 may be recognized specifically or nonspecifically by the pchk1 antibodies used, as significantly reduced amounts of the phosphorylated n protein were detected in cells infected with mutant viruses containing mutations at these two positions, compared to that in cells infected with wild type and other mutants. however, as certain amounts of the phosphorylated n protein were still detected by this antiserum in cells infected with these mutant viruses, other phosphorylation site (s) must exist. multiple cellular kinases are involved in phosphorylation of coronavirus n protein. this may constitute another layer of regulatory mechanisms controlling the phosphorylation status of coronavirus n protein in virus-infected cells. for example, both thr378 and ser379 residues could be phosphorylated by casein kinase ii (ckii), as predicted by computer-aided programs. it was reported that ckii can be stabilized by atr-dependent phosphorylation of chk1 at ser317 in response to stalled dna replication (martin and ouchi, 2008) . this in turn would lead to phosphorylation of phosphatase and tensin homolog protein (pten) at thr383. as ibv infection activates the atr/chk1 pathway, phosphorylation of ibv n protein at multiple sites may be also regulated by a similar mechanism. in addition, phosphorylation of n protein is most likely a dynamic process, timely regulated during the infection cycle. in this study, we showed that the phosphorylated n protein was detected by a pchk1-specific antibody at as early as 8 h postinfection, as well as at 24 h post-infection. analysis of the effects of ala substitutions on the phosphorylation status of ibv n protein in virus-infected cells was carried out at 24 h post-infection in most of the studies shown here. this time point was chosen based on the consideration that slightly delayed growth kinetics was observed in most of the mutant viruses. it was noted that more phosphorylated n protein was detected in cells infected with some mutant viruses (mainly nm1 and nm2 mutant viruses) than that in cells infected with wild type virus. this may reflect this dynamic regulatory process at this time point. the successful recovery of viruses from all the mutant constructs confirms that phosphorylation at these positions is not essential for the viability of ibv in cultured cells. in fact, western blot analysis of viral protein expression and viral growth kinetics analysis showed that most of the mutant viruses grew similarly well as wild type virus. this is confirmed by detailed analysis of the growth properties and kinetics of ibvnm4, showing that the mutant virus grew equally well, if not better, as wild type virus, although a slightly delayed growth kinetics was observed. interestingly, after reaching peak titer at 24 h post-infection, the mutant virus remains at its peak titer until the end of the time course at 32 h post-infection. this is in contrast to wild type virus, which reached the peak titer at 16-24 h post-infection. the titers were gradually declined afterward. the underlying mechanisms for this difference are currently unknown, but it may reflect the difference in virus-host interactions between wild type and the mutant virus. one possibility is that the mutant virus may have an altered ability to induce cell death or cell cycle arrest. a more thorough study would be required to understand this issue more. in conclusion, this study has provided strong evidence that activation of atr/chk1 pathway during ibv infection is responsible for phosphorylation of ibv n protein at two previously identified sites. phosphorylation of n protein at these two positions, however, may play limited, if any, regulatory functions in ibv replication and infectivity in cultured cells. this would meanwhile reinforce our previous conclusion that ibv infection leads to efficient activation of the atr pathway (xu et al., 2011) . vero (atcc ccl-81) cells were maintained in dmem supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ ml) and streptomycin (100 μg/ml) at 37 1c in a 5% co 2 environment. h1299 (atcc crl-5803) cells were maintained in complete rpmi 1640 medium (jrh) supplemented with 10% newborn calf serum. both vero and h1299 cells were routinely used to culture vero-adapted ibv (liu et al., 1995 (liu et al., , 1998 tay et al., 2012) the passage 65 (p65, designated wild type virus (wtibv) of vero-adapted ibv beaudette was described before (fang et al., , 2007 . the ibv strains were propagated in vero cells and the tissue culture infective dose (tcid 50 ) of viral stocks was calculated by the reed-muench method (yamada and liu, 2009 ). viral stocks were kept at −80 1c until use. schisandrin b (schb, shanghai tauto biotech co. ltd., china) was dissolved in dmso and stored at −80 1c, as previously described (xu et al., 2011) . antibodies against chk1ser317 and phospho-(ser/thr) atm/ atr substrates were purchased from cell signaling technology (beverely, ma, usa). antibody against flag was from sigma-aldrich, and antibodies against actin and tubulin were from santa cruz (santa cruz, ca). polyclonal antibodies against ibv n and s were raised in rabbits (xu et al., 2010; yamada and liu, 2009 ). horseradish peroxidase (hrp)-linked goat anti-rabbit secondary antibodies, hrp-linked goat anti-mouse secondary antibodies were purchased from dako (glostrup, denmark). inhibitors schb was added to cells 1 h prior to infection or 8 h post-transfection, and kept in the media during the infection. cells of 90-95% confluency grown on 12-well plates were infected with ibv at a multiplicity of infectivity of approximately 1, and were harvested at the indicated time points post-infection. plasmid pkt-0 which has t7 promoter for transcription was used for transient transfection using the vaccinia/t7 recombinant virus liu, 2000, 2002) . the pkt-n construct that encoded full-length n protein was previously reported (li et al., 2005) . epitope-tagged constructs were made by overlap pcr and cloned into the pkt-0. point mutations were made by site-directed mutagenesis using the quikchange™ kit (stratagene). cells at about 90% confluency were infected with the recombinant vaccinia/t7 virus for 1 h followed by transfection of plasmid dna using the effectene transfection reagent (qiagen). each sample was lysed with 1 â sds sample loading buffer without bromophenol blue and the protein concentration was determined by the bio-rad protein assay kit. equal amounts of total protein were separated by sds-page and transferred to pvdf membranes. membranes were incubated with a primary antibody, subsequently with hrp-conjugated secondary antibody, and detected using the ecl advance western blotting detection kit (amersham). vero cells were cultivated in 4-well chamber slides (iwaki). ibv-infected cells were washed with phosphate buffered saline (pbs) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in pbs for 15 min, and permeabilized with 0.2% triton x-100 for 10 min. if staining was performed by incubating cells with polyclonal antibodies against ibv n protein and subsequently with the fitc-conjugated anti-rabbit igg. cells were examined by fluorescent microscopy. the detailed procedure to create a full-length ibv cdna clone was previously reported (tan et al., 2006; fang et al., 2007) . the region of the ibv cdna covering the n gene was replaced with mutant n genes, and subsequently ligated into full-length ibv cdna. full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tex). wild type ibv n gene transcripts were also generated to enhance the recovery of virus. vero cells at about 90% confluency were trypsinized, washed twice with ice-cold pbs, and resuspended in pbs. rna transcripts were introduced into vero cell by using the bio-rad gene pulser ii electroporator. cells were cultured overnight in 1% fbs-containing dmem in a 6-well plate and further incubated in dmem without fbs. at 48 h post-electroporation, viral rna replication was investigated by rt-pcr of the negative strand grna. transcription of subgenomic mrnas was investigated by rt-pcr as described before (tan et al., 2006; fang et al., 2007) . the n gene of recovered ribv clones (at the 3rd passage in vero cells) was amplified by rt-pcr and subsequently confirmed by the dna sequencing analysis. characterization was carried out with the 3rd passage of viruses in vero cells. the intensities of rna and protein bands were quantified using imagej program according to the manufacturer's instruction. the nucleoprotein is required for efficient coronavirus genome replication interactions between coronavirus nucleocapsid protein and viral rnas: implications for viral transcription phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells coronavirus avian infectious bronchitis virus multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging cis requirement for n-specific protein sequence in bovine coronavirus defective interfering rna replication mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance interaction of the coronavirus nucleocapsid with nucleolar antigens and the host cell ribonucleoprotein of avian infectious bronchitis virus the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability crystal structure of the n-terminal domain from the nucleocapsid protein of coronavirus infectious bronchitis virus functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3c-like proteinase proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of coronavirus infectious bronchitis virus 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polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products cellular commitment to reentry into the cell cycle after stalled dna is determined by site-specific phosphorylation of chk1 and pten characterization of the coronavirus m protein and nuclocapsid interaction in infected cells further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site membrane association and dimerization of a cysteine-rich, 16-kilodalton polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: the effect of phosphorylation on immunoreactivity and specificity unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein specific interaction between coronavirus leader rna and nucleocapsid protein the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation coronavirus translational regulation: leader affects mrna efficiency amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the replication and infectivity of coronavirus in cultured cells binding of the 5′-untranslated region of coronavirus rna to zinc finger cchc-type and rna binding motif 1 enhances viral replication and transcription mechanisms and enzymes involved in sars coronavirus genome expression characterization of cellular furin content as a potential factor determining the susceptibility of cultured human and animal cells to coronavirus infectious bronchitis virus infection identification of mouse hepatitis coronavirus a59 nucleocapsid protein phosphorylation sites viral interaction with the host cell sumoylation system glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division coronavirus infection induces dna replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of dna polymerase delta the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation and infectivity of coronavirus infectious bronchitis virus in cultured cells the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 3′ terminus of the genome coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was partially supported by a competitive research programme (crp) grant (r-154-000-529-281), the national research foundation, singapore. key: cord-263178-lvxxdvas authors: shan, dan; fang, shouguo; han, zongxi; ai, hui; zhao, wenjun; chen, yuqiu; jiang, lei; liu, shengwang title: effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus date: 2018-05-02 journal: virus res doi: 10.1016/j.virusres.2018.04.013 sha: doc_id: 263178 cord_uid: lvxxdvas to study the roles of hypervariable regions (hvrs) in receptor-binding subunit s1 of the spike protein, we manipulated the genome of the ibv beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three hvrs of the beaudette strain with the corresponding fragments from a qx-like nephropathogenic isolate ck/ch/ldl/091022 from china. we characterized the growth properties of these recombinant ibvs in vero cells and embryonated eggs, and their pathogenicity, tropism, and serotypes in specific pathogen-free (spf) chickens. all seven recombinant ibvs proliferated in vero cells, but the heterogenous hvrs could reduce their capacity for adsorption during in vitro infection. the recombinant ibvs did not significantly increase the pathogenicity compared with the beaudette strain in spf chickens, and they still shared the same serotype as the beaudette strain, but the antigenic relatedness values between the recombinant strain and beaudette strain generally decreased with the increase in the number of the hvrs exchanged. the results of this study demonstrate the functions of hvrs and they may help to develop a vaccine candidate, as well as providing insights into the prevention and control of ibv. ibv primarily replicates in the epithelial surface of the respiratory tract, although some strains are nephropathogenic (cavanagh, 2003) . ibv infections reduce egg production, quality, and hatchability, as well as increasing the feed conversion ratio and carcass condemnation in slaughterhouses. thus, infectious bronchitis causes severe economic losses in the poultry industry worldwide. the enveloped ibv belongs to the genus coronavirus, family coronaviridae, order nidovirales (cavanagh, 2003) . the main structural protein is the spike (s) glycoprotein, which comprises a divergent s1 subunit and conserved s2 subunit (de groot et al., 1987; shil et al., 2011) . the s1 subunit contains the receptor-binding domain (wickramasinghe et al., 2011) , and it carries virus-neutralizing and serotype-specific determinants. the s1 domain exhibits high sequence diversity, where 20%-25% (even up to 50%) of the amino acids differ within the s1 subunit among ibv serotypes. after comparing the s1 subunit from a number of distinct ibv isolates, a previous study defined particularly variable segments at the amino terminus of the s1 subunit as hypervariable regions (hvrs) . the hvrs possibly account for the antigenicity and serotypic variation, and evidence indicates that five neutralizing peptides mainly mapped onto the s1 subunit are co-located within the hvrs (cavanagh et al., 1992 (cavanagh et al., , 1988 moore et al., 1997; niesters et al., 1987) . in addition, the hvrs are possibly associated with receptor binding. it has been reported that the critical amino acids for attachment of the m41 spike overlap with a hvr in the s1 subunit (promkuntod et al., 2014) . thus, the hvrs may affect the tropism, serotype, and pathogenicity of ibv, and it is important to elucidate their biological functions. previous studies applied forward genetics methods to determine the roles of the hvrs by characterizing their phylogenetically closely related isolates with mutations in other parts of the ibv genome, but it is difficult to establish a precise model for further analysis based on these studies. thus, in this study, we manipulated the ibv beaudette genome by reverse genetics to exchange the hvrs in the beaudette strain with those in ck/ch/ldl/091022 in order to precisely determine their biological functions. we also explored the possibility of developing a reverse genetic vaccine candidate cultured in vero cells to provide protection from the prevalent field strains. the results of this study contribute to our understanding of the prevention and control of ibv. vero cells were maintained in dulbecco's modified eagle's medium (gibco, grand island, ny, usa) supplemented with 10% fetal bovine serum (sigma-aldrich, saint louis, mo, usa), penicillin (100 units/ ml), and streptomycin (100 μg/ml) at 37°c with 5% carbon dioxide. the ck/ch/ldl/091022 strain was isolated from h120-vaccinated chickens with renal lesions in 2009 . the beaudette strain was routinely propagated in vero cells (liu et al., , 1998 tay et al., 2012) and the 50% tissue culture infection dose (tcid 50 ) of the viral stock was calculated using 96-well plates with 10-fold serial dilutions. the 50% egg infection dose (eid 50 ) of the viral stock was determined by inoculating 10-fold dilutions into groups of 9-day-old embryonated chicken eggs. five fragments spanning the entire ibv genome were obtained by rt-pcr from vero cells infected with the ibv beaudette strain, as described previously (fang et al., 2007) . the three hvrs in the isolate ck/ ch/ldl/091022 were identified based on alignments of the amino acid sequences, as described previously . the sequences of the ibv cdna covering the three hvrs in the s gene were replaced separately or simultaneously with those from ck/ch/ldl/091022, and subsequently ligated into the full-length ibv cdna ( fig. 1a and table 1 ). full-length transcripts generated in vitro were introduced into vero cells by electroporation. ibv n gene transcripts were also generated to enhance the efficiency of viral recovery. total rna was prepared from the electroporated vero cells or infected allantoic liquid. viral rna replication was investigated based on rt-pcr of negativestrand genomic rna (tan et al., 2006) . the s gene in the recovered ibv clones (third passage in vero cells and embryonated eggs) was amplified by rt-pcr and subsequently confirmed by dna sequencing analysis. the gene was characterized during the third passage of viruses in specific pathogen-free (spf) embryonated eggs or vero cells. the seven rescued recombinant ibvs were designated as rhvr i, rhvr ii, rhvr iii, rhvr i/ii, rhvr i/iii, rhvr ii/iii, and rhvr i/ii/iii. to determine the growth kinetics of the rescued recombinant ibvs, a dose of 100 × eid 50 was inoculated into the allantoic cavities of 9-dayold embryonated eggs, and the allantoic fluid was harvested from three eggs in each group at 12, 24, 36, 48, and 60 h post-inoculation, where the fluids from three eggs were pooled for eid 50 determination with three replicates. vero cells were infected with beaudette and recombinant ibvs, and three wells were harvested at 4, 8, 12, 16, 24, and 36 h post-infection. viral stocks were prepared by freezing/thawing the cells three times to determine the tcid 50 with three replicates for each time. ibv infected cells cultured in six-well plates were washed with phosphate-buffered saline (pbs), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% triton x-100 for 10 min. if staining was performed with a monoclonal antibody 6d10 against ibv n protein and subsequently with fitc-conjugated anti-mouse igg (sigma-aldrich). cells were examined by fluorescence microscopy. all of the animal experimental procedures were approved by the ethical and animal welfare committee of heilongjiang province, china (license no. sq20160408). we randomly assigned 150 7-day-old spf layer chickens to 10 groups, i.e., the seven recombinant strains, beaudette, ck/ch/ldl/ 091022, and pbs groups. there were 15 spf chickens in each group. the challenge strain (10 5 × eid 50 per bird) was applied via the intranasal and ocular routes. the clinical signs were monitored in 10 randomly selected challenged birds from each group, and the morbidity and mortality rates were recorded daily. we evaluated the challenged chickens blindly for respiratory rales at 5 days after challenge. signs were scored as 0 = absent, 1 = mild, 2 = moderate, or 3 = severe. at 5 days after ibv challenge, the other five birds in each group were killed humanely using carbon dioxide over inhalation, followed by exsanguination. the cranial third of the trachea, lungs, kidneys, and cecum tonsils were collected, partly fixed in formalin, and embedded in paraffin. longitudinal 5-μm sections were stained with hematoxylineosin (h&e stain). the mucosal thickness, deciliation, goblet cells, and lymphocytes scores for the tracheal mucosa were evaluated blindly and scored from 1 to 5 based on their severity (i.e., normal, mild, moderate, marked, and severe) (van ginkel et al., 2015) . moreover, viral shedding was quantified in the oropharyngeal secretions from 10 infected chickens at 4, 8, 12, 16, and 20 days after challenge by real-time rt-pcr (jones et al., 2011) . the tissues collected at 5 days post-ibv challenge, i.e., tracheas, lungs, kidneys, and cecum tonsils, were also subjected to immunohistochemistry (ihc) using monoclonal antibody 6d10, as described previously (de wit et al., 2011; xu et al., 2016) . viral loads in selected tissues were determined based on ibv rna detection by realtime rt-pcr, as described previously . the seven ibv recombinant strains, beaudette, and ck/ch/ldl/ 091022 were analyzed in cross-virus neutralization tests. sera against the ibv strains were prepared as previously described . in the virus neutralization tests, sera were serially diluted two times with sterile pbs and mixed with 200 × eid 50 or tcid 50 for the ibv strains. the two-way cross-neutralization test between the beaudette and recombinant ibvs was performed in vero cells. after incubation for 1 h at 37°c, the virus-serum mixtures were cultured in 96-well microplates for 5 days. we could not detect the replication with ck/ch/ ldl/091022 in vero cells, so the neutralization tests were performed in 9-day-old spf embryonated eggs to confirm whether the serotypes of the recombinant ibvs belonged to ck/ch/ldl/091022. the end-point titer for each serum sample was calculated using the reed-muench method. antigenic relatedness values were calculated (archetti and horsfall et al., 1950; wadey and faragher, 1981) . the celisa protocol used in this study is similar to the conventional elisa. the viral solution for each strain was serially diluted two times with sterile pbs and coated on the plate. standard curves were drawn according to the standard elisa method. vero cells were cultured overnight in 96-well microplates (corning, usa) (10 5 cells/well). for the adsorption assay, each strain was incubated in cells at a multiplicity of infection (moi) of 1 for 1 h at 4°c with three replicates (fang et al., 2010; sun et al., 2017) . after two washes with pbs, the microplates were fixed with methanol and 1% hydrogen peroxide for 30 min, and then permeabilized with 0.2% triton x-100 for 10 min at room temperature. next, the microplates were washed three times and blocked with 8% skim milk for 30 min at room temperature. after washing three times, the microplates were incubated with the primary mouse monoclonal antibody 6d10 for 1 h at 37°c. a similar procedure was then performed where the plates were incubated for 40 min with the peroxidase-conjugated anti-mouse igg. peroxidase substrate solution (tmb; sigma-aldrich) was added and the plates were developed in darkness. the color reaction was stopped with stop solution (sigma-aldrich) and the absorbance was read at 630 nm. for the internalization assay, cells were seeded into 96-well microplates and inoculated with each strain for 1 h at 37°c. after incubation, the cells were washed twice with pbs, before treating the infected cells with citrate buffer for 1 min to inactivate the adsorbed but not internalized virus. the cells were washed with pbs to remove the citrate buffer. the celisa process was performed as described above. the celisa results were expressed as the relative viral load adsorbed or internalized into cells relative to the beaudette control. vero cells were seeded in 24-well plates (corning) and infected with each separate strain at an moi of 1. in the adsorption and internalization stage, total rna was extracted from the cells, before storing at −80°c for the subsequent quantification of viral loads, as described previously (liu et al., 2006a; sun et al., 2014) . three replicates were analyzed for each sample. first, the stable expression levels of three candidate reference genes comprising 18s rna, β-actin, and gapdh were compared by real-time rt-pcr in vero cells infected with the beaudette strain at 4°c or 37°c for 1 h. the results were analyzed using genorm software, normfinder software, and delta ct (http://150.216. 56.64/referencegene.php). the viral mrna levels of all the samples were calculated by using the most stably expressed genes as an internal reference for normalization (fung et al., 2014) . the real-time rt-pcr results were expressed as the relative viral load adsorbed or internalized into cells relative to the beaudette control. we randomly assigned 200 7-day-old spf layer chickens to 10 groups, with 20 birds in each group. birds in groups 1-9 were inoculated with the seven recombinant ibvs, beaudette, and ck/ch/ldl/ 091022 (10 5 × eid 50 per bird) via intranasal and ocular routes. birds in group 10 were inoculated with pbs and treated as the negative control. half of the surviving birds in each group were randomly selected and challenged after 21 days with a 10 6 × eid 50 dose of ck/ch/ldl/ 091022 via intranasal and ocular routes. the clinical signs were monitored in the challenged birds in each group, and the morbidity and mortality rates were recorded daily. viral shedding was also quantified using oropharyngeal swabs from the challenged chickens at 4, 8, 12, 16, and 20 days after challenge by real-time rt-pcr (jones et al., 2011) . the remaining chickens in each group were investigated for 15 days. were conducted based on three replicates. anova followed tukey's multiple comparison tests were performed to compare the virus titers of different strains at the same time point, and differences were considered significant at p < 0.05. one-way anova followed tukey's multiple comparison tests were used to compare differences in the viral loads in selected tissues, as well as the results of the adsorption and internalization assays. all p-values were two-tailed and differences were considered significant when p < 0.05. in addition, kruskal-wallis anova followed dunn's multiple comparison tests in spss statistics were used to compare the trachea lesion scores and clinical scores. differences in the survival rates were analyzed using the log-rank test. we constructed seven full-length cdna clones where the hvrs from beaudette were substituted with those from ck/ch/ldl/091022 either separately or simultaneously (fig. 1a and table 1 ). the rna transcripts generated from the full-length cdna clones were introduced separately into vero cells together with the n transcripts by electroporation (fang et al., 2005) . after 48 h, the virus present in each medium was collected and used to inoculate 9-day-old spf embryonated eggs. to differentiate the rescued viruses from the parental viruses, viral rna was extracted from allantoic fluid and the s1 genes were amplified using rt-pcr, where the rt-pcr products covering the three hvrs were sequenced. viral antigen was observed in vero cells infected with the beaudette strain and the seven recombinant ibvs (fig. 1b) . no virus antigen was found in cells infected with ck/ch/ldl/091022. rt-pcr analysis of subgenomic mrna 3 and mrna 4 was performed to confirm the virus replication (fig. 1c) (fang et al., 2007) . the results confirmed that the recombinant ibvs were adapted to vero cells in a similar manner to the beaudette strain. to test whether exchanging the hvrs affected the growth properties and genetic stability of the rescued viruses, the recombinant ibvs were propagated on embryonated eggs or vero cells for three passages. the vero cells were cultured in 96-well plates and infected with the recombinant ibvs and two parental strains (10 5 × eid 50 ) for 1 h at 4°c or 1 h at 37°c. the experiments were conducted based on three replicates. (a) infected cells were fixed and celisa was performed as described in the materials and methods. results are shown as viral load adsorption on cell equivalents relative to eid 50 compared with that in the beaudette strain (100%). (b) vero cells were cultured in 24-well plates and infected with the recombinant ibvs and parental strains (10 6 × eid 50 ) for 1 h at 4°c. total rna was extracted from the infected cells before quantifying the viral loads using real-time rt-pcr. data were normalized against the gapdh expression levels and viral rna was calculated relative to that in the beaudette strain (100%). the experiments were conducted based on three replicates. (c) celisa was performed to evaluate the viral load internalized into cells. (d) real-time rt-pcr was also used to evaluate the viral load internalized into cells. the experiments were conducted based on three replicates. differences were considered significant at p < 0.05 using anova followed tukey's multiple comparison tests. s1 gene sequencing results confirmed that the heterogenous hvrs were stably maintained in the recombinant ibvs (sup fig. 1) , and no additional mutations were detected in the s protein after three passages in cells or eggs. the growth properties of the recombinant ibvs were then determined in vero cells and embryonated eggs. in vero cells (fig. 1c) , the growth kinetics of the recombinant ibvs were similar to those of the beaudette strain, and they all reached their peak titer at 16 h post-infection, and there was no significant difference between the titers for the recombinant ibvs and beaudette at different time points (fig. 2) . in embryonated eggs (fig. 3a) , although there were no significant difference between the virus titers for the recombinant ibvs and beaudette at different time points, the titers of ck/ch/ldl/091022 was significantly higher than those of the recombinant ibvs and beaudette at 12, 36, 48, 60 h (fig. 3b ). in conclusion, the recombinant ibvs exhibited similar growth phenotypes compared to the beaudette strain in both cells and eggs. the if staining results showed the ibv n antigens in vero cells infected with the recombinant ibvs (fig. 1b) and they have the similar ability to replicate in vero cells with ibv beaudette. we then evaluated the effects of the heterogenous hvrs on virus adsorption and internalization. the celisa results for the adsorption assay showed that compared with beaudette, the recombinant ibvs exhibited significantly reduced adsorption at 4°c for 1 h (fig. 4a) . the real-time rt-pcr results were consistent with the celisa results in the adsorption assays (fig. 4b) . the results indicated that the heterogenous hvrs could reduce the adsorption capacity of the recombinant ibvs. in addition, the celisa results from the internalization assay showed that the viral yields of the recombinant ibvs internalized into cells did not differ significantly from those with beaudette (fig. 4c) . the real-time rt-pcr results were consistent with the celisa results from the internalization assay (fig. 4d) . in order to test the pathogenicity of the recombinant ibvs, 7-day-old spf chickens were inoculated via intranasal and ocular routes at a dose of 10 5 × eid 50 per bird (sun et al., 2011) . we found that some of the chickens only had very low levels of snicking at 6 and 7 days postinfection, and there were no obvious clinical signs in the groups treated with the recombinant ibvs and beaudette. however, 5/10 birds (50%) died during 5-9 days post-challenge with ck/ch/ldl/091022 and the morbidity rate was 100% (fig. 5a) . the mean scores for the clinical signs in the birds inoculated with the recombinant ibvs and beaudette were much lower than those treated with ck/ch/ldl/091022 (fig. 5b) . the autopsy results showed that ck/ch/ ldl/091022 caused obvious swollen pale kidneys, where the tubules and ureters were distended with urates, thereby indicating the nephropathogenic potential of the virus. however, the autopsies detected almost no changes in the groups treated with the recombinant ibvs and beaudette. the h&e staining results also showed that there were no obvious pathological change in the lungs, cecum tonsils, and kidneys in the groups treated with the recombinant ibvs and beaudette (sup fig. 2) . we also assessed the severity scores for histopathological lesions in the trachea with deciliation (fig. 6a) , goblet cells (fig. 6b) , mucosal thickness (fig. 6c) , and lymphocytes (fig. 6d) at 5 days after challenge with the ibvs (van ginkel et al., 2015) . the scores were lowest in the beaudette group and highest in the ck/ch/ ldl/091022 group, and there were no differences in the scores between the groups treated with the recombinant ibvs and beaudette. however, the scores in the groups treated with the recombinant ibvs and beaudette differed significantly from those with ck/ch/ ldl/091022. finally, no ibv rna was detected in the oropharyngeal swabs from chickens inoculated with the recombinant ibvs and beaudette after 4, 8, 12, 16, and 20 days by real-time rt-pcr, where the results were considered positive when the ct value was less than 32 zhao et al., 2017) . the results confirmed that the recombinant ibvs were avirulent according to the histomorphometry and histopathology findings. using a previously described method (archetti and horsfall et al., 1950) , the serotype relatedness values were calculated based on the cross-virus neutralization studies using vero cells (table 2) or embryonated chicken eggs (table 3) . viruses with an archetti and horsfall relatedness value greater than 25 were considered to be related serotypes. our results showed that the recombinant ibvs and beaudette belonged to the same serotype (table 2) , whereas the recombinant ibvs and ck/ch/ldl/091022 belonged to different serotypes (table 3 ). in general, we found that the serotype relatedness values decreased between the recombinant strain and beaudette (table 2 ) as the number of substituted heterogenous hvrs increased. these results confirmed that the replacement of the heterogenous hvrs had not caused serotype switches in this case. the ihc results showed that the viral antigen of ck/ch/ldl/ 091022 was detected in trachea (sup fig. 3a) , cecal tonsil (sup fig. 3b) , and kidney samples (sup fig. 3c ), but the results were negative in the tissues infected with the recombinant ibvs and beaudette. the results were also negative in all of the lung samples (sup fig. 3d) . the viral rna could be detected by real-time rt-pcr in the selected tissues from chickens infected with ck/ch/ldl/091022, with high viral loads in the kidneys. however, very low rna levels were detected in only a few (a) mortality was recorded daily for 10 randomly selected infected birds and the survival curve was drawn. (b) at 5 days after challenge, we evaluated the challenged chickens blindly and clinical signs were scored as: 0 = absent, 1 = mild, 2 = moderate, or 3 = severe. kruskal-wallis anova followed dunn's multiple comparison tests were employed to perform comparisons of the clinical scores using spss. d. shan et al. virus research 250 (2018) 104-113 trachea samples from the birds infected with the recombinant ibvs and beaudette (fig. 7) . these results confirmed that the recombinant ibvs had the same tissue tropism as the beaudette strain. inoculating chickens with the seven recombinant ibvs induced clinical protection against challenge with ck/ch/ldl/091022 (table 4 ). viral shedding was detected in some of the chickens challenged with ck/ch/ldl/091022, but inoculating chickens with the seven recombinant ibvs and beaudette reduced the morbidity and mortality rates compared with birds in the pbs group. however, the morbidity and mortality of the recombinant ibvs and beaudette vaccinated groups was not as low as that of ck/ch/ldl/091022 inoculated group. these results suggest that although the recombinant ibvs could fig. 6 . trachea lesion scores. at 5 days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation. tissue samples were collected from the trachea and analyzed by h&e staining. mucosal thickness (a), deciliation (b), goblet cells (c) and lymphocyte scores (d) for the trachea samples were evaluated blindly and scored from 1 to 5 based on severity. kruskal-wallis and dunn-bonferroni tests were employed to perform post hoc comparisons of trachea lesion scores using spss. virus replication was found in vero cells infected with beaudette and the recombinant ibvs, so the neutralization tests between them were performed in vero cells. not offer complete protection against challenge with ck/ch/ldl/ 091022, inoculation with the seven recombinant ibvs did confer some cross protection against ck/ch/ldl/091022 challenge. coronavirus ibv s protein has multiple biological functions during the viral replication cycle. previous studies suggest that some of these functions may be associated with hvrs in the s1 subunit of the s protein (cavanagh and davis, 1986; ignjatovic and galli, 1994; johnson et al., 2003; song et al., 1998) . these previous studies employed forward genetics methods to determine the effects of the hvrs on serotypes by characterizing phylogenetically closely related isolates. however, these strains inevitably had mutations in other parts of the ibv genome, thereby making it difficult to establish a precise model for further studies based on these previous findings. in the present study, we manipulated the ibv rna genomes by reverse genetics to produce site-directed mutations in order to elucidate the specific biological functions of the hvrs. we used the beaudette and ck/ch/ldl/091022 strains in our study. ibv beaudette is a well-known apathogenic lab strain (geilhausen et al., 1973) , which was adapted to vero cells from chicken embryos (fang et al., 2005) . the ck/ch/ldl/091022 ibv strain is an epidemic strain isolated from h120-vaccinated layers in china. this strain mainly causes gross lesions in chicken kidneys, with high morbidity and mortality rates (liu et al., 2008 (liu et al., , 2009 liu et al., 2006b) . the ck/ch/ldl/ 091022 strain can only proliferate in chicken embryos and not vero cells. in addition, the beaudette and ck/ch/ldl/091022 strains belong to different serotypes. thus, the differences in these two strains facilitate research into the effects of the hvrs in intact viral particles on serotypes, virulence, and tropisms. in order to determine the roles of individual hvrs and the accumulated effects of hvrs, we constructed seven recombinant ibvs where the three hvrs in the beaudette strain were separately or simultaneously substituted with those from ck/ch/ldl/091022. all of the recombinant ibvs proliferated in vero cells (figs. 1b and figure 2) , and had the similar growth kinetics as beaudette in vero cells (fig. 2) and embryonated eggs (fig. 3b) . the accumulation of heterogenous hvrs weakened the capacity for adsorption during infection by the recombinant viruses in vitro ( fig. 4a and b) . the hvrs may be associated with the receptor-binding domain, so the heterogenous hvrs weakened the interaction between the viruses and their receptors on the host cells. with the same sequences of fusion subunit s2, the recombinant ibvs and beaudette did not differ significantly in the internalization assay. it is unclear whether the recombinant viruses have more efficient internalization than beaudette and the s proteins assemble correctly in the recombinant viruses, and whether there is a comparable amount of s on the virus particle. these problems require for further investigation. both the recombinant ibvs and beaudette only weakly infected the trachea in 7-day-old spf chickens (fig. 5e) , whereas ck/ch/ldl/ 091022 infected multiple chicken tissues, including the trachea, lungs, kidneys, and cecal tonsils. thus, the replication capacity was similar for the recombinant ibvs and the beaudette strain in spf chickens. it appears that only swapping the hvrs did not greatly affect the cell and tissue tropisms, although replacing the ectodomain of the s glycoprotein in the beaudette strain can alter the growth characteristics (casais et al., 2003) . virus binding to host cells is the first step in tropism determination and hvrs within the s1 subunit may affect ibv binding (wickramasinghe et al., 2011) , but s2 is responsible for membrane fusion , and thus exchanging the hvrs did not change their tropism. fig. 7 . detection of ibv replication in challenged chickens. at 5 days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation, and tissue samples of the trachea, lungs, kidneys, and cecum tonsils were collected to determine the presence of ibv by real-time rt-pcr. a ct value less than 32 was considered to be ibv-positive using real-time rt-pcr. the numbers of tissue samples positive for ibv rna/the number detected are presented at the bottom. bars in different colors represent different tissues from chickens, as indicated in the graphical representation. the average copy numbers of ibv rna in the positive samples are shown. differences were considered significant at p < 0.05 using anova followed tukey's multiple comparison tests. 8d 12d 16d 20d 4d 8d 12d 16d 20d 4d 8d 12d 16d 20d rhvr i 7/10 1/10 10/10 4/9 2/9 1/9 0/9 0/10 2/10 3/10 7/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr ii 7/10 1/10 10/10 7/9 4/9 2/9 0/9 0/10 1/10 3/10 5/10 10/10 7/10 9/9 9/9 9/9 9/9 rhvr iii 8/10 1/10 10/10 5/9 2/9 1/9 0/9 0/10 2/10 4/10 5/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr i/ii 7/10 0/10 10/10 6/10 4/10 0/10 0/10 0/10 3/10 5/10 7/10 10/10 7/10 10/10 10/10 10/10 10/10 rhvr i/iii 7/10 1/10 10/10 7/9 2/9 0/9 0/9 0/10 2/10 4/10 6/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr ii/iii 8/10 2/10 10/10 6/8 1/8 0/8 0/8 0/10 1/10 3/10 6/10 10/10 7/10 8/8 8/8 8/8 8/8 rhvr i/ii/iii 7/10 0/10 10/10 6/10 5/10 0/10 0/10 0/10 2/10 5/10 7/10 10/10 8/10 10/10 10/10 10/10 10/10 beaudette 9/10 1/10 10/10 4/9 4/9 1/9 0/9 0/10 2/10 4/10 6/10 10/10 6/10 9/9 9/9 9/9 9/9 ck/ch/ldl/091022 2/5 c 0/5 5/5 3/5 2/5 0/5 0/5 0/10 5/6 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 pbs 10/10 3/10 10/10 8/9 3/7 0/7 0/7 0/10 0/10 0/10 0/10 0/10 3/10 4/9 5/7 7/7 7/7 a viral rna was detected in oral swab samples by real-time rt-pcr and samples with a ct value less than 32 were considered positive. b days after challenge. c five birds died 7 days after inoculation with ck/ch/ldl/091022. previous studies have shown that the s protein has major effects on pathogenicity in coronaviruses (leparc-goffart et al., 1997; phillips et al., 2001) , but the specific effects of hvrs in the s protein on pathogenicity are not clear. in the present study, the recombinant ibvs remained avirulent with the hvrs from pathogenic ck/ch/ldl/ 091022, thereby indicating that only exchanging the hvr sequences did not have major impacts on pathogenicity (figs. 5 and 6, sup. fig. 2 ). according to hodgson et al., the apathogenic nature of the recombinant ibv beaur-m41(s) indicates that the s protein ectodomain from a virulent strain is not necessarily sufficient to overcome the attenuating mutations in other genes in the apathogenic beaudette strain (hodgson et al., 2004) . it is possible that the replicase gene also contributes to the pathogenicity of ibv (armesto et al., 2009; hodgson et al., 2004) . the functions of non-structural proteins (nsp) in the context of pathogenesis are still not well understood, but some of the nsps in other coronaviruses have been linked to loss of pathogenicity (eriksson et al., 2008; sperry et al., 2005) . it is considered that the hvrs of the ibv s1 subunit may induce abundant virus neutralizing antibodies koch et al., 1990; moore et al., 1997; niesters et al., 1987) . however, in our study, the introduction of heterogenous hvrs did not develop an independent serotype, although the antigenic relatedness values for the recombinant ibvs and beaudette generally decreased as the number of heterogenous hvr exchanges increased (table 2 ). this result is consistent with the conclusion of santos fernando et al. who found that three ibv isolates mainly exhibited mutations in the hvrs but they belonged to the same serotype (santos fernando et al., 2017) . the repertoire of neutralizing polyclonal antibodies may react with the many epitopes of an antigen. exchanging the hvrs could change several antigen determinants but it is not necessarily sufficient to change the serotype because other neutralizing epitopes have been identified in other parts of the s1 subunit and s2 subunit (ignjatovic and sapats, 2005; kusters et al., 1989; lenstra et al., 1989) . in particular cases, a very low number of critical amino acid changes is sufficient to greatly alter the antigenicity (cavanagh et al., 1992) , although this view does not apply in all cases (chen et al., 2015) . in conclusion, the recombinant ibvs exhibited differences in antigenicity, but exchanging only the three hvrs between the two parental strains did not change the serotype. vaccination is considered the most cost-effective approach for controlling ibv infection. however, current commercial vaccines have been challenged by the emergence of new ibv serotypes. recently, reverse genetic techniques have been employed to modify ibv vaccine candidates. qx-like strains (lx4-type) emerged in china and spread to asia (mahmood et al., 2011) , russia (bochkov et al., 2006) , and europe (beato et al., 2005; worthington et al., 2008) . in this study, using ck/ ch/ldl/091022 as a representative of epidemic qx-like strains, we modified the genome of the beaudette strain to construct seven recombinant ibvs. we determined the effects of the hvrs and explored the possibility of developing a vaccine candidate that could proliferate in cells and provide protection against the prevalent field strains. the introduction of a few heterogenous peptides rather than the whole s protein may facilitate the development of multiepitope peptide vaccines to protect against a wide range of ibv serotypes (yang et al., 2009 ). in the present study, the virus cross-neutralization and vaccination challenge tests indicated that only swapping the hvrs did not provide sufficient cross-protection, but further exploration of the recombinant ibvs may provide insights into novel vaccine candidates. in conclusion, we manipulated the genome of the ibv beaudette strain using a reverse genetics system to construct seven recombinant strains by replacing the hvrs in the beaudette strain with the corresponding fragments from a qx-like nephropathogenic isolate ck/ch/ ldl/091022 from china. the results showed that the heterogenous hvrs could weaken the capacity for adsorption 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recombination recombinant newcastle disease virus expressing the infectious bronchitis virus s1 gene protects chickens against newcastle disease virus and infectious bronchitis virus challenge this work was supported by grants from the china agriculture research systerm (no. cars-41-k18), the provincial supported science foundation of heilongjiang province for the national key technology r&d program (gx16b003) and national "twelfth five-year" plan for science & technology support (2015bad12b03). the authors declare that they have no competing interests. all authors declare that they have no conflict of interest. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2018.04.013. key: cord-255141-55ho9av4 authors: abolnik, celia title: genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus date: 2015-04-03 journal: infect genet evol doi: 10.1016/j.meegid.2015.03.033 sha: doc_id: 255141 cord_uid: 55ho9av4 infectious bronchitis virus (ibv) is a gammacoronavirus that causes a highly contagious respiratory disease in chickens. a qx-like strain was analysed by high-throughput illumina sequencing and genetic variation across the entire viral genome was explored at the sub-consensus level by single nucleotide polymorphism (snp) analysis. thirteen open reading frames (orfs) in the order 5′-utr-1a-1ab-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′utr were predicted. the relative frequencies of missense: silent snps were calculated to obtain a comparative measure of variability in specific genes. the most variable orfs in descending order were e, 3b, 5′utr, n, 1a, s, 1ab, m, 4c, 5a, 6b. the e and 3b protein products play key roles in coronavirus virulence, and rna folding demonstrated that the mutations in the 5′utr did not alter the predicted secondary structure. the frequency of snps in the spike (s) protein orf of 0.67% was below the genomic average of 0.76%. only three snps were identified in the s1 subunit, none of which were located in hypervariable region (hvr) 1 or hvr2. the s2 subunit was considerably more variable containing 87% of the polymorphisms detected across the entire s protein. the s2 subunit also contained a previously unreported multi-a insertion site and a stretch of four consecutive mutated amino acids, which mapped to the stalk region of the spike protein. template-based protein structure modelling produced the first theoretical model of the ibv spike monomer. given the lack of diversity observed at the sub-consensus level, the tenet that the hvrs in the s1 subunit are very tolerant of amino acid changes produced by genetic drift is questioned. coronaviruses (family coronaviridae, order nidovirales) are enveloped, single-stranded rna viruses with large genome sizes of $25-30 kb. the family is split into four genera: alpha-, beta, gamma and deltacoronaviruses, each containing pathogens of veterinary or human importance. a current evolutionary model postulates that bats are the ancestral source of alpha-and betacoronaviruses and birds the source of gamma-and deltacoronaviruses (woo et al., 2012) . the alphacoronaviruses infect swine, cats, dogs and humans. betacoronaviruses infect diverse mammalian species including bats, humans, rodents and ungulates. the sars coronavirus (sars-cov), which verged on a pandemic in 2003 with 8273 cases in humans and 755 deaths is a betacoronavirus. another member of this genus, the recently-discovered middle east respiratory syndrome (mers) coronavirus (mers-cov) has claimed 88 human lives from 212 cases since april 2012, and dromedary camels are the suspected reservoir (briese et al., 2014) . genus gammacoronavirinae includes strains infecting birds and whales (woo et al., 2012; mcbride et al., 2014; borucki et al., 2013) and deltacoronaviruses have been described in birds, swine and cats (woo et al., 2012) . the diversity of hosts and genomic features amongst covs have been attributed to their unique mechanism of viral recombination, a high frequency of recombination, and an inherently high mutation rate (lai and cavanagh, 1997) . infectious bronchitis virus (ibv) is a gammacoronavirus which causes a highly contagious respiratory disease of economic importance in chickens (cook et al., 2012) . ibv primarily replicates in the respiratory tract but also, depending on the strain, in epithelial cells of the gut, kidney and oviduct. clinical signs of respiratory distress, interstitial nephritis and reduced egg production are common, and the disease has a global distribution (cavanagh, 2007; cook et al., 2012) . the ibv genome encodes at least ten open reading frames (orfs) organised as follows: 5 0 utr-1a-1ab-s-3a-3b-e-m-5a-5b-n-3a-3 0 utr. six mrnas (mrna 1-6) are associated with production of progeny virus. four structural proteins including the spike glycoprotein (s), small membrane protein (e), membrane glycoprotein (m), and nucleocapsid protein (n) are encoded by mrnas 2, 3, 4 and 6, respectively (casais et al., 2005; hodgson et al., 2006) . messenger rna (mrna) 1 consists of orf1a and orf1b, encoding two large polyproteins via a ribosomal frameshift mechanism (inglis et al., 1990) . during or after synthesis, these polyproteins are cleaved into 15 non-structural proteins (nsp2-16) which are associated with rna replication and transcription. the s glycoprotein is post-translationally cleaved at a protease cleavage recognition motif into the amino-terminal s1 subunit (92 kda) and the carboxyl-terminal s2 subunit (84 kda) by the host serine protease furin (de haan et al., 2004) . the multimeric s glycoprotein extends from the viral membrane, and the globular s1 subunit is anchored to the viral membrane by the s2 subunit via non-covalent bonds. proteins 3a and 3b, and 5a and 5b are encoded by mrna 3 and mrna 5, respectively and are not essential to viral replication (casais et al., 2005; hodgson et al., 2006) . a confounding feature of ibv infection is the lack of correlation between antibodies and protection, and discrepancies between in vitro strain differentiation by virus neutralization (vn) tests and in vivo cross-protection results. taken with the ability for high viral shedding in the presence of high titres of circulating antibodies, the involvement of other immune mechanisms are evident, and the roles of cell-mediated immunity and interferon have been experimentally demonstrated (timms et al., 1980; collisson et al., 2000; pei et al., 2001; cook et al., 2012) . dozens of ibv serotypes that are poorly cross-protective have been discovered and studied by vn tests and molecular characterisation of the s protein gene. most of these serotypes differ from each other by 20-25% at amino acid level in s1, but may differ by up to 50%. s1 contains the epitopes involved in the induction of neutralizing, serotype-specific and hemagglutinaton inhibiting antibodies (cavanagh, 2007; darbyshire et al., 1979; farsang et al., 2002; ignjatovic and mcwaters, 1991; meulemans et al., 2001; gelb et al., 1997) . most of the strain differences in s1 occur in three hypervariable regions (hvrs) located between the amino acid residues 56-69 (hvr1), 117-131 (hvr2) and 274-387 (hvr3) (moore et al., 1997; wang and huang, 2000) . monoclonal antibody analysis mapped the locations of many of the amino acids involved in the formation of vn epitopes to within the first and third quarters of the linear s1 polypeptide (de wit, 2000; kant et al., 1992; koch et al., 1990) , which is where closely-related stains (>95% amino acid identity) also differ (bijlenga et al., 2004; farsang et al., 2002) . cavanagh (2007) proposed that these parts of the s1 subunit are very tolerant of amino acid changes, conferring a selective advantage. recently, the receptor-binding domain of the ibv m41 strain was mapped to residues 16-69 of the n terminus of s1, which overlaps with hvr1 (promkuntod et al., 2014) . the s2 subunit, which drives virus-cell fusion, is more conserved between serotypes than s1, varying by only 10-15% at the amino acid level (bosch et al., 2005; cavanagh, 2005) . although it was initially thought that s2 played little or no role in the induction of a host immune response, it has since been shown that an immunodominant region located in the n-terminal half of the s2 subunit can induce neutralizing, but not serotype-specific, antibodies demonstrated by the ability of this subunit to confer broad protection against challenge with an unrelated serotype (kusters et al., 1989; toro et al., 2014) . ibvs are continuously evolving as a result of (a) frequent point mutations and (b) genomic recombination events (cavanagh et al., 1992; kottier et al., 1995; jackwood et al., 2005; zhao et al., 2013; kuo et al., 2013; liu et al., 2014) . multiple studies on ibv diversity have focused on inter-serotypic and inter-strain variation, and a few have focused on sub-populations within the s1 subunit in vaccine strains (gallardo et al., 2012; ndegwa et al., 2014) . the present study aimed to explore genetic variation across the entire viral genome at the sub-consensus level. it was anticipated, based on the published literature, that certain regions, and the s1 subunit hvrs in particular, would display significant sub-genomic variation. this study focused on a qx-like strain, a serotype currently causing significant poultry health problems across europe, asia, south america and south africa. 2.1. origin and isolation of qx-like strain ck/za/3665/11 twenty-eight-day old chickens in a commercial broiler operation presented with acute lethargy, reduced feed consumption and mortality. tracheitis and swollen kidneys were noted on post mortem, as well as a secondary escherichia coli infection. the worst affected houses had mortality rates of 19.8%, 11.9% and 10.2%. ibv was isolated in specific pathogen free (spf) embryonated chicken eggs (ece) as described in knoetze et al. (2014) . after an initial two passages in ece, the virus was passaged twice further at the university of pretoria. rna was extracted from allantoic fluid using trizol ò reagent (ambion, life technologies, carlsbad, usa) according to the manufacturer's protocol. the genome was transcribed to cdna and amplified using a transplex ò whole transcriptome amplification kit (sigma-aldrich, steinheim, germany). illumina miseq sequencing on the cdna library was performed at the arc-biotechnology platform, onderstepoort, pretoria. illumina results were analysed using the clc genomics workbench v 5.1.5. paired-end reads were trimmed and a preliminary de novo assembly was performed. the larger segments were analysed by blast to identify the closest genomic reference strain (ita/90254/2005, caz86699) . this strain was retrieved and used as a scaffold for assembly-to-reference, generating a consensus sequence for 3665/11. trimmed paired-end reads were also mapped against other ibv serotype genomes, subsequently confirming that strain 3665/11 was a pure culture of a qx-like ibv. the genome was deposited in genbank under the accession number kp662631. rna folding was predicted using the clc genomics workbench v 5.1.5. genetic recombination in the consensus sequence was evaluated using the recombination detection program rdp v4.31. coding sequence and orf prediction was carried out in vigor (wang et al., 2010) . trimmed paired-end reads were re-mapped against the 3665/11 consensus sequence for snp detection. a snp detection table generated in the clc genomics workbench was manually edited to eliminate all snps with a frequency of <5%. this conservative cutoff was selected to eliminate any nonspecific pcr errors introduced during preparation of the transcriptome library or deep sequencing, and excluded most of the point insertions producing gaps and frameshift mutations across the genome. nucleotide substitutions in coding regions were manually inspected for changes to the consensus amino acid (table 1 , supplementary data). motifs were predicted using the elm eukaryotic linear motif resource for functional sites in proteins (dinkel et al., 2014) . protein structures for s1 and s2 were predicted in raptorx, a structure prediction server that predicts three dimensional (3d) structures for protein sequences without close homologs in the protein data bank (pdb) (kallberg et al., 2012) . s1 and s2 3d structures were annotated and superposed in ccp4mg v2.9.0 using the secondary structure (ssm) superposition method. this method superimposes pairs of structures by: (1) finding the secondary structure elements (sses) and representing them as one simple vector spanning the length of the sse; (2) finding equivalent sses in the two structures using graph-theory matching by geometric criteria of distances and angles between the vectors; (3) superimposing vectors representing equivalent sses; (4) finding the most likely equivalent residues in the superposed sses; (5) superimposing ca atoms of equivalent residues; and (6) iterating the last two steps. the genome sequence of qx-like strain ck/za/3665/11 was assembled from 74, 578 ibv-specific paired-end reads of 144 bp each. the genome was 27, 388 nt in length with the 5 0 utr incomplete by $139 nt. thirteen orfs were predicted by vigor in the order fig. 1 ). this genome organisation including 4b, 4c and 6b was similar to that of turkey coronavirus (tcov; cao et al., 2008) , and the orfs 4b, 4c and 6b were also predicted in australian ibv strains (hewson et al., 2011) . when the sequences for a qx-like sequence (jq088078) and arkdpi (eu418976) were analysed using vigor, a similar genome arrangement was detected. mass41 (ay851295) did not however contain the predicted 4b, 4c and 6b orfs (data not shown). orf 4b was 94 amino acids (aa) in length and no smart domains were predicted, whereas orf 4c was 56 aa in length and a low complexity region was identified. the 6b orf encoded a 74 aa protein with a signal peptide predicted from residues 1 to 24 and two transmembrane domains from residues 2 to 25 and 35 to 57. no recombination was detected across the genome of qx-like strain ck/za/3665/11. a gap was present between nucleotides 1666 and 1667 (table 1 , supplementary data) ($aa 370 in the 1a orf). although the gap was present in the majority (74.6%) of reads, the sequence for strain 3665/11 deposited in genbank contains the minority adenine residue because the gap introduced a frame shift, splitting orf 1a into two. it may be a legitimate mutation, but until further transcriptional analyses are conducted, the orf 1a gene has been reported intact here. two hundred and eight snps across the ibv qx-like genome were evaluated at the selected cut-off value. in table 1 the consensus reference is juxtaposed with the allele variations, the relative frequencies of these point mutations, the actual number of counts and coverage at that position, the corresponding orf or region and the mutational effect. coverage ranged from 4-fold (position 11,540) up to 4587 fold (position 26,637). the relative frequencies of missense: silent snps in relation to orf length were calculated in order to obtain a comparative measure of variability in specific genes (table 2) . results for the structural genes and polymerase are illustrated in fig. 2 , and the results for the non-structural protein orfs and non-coding regions, which were much shorter in length, are presented in fig. 3 . overall the most variable orfs in terms of total snps, in descending order, were: e, 3b, 5 0 utr, n, 1a, s, 1ab, m, 4c, 5a, 6b (no snps were detected at the 5% cut-off in the 3a and 3 0 utr regions). the most variable, as assessed by snps leading to missense mutations, in descending order, were: 3b, e, 5 0 utr, 1a, n, m, 5a, 1ab, s, 4b, 3a/ 4c/6b. these mutations presumably did not affect the tertiary protein structure and might be advantageous to the virus. the orfs under the strongest positive selection pressure as indicated by the proportion of synonymous mutations, were, in descending order, 4c, 1ab, n, s, e, 3a/3b/m/4b/5a/6b. the e protein orf had significantly more missense mutations on average, at a frequency of 1.5% of the orf, which is more than threefold higher than the average value (0.55) for the 1a, 1ab, s, m and n genes. the e protein gene was the most variable at the sub-consensus level, with 5 missense mutations and only one silent mutation across its 333 bp orf. despite its small size, the cov e protein drastically influences the replication of covs and their pathogenicity. in the sars-cov, it was experimentally demonstrated that the e protein is not essential for genome replication or subgenomic mrna synthesis, but it does affect morphogenesis, budding, assembly, intracellular trafficking and virulence. in fact, in sars-cov the e protein is the main antagonist associated with induction of inflammation in the lung, which causes the acute respiratory distress syndrome from which the virus derives its name (dediego et al., 2014) . no studies have been published for the ibv e protein, but the high variability demonstrated here suggests that it may be an important virulence factor in poultry, and that a higher mutation rate possibly provides an evolutionary advantage in overcoming host cellular immune responses. although the n protein gene contained one of the highest overall frequencies of snps (1.06%), the n gene is evidently under greater selective pressure, since 38.9% of these mutations (0.41% as a total of the gene) were silent. the coronavirus n protein is multifunctional, playing vital roles in viral assembly and formation of the complete virion and is required for optimal viral replication. additionally, the cov n protein is implicated in cell cycle regulation and host translational shutoff, displays chaperone activity, activates host signal transduction and aids viral pathogenesis through the antagonism of interferon induction (reviewed by mcbride et al., 2014) . given its fundamental roles in rna binding, formation of the ribonucleoprotein complex and in the virion, it is not surprising that this structural protein is the most conserved, as evidenced by its gene having the highest ratio of silent mutations of all the genes analysed. the importance of maintaining the fig. 1 . genome organisation of qx-like ibv strain 3665/11. sequence integrity in the n protein in ibv was demonstrated by kuo et al. (2013) , who reported that two residues within the nterminal domain of a taiwanese ibv strain were positively selected, and that mutation of either of these significantly reduced the affinity of the n protein for the viral transcriptional regulatory sequence. the glycosylated amino terminus of the m protein lies on the outside of the virion and m spans the membrane structure three times (collisson et al., 2000) . all four snps in the m gene resulted in missense mutations, two of which were located in the predicted transmembrane region. the m protein plays an important role in cov virion formation. ibv m protein co-expressed with s assembled into virus-like particles confirming its major role in virion formation, but cov m proteins also interact with other proteins and perform other roles in the infected cell. for example, m together with the accessory proteins 4a, 4b and 5 were all found to prevent the synthesis of ifn-b through the inhibition of interferon promotor activation and irf-3 function, thus influencing disease outcome (yang et al., 2013) . coronavirus accessory proteins are generally dispensable for virus replication, but they play vital roles in virulence and pathogenesis by affecting host innate immune responses, encoding pro-or anti-apoptotic activities, or by effecting other signalling pathways that influence disease outcomes (susan & julian, 2011) . ibv was demonstrated to induce a considerable activation of the type i ifn response, but it was delayed with respect to the peak of viral replication and accumulation of viral dsrna (kint et al., 2014) . ibv accessory proteins 3a and 3b play a role in the modulation of this delayed ifn response, by regulating interferon production at both the transcriptional and translational levels. interestingly, ibv proteins 3a and 3b seem to have opposing effects on ifn production in infected cells: 3a seems to promote ifn production, and 3b is involved in limiting ifn production, antagonising each other to tightly regulate ifn production (kint et al., 2014) . field isolates lacking 3a and 3b displayed reduced virulence in vitro and in vivo (mardani et al., 2008) . orf 3a in strain 3665/11 lacked snps, but orf 3b in had the highest frequency of snps relative to its size (n = 3; 1.56%). orf 4b is present in many international ibv strains (hewson et al., 2011; bentley et al., 2013) but is rarely mentioned in the literature since a canonical transcription regulatory sequence (trs-b) could not be identified upstream of the encoding rna. however, bentley et al. (2013) demonstrated that ibv was capable of producing subgenomic mrnas from noncanonical trs-bs via a template-switching mechanism with trs-l, the conserved trs in the leader sequence in the 5 0 utr, which may expand the gammacoronavirus repertoire of proteins. they specifically demonstrated the transcription of the 4b orf by this mechanism. although no studies have been performed determining orf 4b 0 s functional role in the pathogenesis of ibv, the homolog in mers-cov is a potent interferon antagonist (yang et al., 2013) . a single snp causing a missense mutation was present in 11.3% of the sub-consensus population of the 4b orf in this study. the single mutation in orf 4c was silent, and the predicted protein contained a low complexity region. low complexity regions are regions of protein sequences with biased amino acid composition, and may be involved in flexible binding associated with specific functions (coletta et al., 2010) . orf 6b, a 73 aa protein with a signal peptide and two transmembrane domains, was identified in the genome of strain 3665/ 11, and orf 6b was also reported in tcov and australian ibv strains (cao et al., 2008; hewson et al., 2011) . the homolog in sars-cov is 63 aa in length and was identified as an endoplasmic reticulum/golgi membrane-localised protein that induces apoptosis. apoptosis may play an important role in promoting cov dissemination in vivo, minimising inflammation and aiding evasion of the host's defence mechanisms (ye et al., 2010) . protein 6 from sars-cov accelerated the replication of murine cov, increasing the virulence of the original attenuated virus (tangudu et al., 2007) . presumably, this accessory protein plays a similar role in ibv pathogenesis, although this remains to be determined experimentally. the 3 0 utrs of cov genomes contain conserved cis-acting sequence and structural elements that play essential roles in rna synthesis, gene expression and virion assembly, and each sub-genomic rna contains a 5 0 leader segment that is identical to this 3 0 utr region of the genome (goebel et al., 2004; sola et al., 2011) . no snps were detected in the 3 0 utr in the sub-consensus sequences of strain 3665/11, which is consistent with the vital regulatory role that this region plays. conversely, the partial 5 0 utr sequence of strain 3665/11 was highly variable. the un-sequenced 139 nucleotides from the 5 0 end of the genome were extrapolated from the most similar genomic sequence, that strain ita/90254/2005, and the secondary rna structure of the 5 0 utr for 3665/11 was predictively folded (fig. 5) . the snps were then systematically substituted into the consensus sequence and rna folding repeated. delta g values for the predicted rna secondary structures in fig. 4(a) -(h) varied from à182.9 kcal/mol to à185.5 kcal/mol. apart from the 148 c to g mutation (fig. 4(c) ), effects on rna secondary structure were minor and the structures in fig. 4(b) and (d)-(h) were similar. to assess the effect of combining mutations, an rna containing 148 t(u), 164 t(u), 169 g and 170 c was folded, and this resulted in a similar stem-loop structure to those in fig. 4(a), (b) and (d)-(h) (data not shown). apart from the mutation 148 c to g, the snps had little effect on the secondary rna structure in the 5 0 utr. twenty-three snps were identified in the 3438 bp spike protein orf; 15 of these resulted in missense mutations at the amino acid level, and 8 were silent mutations. the frequency of total snps in the s protein orf was below average, at 0.67%, compared to the genome average of 0.76%. it was anticipated that the majority of mutations in the s orf would be in the s1 gene, particularly in the hvrs, but, surprisingly, this was not the case. only three of these snps (two missense and one silent) were found in the s1 gene, and all three were located in the cooh-terminal half of the s1 protein (fig. 5) . only one mutation, a missense mutation, 6 . predicted structure of the spike protein monomer of qx-like ibv strain 3665/11. missense mutations in s1 (blue) and s2 (yellow) are indicated as coloured side chains. mapped to hvr3. no snps were detected in hvr1 or hvr2. the s2 subunit was considerably more variable, containing 87% of the polymorphisms detected across the entire s protein. two other notable features of s2 were detected: the first was a multi-a insertion site located between nucleotides 22,794 and 22,795 in the genome. the polymorphism involved the insertion of either one or two adenine nucleotides, possibly via a mechanism of polymerase stuttering. the second region of interest was located in close proximity, just downstream of the multi-a insertion site: a stretch of three consecutive mutated amino acids, namely 889 c ? w, 890 g ? d, 891 s ? c followed by silent mutation 892 g (fig. 5) . template-based protein structure modelling was used to predict the secondary structure of the ibv spike monomer, based on the available crystal structure for the mers-cov s1 and s2 subunits (fig. 6 ). s1 and s2 were modelled separately in raptor x and then superposed. the ibv s1 structure was arranged as two beta barrels and s2 formed packed a-helices. the s2 protein was not complete and the transmembrane domain was not represented since there were no sufficiently similar structures on which to model this region, but this is the first model of the spike protein monomer for ibv. hvr1 and the putative receptor binding domain maps to the apical beta barrel (fig. 7(a) ) and hvr2 is located on the flat plane on the base of the apical beta barrel and the peptide connecting it to the basal beta barrel (fig. 7(b) ). hvr3 maps to a region in the basal beta barrel of s1 that was predicted to contact or interact with s2 ( fig. 7(c) ). the locations of the missense mutations detected by snp analysis in the s1 and s2 subunits are indicated in fig. 6 . many of these snps mapped to codons encoding amino acids on the surface of the predicted structure, but two regions were notable. firstly, the highly variable region in s2 spanning amino acids 889-901 was exposed on the s2 stalk, although folding of the remainder of the cooh domain may have influenced this conformation. secondly, 668 ile was exposed on a projection at the top of the monomer. this residue precedes the second furin cleavage site in the s2 subunit with the sequence 667 pisssgr/s 674 . the cleavage of the s1/s2 furin motif ( 517 rrrr/s 521 in strain 3665/11) was found to be non-essential for attachment of ibv to the cell. rather, it promotes infectivity within the cell. in studies with the beaudette ibv strain, the second furin cleavage site in the s2 subunit was required for furin-dependent entry and syncytium formation, and the current hypothesis is that interplay between the s1 and s2 subunits determines virus attachment to specific receptors, determining tissue tropism of the virus (promkuntod et al., 2013) . the exact biological roles of these areas in s2 that are prone to mutation remain to be experimentally determined. archaeological remains of domestic chickens in northeast china and the indus valley date back $8000 years (west & zhou, 1988) . 7 . the predicted locations of hvr1 (7a), hvr2 (7b) and hvr3 (7c) of qx-like ibv strain 3665/11, indicated as coloured side-chains on the s1 subunit. the cov group has been estimated to have arisen around 8100 bc, and the gammacoronaviruses diverged from the cov group around 2800 bc (woo et al., 2012) . covs have probably been co-evolving with their gallinaceous hosts for several thousand years. indeed, cook and co-authors (2012) state that ''ibv is found everywhere that commercial chickens are kept''. the implication is that although ibv was only discovered some 80 years ago, the variety of serotypes we now observe are the results of hundreds if not thousands of years of genetic drift and recombination, accelerated by modern poultry farming practises where chickens are kept in high densities, and inter-regional trade in poultry and other avian species. studies on antigenic diversity of ibvs are heavily biased towards studies of the s1 gene, and the hvrs in particular (cavanagh, 2007; ducatez et al., 2009; kant et al., 1992; mork et al., 2014) . many of these studies cite frequent point mutations in the s1 gene, but this was not the finding of the present study. the discovery of a novel 3 0 -to-5 0 exoribonuclease activity in cov nsp14, which regulates replication fidelity and diversity in coronaviruses (denison et al., 2011) , lends weight to the theory that genetic drift is not primarily responsible for the degree of variation and serotypes we observe in poultry nowadays. instead, generation of variation by recombination is likely the main mechanism of serotypic diversity. the high frequency of rna recombination in coronaviruses is likely caused by their unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (jeong et al., 1996) . sequencing of many field strains has provided convincing evidence that many, possibly all, ibv strains are recombinants between different field strains (cavanagh, 2007; kuo et al., 2013; liu et al., 2013; hewson et al., 2011) , driving ibv evolution at a population level. recombination of distinct ibv strains has been experimentally demonstrated in vitro, in ovo and in vivo (kottier et al., 1995; wang et al., 1997) . the s1 subunit hvr1 contains the ibv receptor-binding site. therefore despite the sequence variability in this region (which includes insertions and deletions), diverse strains must retain this critical biological function. all three hvrs may represent ancient artefacts of recombination, which have been perpetuated because they retain receptor-binding properties, with minimal permissive amino acid changes. this theory contrasts the tenet that the hvrs in the s1 subunit are very tolerant of amino acid changes produced by genetic drift, thereby conferring a selective advantage (cavanagh, 2007; de wit, 2000; kant et al., 1992; koch et al., 1990) . whereas s1 fulfils a primary role in receptor binding (promkuntod et al., 2014) , a broader role of s2 in antigenicity and attachment to receptors is emerging. chickens primed with a recombinant-expressed s2 subunit of a virulent arkdpi strain and boosted with a live mass-type vaccine were protected against challenge with live virulent arkdpi virus . although s2 subunits most likely do not contain an additional independent receptor-binding site, s2 in association with s1 forms part of a specific ectodomain which is critical to the binding of the virus to chicken tissues, which implies that both s1 and s2 contain determinants important to viral host range (promkuntod et al., 2013) . the results of the present study demonstrate that s2 is more predisposed to mutations than s1, providing an adaptive advantage and at least one other study has reported higher variability in s2 compared to s1 (mo et al., 2012) . ibv has not been as extensively studied as other covs, and little progress has been made in effectively controlling or eradicating the disease in poultry. experimental and field studies provide substantial evidence that use of a homologous ibv vaccine is best, but sometimes, intriguingly, protection can be offered by an unrelated vaccine, or by the use of two heterologous vaccines (jones, 2010) . genotyping and phylogenetic analysis of ibv are typically focused on the s1 subunit sequence, and liu et al. (2014) caution against drawing conclusions based on a single gene sequence, particularly a partial gene sequence. the roles of the ibv e and accessory proteins and their roles in the pathogenesis of ibv have been completely overlooked, even when the roles of the homologs in other covs have been proven significant. accessory proteins of ibv and other covs may also offer a new generation of vaccine targets: the use of codon-deoptimization of non-structural virulence genes in influenza a virus and respiratory syncytial virus resulted in genetically stable viruses that retained immunogenicity but were attenuated (nogales et al., 2014; meng et al., 2014) . evidently virulence and immunogenicity in ibv is a multi-genic trait, and future studies must aim to pursue a better understanding and exploitation of the roles of various viral proteins in the host, if any advances are to be made in controlling the disease in poultry. identification of a noncanonically transcribed subgenomic mrna of 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gene sequences adrian knoetze and rainbow veterinary laboratory are thanked for providing strain 3665/11 for this study. funding was provided by the poultry section, department of production animal studies. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.03. 033. key: cord-002407-25cawzi0 authors: nogales, aitor; martínez-sobrido, luis title: reverse genetics approaches for the development of influenza vaccines date: 2016-12-22 journal: int j mol sci doi: 10.3390/ijms18010020 sha: doc_id: 2407 cord_uid: 25cawzi0 influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. however, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. the segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. with the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary dna copies of the viral genome by transfection of susceptible cells. these reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. in addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. in this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. influenza a (iav) and b (ibv) viruses belong to the orthomyxoviridae family of enveloped viruses [1] . iav is able to infect several species and mostly exists in the wild aquatic fowl reservoir [2] [3] [4] . on the other hand, ibv is mainly restricted and adapted to humans, although sporadic infections of seals have been documented [5, 6] . iav and ibv genomes contain eight negative sense, single-stranded viral (v)rna segments [1] (figure 1 ). iav and ibv vrnas contain a central coding region that is flanked at both terminal ends by non-coding regions (ncrs), which serve as promoters to initiate genome replication and gene transcription by the viral polymerase complex [1, 7] . influenza vrnas in the virion are found as viral ribonucleoprotein (vrnp) complexes encapsidated by the viral nucleoprotein (np) and a single copy of the viral polymerase complex. influenza virus-encoded rna-dependent rna polymerase (rdrp) [8] is a trimeric complex consisting of the polymerase basic 1 (pb1) and 2 (pb2) and acidic (pa) proteins and, together with the viral np, are the minimal components involved in viral replication and transcription [9] . iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and noncoding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and non-coding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from sialyloligosaccharides and facilitates the release of newly produced virions from infected cells [17, 18] . iavs are classified on the basis of the antigenic properties of ha and na into 18 ha (h1-h18) and 11 na (n1-n11) subtypes [1, 19, 20] . however, only iav h1n1 and h3n2 subtypes are currently circulating in humans. on the other hand, two major lineages of ibv are circulating in humans, the victoria-like and yamagata-like subtypes that are divergent from the ancestral ibv (b/lee/1940) and have been co-circulating in humans since the 1980s [5, 21, 22] . these two subtypes are the predominant circulating virus strains about once every three years [23] [24] [25] . infection with influenza viruses begins when the viral ha protein binds to its cellular receptor, a sialylated glycoprotein containing α-2,3 or α-2,6 linkages [16] . upon the binding to the receptor, the uptake of the virus by receptor-mediated endocytosis is initiated, and the cell membrane engulfs the virus particles in an endosome. after endocytosis and upon acidification of the endosome, influenza viral ha undergoes a conformational change responsible for the fusion of the viral and the endosome membrane [16] . then, the m2 (iav) or bm2 (ibv) ion channel proteins promote the release of the vrnp complexes from the virion core into the cytoplasm of infected cells [14, 26] . the vrnps are translocated from the cytoplasm to the nucleus of infected cells to initiate viral genome replication and gene transcription [27] . the nuclear export (nep) and matrix 1 (m1) proteins are responsible for the nuclear export of newly-synthesized vrnps into the cytoplasm of infected cells. notably, a single copy of each of the eight vrnas is packaged into new virions [28, 29] . selective package of influenza vrnas into nascent virions is mediated by rna-rna interactions of vrna packaging signals present at the terminal ends of each of the vrna segments [30] . finally, the receptor-destroying enzymatic activity of na is responsible for the release of newly-synthesized viral particles from the surface of infected cells [17] . among the first battles between the host and the virus, cellular type i interferon (ifn-i) plays an important role in controlling viral infection [31] . therefore, viruses have developed multiple strategies to hijack the host cellular immune response. influenza vrna segment 8, or the nonstructural (ns) gene, encodes two distinct proteins through a direct or alternative splicing mechanism [1] . influenza virus segment 8 produces the nonstructural protein 1 (ns1) as a primary transcript, whereas nep is produced by alternative splicing of the ns mrna [32] . ns1 has multiple functions during the replication cycle of influenza virus, but most notably inhibits induction of ifn-i response and innate immune activation [31] . influenza viruses pose a threat to human health and are responsible for global epidemics every year [33] [34] [35] [36] [37] . in fact, influenza virus is one of the most significant causes of morbidity and mortality yearly, leading to a significant economic impact [38] . despite the implementation of effective and comprehensive vaccination programs, the world health organization (who) estimates that seasonal influenza virus infections results in about one billion infections, 3-5 million cases of severe disease and between 300,000 and 500,000 deaths around the world annually [39] . moreover, just in the united states (u.s.), influenza viral infections result in an average of 87 billion dollars of cost due to prophylactic, therapeutic and hospitalization costs, as well as missed school or work days [38, [40] [41] [42] . in addition to seasonal influenza, iav can cause sporadic pandemics of great consequences when novel viruses are introduced into humans [43] . the mechanisms responsible for the emergence of seasonal and pandemic iavs are antigenic drift and antigenic shift, respectively [12, [44] [45] [46] . in the case of seasonal influenza, mutations in the viral genome result in the selection of antigenic variants with changes in viral tropism, increased levels of viral fitness or in the ability to escape neutralizing antibody (nab) responses induced upon previous natural infections or vaccinations [44, 45] . moreover, antigenic drift can lead to variant viruses resistant to antivirals [47] . on the contrary, antigenic shift results from co-infection of a host cell or organism with two or more iavs where vrnas in viral progenies are reassorted [12, 46] . by obtaining viral segment constellations that confer influenza virus optimal replication, transmissibility and immunologic escape, reassortant iavs can lead to a pandemic in immunologically-naive populations. iavs and ibvs can reassort intratypically between subtypes or lineages, but intertypic reassortment, or genetic swapping of segments between iav and ibv, has not been reported [21, 48, 49] . the absence of intertypic reassortment is mediated by the lack of compatible packaging signals between iav and ibv [12] . in the last century, three iav pandemics have occurred: the h1n1 spanish flu of 1918, the h2n2 asian flu of 1957 and the h3n2 hong kong flu of 1968 [44, 50] . of these three, the 1918 h1n1 spanish flu was particularly fatal and responsible for approximately 50 million deaths around the world [51] . although not classified as true pandemics, three epidemics of influenza h1n1 viruses in 1947, 1976 and 1977 were feared to have pandemic potential. in 2009, a swine-origin h1n1 iav was responsible for the first influenza pandemic of the 21st century and infected, in less than one year, more than 600,000 individuals around the world [52, 53] . although influenza virus infections are primarily spread by person-to-person transmission via aerosolized droplets, infection with new avian-or swine-origin iav can take place and may have great risk for pandemic potential if the virus acquires the ability to be transmitted between humans [54, 55] . ibvs usually contribute less to seasonal epidemics than iavs of the h3n2 subtype, but they contribute more than type a h1n1 influenza strains and are the predominant circulating virus strains once every three years [23] [24] [25] . moreover, during the last decade, ibv has been the cause of several acute respiratory illness outbreaks [56] [57] [58] [59] [60] . to date, no influenza pandemics have been linked to ibvs. public health concerns posed by influenza virus infections are aggravated by the ability of influenza viruses to efficiently transmit and the limited therapeutic options to treat viral infections [37] . thus, vaccination remains our best medical intervention to protect humans against seasonal influenza virus. however, the efficiency of current influenza vaccines is suboptimal [61] . the segmented genome of influenza viruses provides an evolutionary advantage of reassortment, or the exchange of viral genome segments between different viral strains within the same type. in addition to this exchange of genome material (or antigenic shift), influenza viruses can introduce mutations in their genomes (or antigenic drift), leading to viral mutants with resistance against current antivirals or nabs [62] [63] [64] . because of the antigenic drift, influenza vaccines need to be reformulated yearly to ensure that the ha and na present in the vaccine match those present in circulating seasonal viruses. to date, three types of influenza virus vaccines are approved by the food and drug administration (fda) for human use: recombinant viral ha, inactivated virus and live-attenuated viruses [65] [66] [67] [68] [69] . regardless of the type of vaccine, seasonal influenza vaccines contain antigens from the three circulating influenza virus strains: iav subtypes h1n1 and h3n2; and ibv (victoria-like or yamagata-like lineage) [67, 70] . recently, to improve the efficacy of seasonal influenza vaccines, a quadrivalent influenza vaccine formulation that includes both ibv lineages components (victoria-like and yamagata-like lineage) has been approved by the fda [14] . the most common influenza vaccine is the inactivated influenza vaccine (iiv). iiv, which is administered intramuscularly, has been shown to elicit protective humoral immunity by producing nabs that target epitopes on ha [61, 71] . in contrast to iiv, the live-attenuated influenza vaccine (laiv) and its administration mimic the natural route of virus infection, which consequently having risks and benefits [72] . an advantage is that laiv elicits both more rapid and efficient innate and adaptive immune responses [65] and can provide more efficient cross-reactive t-cell-mediated protection against heterologous influenza viruses [72, 73] . although both iiv and laiv have been shown to be efficient for the treatment of influenza viral infections, there is an unmet need to increase the effectiveness of seasonal influenza vaccines. likewise, there is an urgent need to develop effective vaccines for the treatment of potential pandemic influenza viruses. although vaccination is the main method to prevent influenza infections in humans, antivirals offer an additional countermeasure against new rapidly-spreading and/or potentially pandemic influenza viruses [74] [75] [76] . therapeutic choices to control influenza infection are currently limited to two classes of fda-approved antivirals targeting either the viral m2 ion channel (amantadine, rimantadine) [77] [78] [79] or the sialidase active site of na (oseltamivir, zanamivir) [80] . the first inhibits the initial steps of the virus life cycle, while the second inhibits virus release. however, na inhibitors are the only type of antivirals approved for the prophylaxis and treatment of ibv infections [81] , although data from clinical trials have shown that oseltamivir is less effective against ibvs than against iavs [82] . importantly, mutations in the viral genome can lead to influenza antivirals being ineffective, like in the case of m2 blockers, which are no longer recommended against circulating seasonal influenza viral strains [83] . therefore, the emergence of drug-resistant influenza variants is an increasing concern for controlling influenza infections [76, 84] , and there is a significant need for the identification of novel compounds with antivirals properties. influenza viruses evade human pre-existing immunity by accumulating mutations (antigenic drift), allowing reinfection of individuals previously exposed to natural infections or vaccinated. thus, vaccine companies have to reformulate the composition of influenza vaccines yearly to ensure a good match between the viruses present in the vaccine and those seasonally circulating in humans [33, 34, 85] . the who global influenza surveillance network (gisn) [35] , which includes more than 120 national influenza centers in over 90 countries [86] , tracks the evolution and epidemiology of influenza viruses and uses the collected data for the vaccine strain selection process. these studies also help to understand virus evolution and epidemiology in different geographic areas [19] . (4) the national institute of infectious disease in tokyo, japan. basically, the degree of immunity induced by one influenza strain that is effective against another strain is mainly dependent on the antigenic difference between both viral strains [87, 88] . the influenza viral glycoprotein ha, the primary target of the protective neutralizing immune responses [88, 89] , is the focus of influenza virus surveillance and the primary component targeted by currently licensed influenza vaccines [72, 89] . influenza vaccine production is challenging because of the wait time that is needed to identify the predominant circulating virus and the production time that is needed to manufacture the vaccine. the national influenza centers perform virus isolation on certain samples obtained from patients to identify circulating viruses and to determine if the viruses grow efficiently in culture. normally, influenza viruses are isolated using madin darby canine kidney (mdck) cells instead of chicken embryonated eggs because higher isolation rates, especially for h3n2 iav strains, have been reported using mdck cells [90] [91] [92] . although most influenza viruses are isolated from mammalian cells, viruses must be generated in embryonated eggs for vaccine manufacturing due to regulations [93] . nowadays, the vaccine strains must be selected almost 7-9 months ahead of the influenza season in which they will be used. the recommendation for the strains included in the vaccine composition for the northern hemisphere is made in february to allow time for the~300 million doses of vaccine to be produced in time for vaccinating people in october/november. this allows for influenza season preparation, which typically peaks sometime between december and march [86] . on the other hand, for the southern hemisphere, recommendations are provided in september, and vaccination takes place in march/april of the following year [86] . seasonal influenza vaccines must protect against h1n1, h3n2 and b viral strains currently circulating in humans globally [34, 86] . the main goals of influenza vaccines are the protection against infection and disease caused by influenza infections and to restrict virus transmission within the population [67, 94] . nab responses, commonly assessed by measuring hemagglutination inhibition (hai) titers, are currently used as a serological marker of the efficient immunological response to the vaccine. the effectiveness of influenza vaccines is variable and usually higher in children and in healthy adults under the age of 65. in individuals above 65 years of age, lower effectiveness has been observed [61, 71, 95] . recommendations for influenza vaccination differ between countries, but since influenza vaccines do not induce long-lasting antibody protection, annual influenza vaccinations are recommended. nowadays, the most used influenza vaccines can be divided into iiv and laiv [65, 66, 72, 73, 96 ]. killed virus vaccines or iiv are generally administered intramuscularly and can be classified as whole virus vaccines or split vaccines [66, 71, 97, 98] . whole virus vaccines were the first to be developed. the influenza virus is grown in embryonated chicken eggs, subsequently purified, concentrated and chemically inactivated with formaldehyde [98] (figure 2 ). whole virus vaccines are safe and well tolerated, with an efficacy of 60%-90% in children and adults. on the other hand, the split-virus vaccine exposes all viral proteins and subviral elements upon dissociation of the virions by a nonionic detergent treatment step [97, 99] . most influenza vaccines in the u.s. and europe are egg-produced, formaldehyde-inactivated, then chemically disrupted with nonionic detergents after purification. unlike virus-based vaccines, subunit influenza vaccines consist of purified viral ha or ha/na proteins without the other viral components [68] . these subunit vaccines can be produced in eggs if the viral proteins are prepared from viruses where ha/na have been purified by removal of other viral component. in addition, the subunit vaccines can be generated using novel manufacturing technologies [98, 100] , which allows for the production of large quantities of the viral ha using baculovirus expression systems and recombinant dna technologies [100] . the iav strains that are used in vaccine manufacture are high-growth 6 + 2 reassortants containing the ha and na gene segments from the target strains in the backbone of influenza a/puerto rico/8/1934 h1n1 (pr8) or other high growth virus. influenza pr8 replicates at high titers in eggs and cells and also has a favorable safety profile in humans [98, 101] (figure 2a ). to generate the reassortant viruses, eggs are co-infected with pr8 and seasonal strains (figure 2a ). selection of appropriated seed vaccine viruses is made by amplification in the presence of pr8 ha and na nabs [98] (figure 2a) . then, selected viruses are cloned and sequenced for confirmation. the ibv vaccines are typically wild-type (wt) viruses. however, ibv reassortants are used if wt ibv growth properties are not optimal for efficient growth and vaccine production [98] . the remaining class of vaccines consists of live-attenuated influenza viruses. attenuated human laivs were developed in the 1960s by serial passage of the virus in eggs using suboptimal conditions of temperature. the resulting attenuated viruses displayed a temperature-sensitive (ts) cold-adapted (ca) attenuated (att) phenotype that grew at 25 • c, but not at temperatures found in the lower respiratory tract (>35 • c) [102] [103] [104] [105] . because this ts, ca, att phenotype restricts virus replication to the upper respiratory track, these viruses could induce local protective immunological responses [65, 95, 96, 98] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cellmediated immune response and provide a cross-reactive and longer lasting immune response [106] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cell-mediated immune response and provide a cross-reactive and longer lasting immune response [106] . the current laivs consist of the internal viral segments (pb2, pb1, pa, np, m and ns) of an attenuated master donor virus (mdv) and the ha and na viral segments from the selected seasonal virus strain ( figure 2b ). the mdvs used are a/ann arbor/6/60 (h2n2) and b/ann arbor/1/66 for iav and ibv, respectively [103, 104, [107] [108] [109] [110] [111] [112] [113] . similar to mdv iav, mdv ibv was originally derived by serial passage of the parental wt virus and isolated at successively reduced temperatures in primary chicken kidney (pck) cells [114] . the resulting ibv mdv grows efficiently at 25 • c (ca), but its growth is restricted at 37 • c (ts). the genetic changes in the mdv strains have been recently characterized. the mdv iav includes five mutations in two of the viral polymerases (pb2 n265s; and pb1 k391e, d581g and a661t) and np (d34g) [103, 104, 115] . the mdv ibv has been reported to contain two mutant amino acids in np (a114 and h410) and one in pa (m431) that are responsible for the ts, ca signature [108] . two additional residues in m1 (q159 and v183) provide the mdv ibv an attenuated (att) phenotype [108] . although laivs have been approved for clinical use, to date, their mechanism of attenuation has not been completely understood. however, the tolerability of laivs in specific populations is an important concern because of the inherent risk of immunizing with live viruses. thus, laivs are not recommended for immunocompromised patients or asthmatics [116] and are not approved for use in children under two years of age [72, 117] . moreover, laivs harboring different ha and na viral segments can be unequally safe or immunogenic from year to year as the viral ha and na are different. similar to u.s. laivs, in russia, two ts, ca mdv iavs have been obtained by using a similar temperature adaptation approach. the two russian mdvs were originated from the same parental a/leningrad/134/57 h2n2 (len/57) influenza strain [118, 119] . similarly to the u.s. mdv, the russian mdvs have been selected by growing len/57 in embryonated chicken eggs at lower (25 • c) temperatures. the mdv len/17 was obtained after 17 passages of len/57 at 25 • c and has been used in the preparation of the russian laiv for use in adults. the genetic changes in the mdvs len/17 have been identified, and these include four mutations in three viral proteins (pb2 v478l; pb1 k265n and v591i; nep m100i) [120] . the second donor strain (len/47) was obtained after a total of 47 passages and has been used for vaccinating children less than 16 years of age, although the genetic changes responsible for the further ts, ca phenotype have not been well characterized [121] . in all cases, like for iiv, the mdvs contain six internal genes to generate vaccine strains in combination with the seasonal recommended ha and na genes from the circulating strains ( figure 2b ). laivs are generated either by classical reassortment in eggs (as previously described for the iiv) ( figure 2b ) or by reverse genetics, as indicated below [98] . most of the influenza virus vaccines have been traditionally produced in eggs, but with the progress made in mammalian cell culture technologies, influenza vaccine manufacturers have invested in these novel cell culture systems for the mass production of influenza vaccines without the need of eggs. the majority of the currently licensed influenza vaccines that are made by biotechnology companies use fertilized chicken egg-based production technology, but this process has multiple drawbacks. this form of manufacturing depends on the access to embryonated eggs, relies on the ability of influenza viruses to efficiently grow in eggs and is a resource-and time-intensive process [91, [122] [123] [124] . moreover, the risk of egg contamination by avian pathogens or microbes represents a risk for the production of influenza vaccines [125, 126] . importantly, in the case of an iav pandemic, the egg supplies can be compromised. for iiv, one dose for adults contains approximately 45 µg of ha (15 µg of viral ha for each of the three antigenic h1n1, h3n2 and ibv components), meaning one egg = 7-10 dose of vaccine. laivs share similar egg-based production process steps. the laiv is recovered from infected eggs and then purified and concentrated [90] . importantly, new vaccine production approaches that do not depend on the propagation of influenza viruses in eggs (e.g., cell cultures) represent an excellent option to increase influenza vaccine production. mammalian cell cultures have been used in the biopharmaceutical industry for the production of therapeutic proteins and/or vaccines [127, 128] . in 2012, the fda approved a cell-based production process for influenza vaccines, but the manufacturing process begins with egg-grown vaccine viruses per fda regulations. influenza vaccine production using fda-approved mdck or vero (african green monkey kidney) cells may eventually and completely replace the use of eggs for the production of influenza vaccines in the future. influenza vaccine production in mammalian cell lines offers several advantages over egg-based production: it allows faster and greater production capacity, improved availability of substrate for virus growth [122, 127] and eliminates reliance on the supply of embryonated chicken eggs [129, 130] . in addition, cell cultures can be cryopreserved and scaled up in bioreactors at any time. adjuvants have been shown to enhance the immune response elicited by an antigen and could be used to improve the immunogenicity of iiv [131, 132] . the use of adjuvants could also reduce vaccine dose, stretching antigen and vaccine supplies. currently, fda-licensed adjuvants for influenza vaccine usage include aluminum salt (alum) and the squalene oil-in-water emulsion systems mf59 (wadman 2005 (novartis)) [133] and as03 (glaxosmithkline) [134] . however, most of the current iivs do not contain any type of adjuvant, but many are under investigation. genetics techniques to generate recombinant viruses were first developed for dna viruses and based on the transfection of cells with plasmids encoding the viral genome or by heterologous recombination between plasmids bearing viral sequences with the virus genome and the helper virus [135] . initial genetics approaches for dna viruses were followed by manipulations of positive-sense rna viral genomes [136, 137] . transfection of plasmid dna, or rna transcribed directly in vitro from plasmids, containing the genome of poliovirus into susceptible cells led to the generation of recombinant infectious poliovirus [138] . however, the genomes of negative-sense rna viruses, including influenza, were less suitable to molecular biology manipulations in comparison with dna or positive-sense rna viruses since their genomes are complementary to mrna in their orientation and, therefore, not infectious by themselves [1, 7] . they require the presence of vrna(s) and the viral rdrps to initiate the replication cycle of the virus [33, 44] . the advent of reverse genetics and molecular engineering has transformed the influenza field, allowing multiple questions to be answered using genetically-engineered recombinant influenza viruses [139] . such studies include mechanisms of viral genome replication and gene transcription, pathogenicity and virulence, virus-host interactions or host range and transmissibility [12, 13, 139, 140] . moreover, these technologies have been implemented to develop influenza vaccines [141] and to generate recombinant influenza viruses expressing foreign proteins as vaccine vectors [142] [143] [144] or harboring reporter genes to easily track viral infections [142, [145] [146] [147] . plasmid-based reverse genetics for influenza virus allows for the simultaneous expression of the viral components involved in viral genome replication and gene transcription (pb2, pb1, pa and np) and the eight negative-stranded vrnas in transfected susceptible cells, which together generate de novo, recombinant iavs or ibvs (figure 3 ) [48] . the goal to generate vrna in vivo from cloned complementary (c)dnas was achieved when the rna polymerase i (pol i) system for influenza vrna synthesis was established [148, 149] . pol i is a nuclear enzyme that transcribes ribosomal (r)rna, which like influenza vrna does not contain a cap structure on the 5 or poly (a) structures on the 3 ends [150, 151] . importantly, pol i initiates and terminates transcription at defined promoter and terminator sequences allowing the generation of vrnas without additional nucleotides at their 5 or 3 ends, which is required for efficient generation of recombinant viruses using reverse genetics. nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that only eight ambisense and/or bidirectional plasmids were needed for complete reconstitution of influenza viruses ( figure 3a ) [150, 151] . the eight plasmid-based rescue system is now the most common method for the generation of recombinant influenza viruses. because fewer plasmids are required, the eight-plasmid approach is more successful than the initial twelve-plasmid reverse genetic technique. the core of the eight-plasmid rescue system is that each plasmid contains an "ambisense cassette" that includes rna pol i and/or ii sequences, which drives the transcription of vrnas (pol i) and protein (pol ii) expression from the same viral cdnas ( figure 3b ). using a similar technology in 2002, this reverse genetics technique allowed the recovery of ibv entirely from ambisense plasmids [13, [160] [161] [162] . now, iav and ibv reverse genetics techniques are well established and commonly used in multiple research laboratories for different research purposes. influenza reverse genetics techniques have had an important effect on expanding our knowledge of the molecular biology and pathogenesis of influenza viruses, allowing researchers to answer important questions in the biology of iav and ibv that were not possible using conventional virological or biochemical procedures [12, 144, 147] . scientists can now mutate specific nucleotides in the influenza viral genome to elucidate the nature of regulatory sequences or the contribution of specific amino acids to the function of influenza viral proteins. for instance, reverse genetics technologies have enabled the identification and characterization of the cis-acting elements required for virus cell entry, uncoating, genome replication and gene transcription, encapsidation, packaging and viral release [13, 135, [163] [164] [165] . moreover, by engineering viral vectors suitable for the expression of foreign proteins in infected cells, multiple recombinant influenza viruses harboring reporter fluorescent and/or luminescent genes have been generated and used to identify antivirals or nabs in vitro and/or in vivo [142, 146, 147] . finally, reverse genetics have allowed the creation of single-cycle infectious iavs (sciiavs) that are restricted to one cycle of replication in parental cell lines. however, in complementing cell lines, sciiavs can replicate efficiently and to levels comparable to wt forms of influenza viruses [144] . highly virulent iav have the potential to pose a greater human threat than many other biosafety level (bsl)-3 and bsl-4 pathogens because of their efficient transmission and limited therapeutic options [47] . traditional immunological approaches (e.g., hai or microneutralization assays) to identify the presence of iav nabs rely on the manipulation of live forms of viruses and need the use of special bsl conditions. thus, novel approaches that allow the detection of viral nabs without the use of live forms of iavs and highly contained bsl laboratories would facilitate these serological assays. in this regard and because of their safety profile, sciiavs represent an excellent alternative to identify new antivirals and/or nabs [144] . moreover, several sciiavs have been shown to represent an excellent option for their implementation as safe, immunogenic and protective vaccines and/or vaccine vectors [69, 144] . influenza plasmid-based reverse genetics represent a better alternative to circumvent the process of generating reassortant virus by co-infection of chicken embryonated eggs for the generation of influenza vaccines (figure 2 ) [135] . moreover, the generation of recombinant influenza viruses using plasmid-based reverse genetics approaches is simple, well established and currently in use in several laboratories around the world [139, 166] (figure 4) . briefly, the eight-ambisense plasmids are co-transfected into susceptible fda-approved cells, and viable virus can be recovered from the tissue culture supernatants [139, 166] , then amplified in either embryonic eggs or fda-approved for vaccine production cells (figure 4) . for many years, iivs have been produced by reassortment in eggs ( figure 2) [90, 98, 122] . however, strains with the desired genotype (six internal genes of a high-growth virus and the ha and na glycoproteins of the seasonal influenza virus) could be produced easily and more quickly by implementing reverse genetics approaches. this process would overcome the need of chicken embryonated eggs to generate the desired reassortant virus and, therefore, minimizing the time associated with the selection process of the reassortant iiv ( figure 4a) . moreover, the improvements in surveillance, as well as the de novo gene synthesis for the production of the ha and na viral segments from the selected strains could reduce considerably the time of vaccine production. although influenza reverse genetics could be useful for the production of vaccine seed strains, to date, it has not been possible to predict which gene segment(s) constellations are required for efficient growth of different vaccine viruses. moreover, the time to produce vaccine viruses using reverse genetics versus the time needed to generate them using the classical reassortment approach could be a major consideration for the cost effectiveness in vaccine production and manufacturing. thus, the traditional viral reassortment has remained preferred because it allows the generation of a number of diverse gene(s) combinations in order to select recombinant viruses with better fitness [98] . during the last few decades, considerable improvements have been accomplished in the development of influenza vaccines. however, novel approaches to increase the effectiveness of seasonal influenza vaccines are needed. reverse genetics technologies have proven a valuable tool to develop reassortant strains for the production of laiv candidates ( figure 4b ). currently, seed viruses containing six gene segments from the mdv a/ann arbor/6/60 (h2n2) and the ha and na from the selected seasonal virus can be quickly generated using reverse genetics systems [98, 131] ( figure 4b ). the ha and na from selected seasonal influenza viruses can be amplified by rt-pcr or quickly chemically synthetized and then cloned in ambisense plasmids used for virus rescue. this technology could speed up the development of new laivs, bypassing the need to isolate the exact virus reassortment in eggs ( figure 2 ). the best way to combat influenza virus infection is to prevent it. thus, an urgent need exists to develop novel and more effective influenza vaccines. plasmid-based reverse genetic technologies have allowed the engineering of recombinant influenza viruses that contain single or multiple mutations in the viral genome, which can be potentially implemented as novel or improved vaccine approaches. in fact, several novel vaccine candidates have been developed with promising results in animal models of experimentation. here, we review and discuss some of them. because of ns1's ability to hijack the host innate immune ifn-i response, a variety of potential vaccine strategies have been developed, which are based on the use of modified ns1 proteins as a means for virus attenuation [167] [168] [169] [170] [171] [172] . equine [170] , swine [169, 173, 174] , avian [167, 175, 176] , canine [177] and human [178, 179] iavs with partial truncations in or deletions of the viral ns1 protein are all attenuated in vitro and in vivo [167, 169, 170, 179] . importantly, these recombinant iavs can induce a protective immune response upon a single intranasal vaccination in mice [177, [179] [180] [181] , horses [170] , pigs [169, 173, 174] , birds [167, 175, 176] and macaques [178] ; therefore, they represent excellent laiv candidates to prevent iav infections. in addition, a similar approach has been employed to develop attenuated ibvs with similar results [180] . mice inoculated with the ns1-truncated or -deleted mutants elicited an antibody response and showed protection against wt virus challenge [180] . thus, these ns1-truncated influenza a and b viruses represent excellent candidates as safe, immunogenic and protective laivs for multiple virus strains in different animal models. the genetic code in animals encodes for 20 different amino acids (aa) using 61 codons. this degeneracy of the genetic code allows amino acids, except tryptophan (w) and methionine (m), to be encoded by more than one synonymous codon [46] . viruses, including influenza, rely on the host cell translation machinery to synthesize their viral proteins for the formation of infectious virus progeny. as an evolutionary consequence, viruses have modified their codon usage according to the host they infect [182] . experimentally, protein synthesis can be downregulated by synthetically deoptimizing the codon usage of a gene [141, 182] . the generation of recombinant viruses containing genes with deoptimized codons is now feasible [141, 182] , and their level of attenuation depends on the viral gene targeted and the number of codon changes made during the codon deoptimization process [182] . for influenza viruses, many regions in the viral genome cannot be altered because of their important role in viral replication and transcription (e.g., ncrs), packaging (e.g., packaging signals), the presence of multiple overlapping orfs (e.g., segments 7 and 8), etc. [48] . to date, recombinant iavs that have been attenuated using a codon-pair [183, 184] or a codon bias [182] deoptimization approach to decrease expression levels of the viral pb1, ha and np [183] , na and ha [184] or ns1 and nep [182] have been generated. importantly, influenza viruses generated by codon deoptimization showed similar viral replication kinetics to wt virus in mdck cells, which is important for their effective use for vaccine production. however, to date, the ability of these recombinant iavs containing codon-pair or codon bias deoptimized viral segments to replicate in eggs has not yet been evaluated. importantly, all of the codon deoptimized iavs were attenuated in mice and able to provide, upon a single immunization dose, protection against a lethal challenge with a wt form of the virus, showing that laivs were safe, immunogenic and protective. however, mice do not accurately reflect virus pathogenesis and immunological responses seen in humans [81] and do not have the same codon usage bias as humans. therefore, studies aimed to demonstrate the safety, immunogenicity and protection efficacy of codon deoptimized recombinant iavs in other well-established animal models of influenza (e.g., guinea pigs, ferrets or nonhuman primates) could lead to their implementation as laivs in future vaccinations [141] . other promising approaches for the development of laivs relate to the use of sciiavs [144] . sciiavs based on their safety profile, ability to elicit protective humoral and cellular responses and protective effectiveness represent a feasible alternative to current influenza vaccines for the treatment of influenza viral infections [69, 144] . however, to date, no single-cycle infectious ibvs (sciibv) have been reported. when delivered intranasally, sciiav has been shown to be safe in a mouse model of influenza infection, without signs of illness or mortality [69, [185] [186] [187] [188] [189] . moreover, and similar to the current laivs, intranasal immunization with a single dose of sciiavs elicited localized mucosal immune responses and recruitment of influenza-specific cd8 t-cells into the lungs of vaccinated animals [145, 185, 186, 190] , the latest being the main contributor of immunity against challenge with heterologous influenza viruses [69, 190] . importantly, sciiavs protected mice against lethal influenza virus challenges [69, 144] . moreover, similar safety, immunogenicity and protection efficacy of sciiavs were observed in ferrets [69] . vaccination of pigs with sciiav has also been shown to be immunogenic and protective against challenge with swine influenza viruses [191] . moreover, sciiavs expressing foreign genes represent an excellent option for their implementation as bivalent vaccines. for instance, sciiavs expressing the surface protein a (pspa) of streptococcus pneumoniae, the hemagglutinin-neuraminidase (hn) protein of hpiv-3 or the fusion (f) protein of respiratory syncytial virus (rsv) [192] [193] [194] , were able to induced abs against the foreign polypeptides and reduced the viral load of the heterologous pathogen while retaining their ability to protect against challenge with iav. it is worth indicating that, for vaccine purposes, it is important to consider what influenza viral gene is replaced, since it was shown that sciiav replication is required for protection [69, 185, 189] . although substitution of the polymerase (pb2, pb1 and pa) segments may allow insertion of larger foreign genes, removing the viral polymerase from sciiavs limits their ability to express more polymerase during the single-cycle round of infection, decreasing total viral antigens and, thus, limiting their protective efficacy. thus, sciiavs where the viral ha or na has been removed were able to confer, upon a single immunization, protection against a lethal challenge with influenza. [190] . on the contrary, a single dose of a sciiav where the viral pb2 has been removed was only as efficacious as an inactivated virus [189] . it is worth noting that sciiavs combine the advantages (better immunogenic properties of the laiv and the safety profile of the iiv) and circumvent the disadvantages (safety of the laiv and poor immunogenicity of the iiv) of current influenza vaccine approaches [144] . while sciiavs have been shown to be safe, immunogenic and protective against lethal challenges with wild-type forms of iavs in animal studies, no human trials have yet been performed. more recently, the rearrangement of the influenza virus genome has been shown to have great potential for the development of improved laivs against influenza virus, as well as vaccine vectors against other pathogens [195, 196] . avian influenza virus subtypes h5n1 and h9n2 have pandemic potential [197, 198] . however, h5n1 iiv induces limited adaptive immune responses, and in the case of laiv, there are safety concerns about the possibility of reassortment between the viral segments in the laiv and circulating h5n1 strains. to overcome these concerns, a bivalent laiv against influenza a/vietnam/120320/04 h5n1 and a/guinea fowl/hong kong/wf10/1999 h9n2 was generated using viral genome rearrangement [195] . to that end, the nep was removed from the ns viral segment of the h9n2 virus and substituted by the ha of the h5n1 virus. h9n2 ns1 and h5n1 ha were separated by the foot-and-mouth disease virus (fmdv) 2a autocleavage site to allow co-linear expression of both viral proteins. then, nep was cloned down-strain of the h9n2 pb1 segment separated by another fmdv 2a autocleavage site. the rearranged h9n2 virus expressing the h5n1 ha was able to provide complete protection against challenge with a/vietnam/1203/2004 h5n1 and also against a potential pandemic h9:ph1n1 iav reassortant virus in both mice and ferrets [195] . segments 7 (m) and 8 (ns) of iavs use an alternative splicing mechanism to express two different viral proteins from the same viral segment. recently, we have generated recombinant influenza a/puerto rico/8/34 h1n1 viruses containing modified m and/or ns segments, in which the overlapping orfs of the m1 and m2 viral proteins (m segment) and/or the ns1 and nep proteins (ns segment) were separated with the porcine teschovirus 1 (ptv-1) 2a autocleavage site [196] . recombinant viruses with a rearranged m segment were affected or impaired in replication in vitro at nonpermissive temperatures (37 and 39 • c, respectively), whereas high viral titers were obtained at permissive low temperatures (33 • c) [196] . notably, viruses containing the m split segment were highly attenuated in vivo, but able to confer, upon a single immunization dose, complete protection against a lethal homologous challenge with wild-type pr8 [196] . importantly, viruses with a reorganized m segment were able to confer better protection than a temperature-sensitive, laiv pr8 virus [103, 104, 115, 196] . these studies demonstrate that the rearrangement of the influenza viral genome can be used for the generation of safe, immunogenic and protective laivs. current influenza vaccines induce immunity to the influenza virus strain-specific ha antigen and are not very effective against new pandemic viruses, given that ha is highly susceptible to frequent changes by antigenic drift and shift [12, [44] [45] [46] [74] [75] [76] . to overcome these drawbacks, different approaches aimed to develop a universal influenza vaccine able to induce cross-protective broadly neutralizing immunity against conserved viral antigens, such as the ectodomain of m2 (m2e) [199] , the ha stalk domain [200] or na [201] , have been explored. the m2e antigen is a linear peptide that is very well conserved across iav strains. although the mechanism of m2e-specific immunity is unclear, protective anti-m2 antibodies have been observed in multiple animal models, including mice, ferrets and primates [36] . however, these conserved antigenic targets need to be presented in a carrier system or conjugated to adjuvant molecules. promising results have been obtained with virus-like particles (vlps), which are morphologically similar to the virus and present surface proteins in a highly immunogenic form. because vlps do not contain the viral genome, they are considered safer than viral vaccines, yet still induce strong humoral and cellular immune responses [202] . vlps are most commonly made by expression of ha, na and m1 [203, 204] , although ha and na alone may be sufficient for vlp production [205] . reverse genetics approaches to generate recombinant viruses, including influenza, have been described for representative family members of negative-sense, single-stranded rna viruses. these plasmid-based reverse genetics methods have provided scientists with a unique opportunity to study different aspects of the biology and pathogenesis of these viruses, both in vitro and in vivo, as well as to generate attenuated forms to be used as vaccines [135] . in this review, we discussed the use of reverse genetics for the generation of influenza vaccines with a special focus on laivs. in general, laivs are highly immunogenic, and immunization usually induces faster and substantially higher levels of both systemic and local mucosal antibody and t-cell responses, providing better protection than their inactivated counterparts. prevention of influenza virus infection requires seasonal vaccinations, and identification of the correct virus subtype to include in the vaccine leaves little time for vaccine development, scale up and distribution. although influenza vaccines work well most of the years, if a new viral variant emerges after the strain to be included in the vaccine has been selected, the efficacy of the vaccine would be sub-optimal for this new variant strain. this usually results in decreased efficacy of the vaccine. reverse genetics approaches have also allowed the development of live-attenuated viruses that could be implemented, in the near future, as laivs. these new advances in reverse genetics approaches are reducing the potential time of recovery and production from months to weeks and represent an excellent alternative for the rapid development and implementation of laivs for the treatment of both seasonal and potentially pandemic influenza strains. acknowledgments: influenza virus research in the luis martínez-sobrido and aitor nogales laboratory was partially funded by the national institute of allergy and infectious diseases (niaid) centers of excellence for influenza research and surveillance (ceirs hhsn266200700008c). the authors declare no conflict of interest. the viruses and their replication transmission of influenza virus in a mammalian host is increased by pb2 amino acids 627k or 627e/701n the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses evolution of 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authors: brownsword, matthew j.; doyle, nicole; brocard, michèle; locker, nicolas; maier, helena j. title: infectious bronchitis virus regulates cellular stress granule signaling date: 2020-05-14 journal: viruses doi: 10.3390/v12050536 sha: doc_id: 273366 cord_uid: xd84f8ct viruses must hijack cellular translation machinery to express viral genes. in many cases, this is impeded by cellular stress responses. these stress responses result in the global inhibition of translation and the storage of stalled mrnas, into rna-protein aggregates called stress granules. this results in the translational silencing of the majority of mrnas excluding those beneficial for the cell to resolve the specific stress. for example, the expression of antiviral factors is maintained during viral infection. here we investigated stress granule regulation by gammacoronavirus infectious bronchitis virus (ibv), which causes the economically important poultry disease, infectious bronchitis. interestingly, we found that ibv is able to inhibit multiple cellular stress granule signaling pathways, whilst at the same time, ibv replication also results in the induction of seemingly canonical stress granules in a proportion of infected cells. moreover, ibv infection uncouples translational repression and stress granule formation and both processes are independent of eif2α phosphorylation. these results provide novel insights into how ibv modulates cellular translation and antiviral stress signaling. during replication within a host cell, all viruses must regulate a variety of cellular processes to generate an environment that allows progeny virus to be produced to continue the infection cycle. this includes promoting pathways that are favorable to replication and overcoming intrinsic immune pathways. cellular stress granules (sg) play an important role in the regulation of gene expression by regulating mrna translation and location, as well as integrating intracellular signaling and antiviral responses, and are therefore often targeted by viruses [1, 2] . sg are cytoplasmic, non-membrane bound aggregations of mrna associated with translation initiation factors, the 40s ribosome and rna binding proteins. they primarily form under stress conditions that trigger the phosphorylation of translation initiation factor eif2α [3] . there are four eif2α kinases; protein kinase r (pkr), recognizing dsrna, pkr-like endoplasmic reticulum kinase (perk), sensing er stress, heme regulated eif2α kinase (hri) and general control nonderepressible 2 (gcn2), activated by oxidative stress and amino acid deprivation [4] [5] [6] [7] . despite pkr being the assumed major kinase to activate the integrated stress response (isr) during viral infection, perk [8] and gcn2 [9] have also been found to play an important role. phosphorylation of eif2α prevents delivery of the initiator trna to initiating ribosomes, therefore inhibiting translation initiation and leading to the accumulation of stalled 48s mrnps. sg can also be vero cells were maintained in 1× eagle's modified essential medium (sigma) supplemented with 1× l-glutamine (gibco) and 10% fetal bovine serum (sigma). recombinant ibv strain beau-r has been described previously [35] . inactivated ibv was generated by treatment with binary ethylenimine (bei). briefly, the virus was incubated in 0.1 m bei for 48 h at 37 • c, followed by inactivation of bei by addition of 1 m sodium thiosulfate. inactivation of virus was confirmed by rt-qpcr following the infection of cells. sodium arsenite, cycloheximide, puromycin and emetine were purchased from sigma. vero cells seeded onto glass coverslips were mock infected or infected with undiluted ibv (moi 0.02) and incubated at 37 • c. after 1 h, 1× bes (mem, 0.3% tryptose phosphate broth, 0.2% bovine serum albumin, 20 mm n,n-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (bes), 0.21% sodium bicarbonate, 2 mm l-glutamine, 250 u/ml nystatin, 100 u/ml penicillin, and 100 u/ml streptomycin) were added and cells were incubated for the indicated time. where indicated, cells were treated for 1 h prior to fixation with 500 µm sodium arsenite or 35 µm cycloheximide, or for 2 h prior to fixation with 2 µm hydrogen peroxide. cells were fixed in 4% paraformaldehyde in pbs, permeabilized in 0.1% triton x-100 in pbs and blocked in 0.5% bovine serum albumin (bsa) in pbs. primary and secondary antibodies were diluted in blocking buffer. nuclei were stained with 4 ,6-diamidino-2-phenylindole (dapi). anti-dsrna j2 (english and scientific consulting) was diluted 1:1000, anti-nsp12 [36] was diluted 1:1000, anti-s2 (26.1, wageningen university and research) was diluted 1:500, anti-ibv (abcam) was diluted 1:1000, anti-g3bp1 (bd biosciences) was diluted at 1:500, anti-g3bp1 (sigma) was diluted 1:500, anti-eif3η (santa-cruz) was diluted 1:500 and anti-eif4g (santa-cruz) was diluted 1:500. alexa fluor-conjugated secondary antibodies (invitrogen) were diluted 1:500. cells were visualized using a leica sp5 or nikon ti eclipse confocal microscope. to determine the percentage of the cells which were positive for sg, cells were counted manually, with at least 50 cells counted over three independent biological replicates. vero cells seeded onto glass coverslips were mock or ibv infected (moi 0.02). after 24 h, cells were fixed and labelled using the stellaris rna fish simultaneous labelling protocol (biosearch technologies). briefly, cells were fixed in 10% formaldehyde in pbs and permeabilized in 70% ethanol at 4 • c. cells were incubated overnight at 37 • c in a humidified chamber, with hybridization buffer containing 125 nm probe and primary antibody. cells were then washed and labelled with alexa fluor-conjugated secondary antibody and dapi. finally, cells were mounted onto glass coverslips using vectashield and sealed with nail varnish. cells were visualized using a leica sp5 confocal microscope. stellaris fish probes with a quasar 570 label were designed specifically for the nsp15 and nsp16 region of the ibv beau-r genome. vero cells seeded in 6-well plates were mock infected or infected with undiluted ibv (moi 0.005). at the indicated time points, cells were washed once with cold pbs and lysed in 1× sample buffer (biorad) containing dtt. cell lysates were heated to 95 • c for 3 min and briefly sonicated. proteins were separated on a 4%-20% bis-tris gel (biorad) and transferred onto nitrocellulose membranes. these were blocked in 0.5% bsa or 5% milk in tbs-tween (tbs-t), then incubated with primary antibody diluted in blocking buffer. following three washes in tbs-t, membranes were incubated with hrp labelled secondary antibodies (dako), diluted in blocking buffer. after three further washes in tbs-t, blots are incubated chemiluminescence substrate using the clarity western ecl substrate (bio-rad). labelled protein bands were visualized, using a vilber imaging system. anti-ibv was viruses 2020, 12, 536 4 of 20 diluted 1:1000, anti-eif2α (cell signaling technologies) was diluted 1:1000, anti-eif2α-p (cell signaling technologies) was diluted 1:2000 and anti-gapdh (invitrogen) was diluted 1:10000. vero cells seeded onto glass coverslips were mock or ibv infected (moi 0.02), as before. rpm was performed as described by david et al. [37] briefly, one hour prior to processing, control wells were treated with 500 µm sodium arsenite. at the indicated times post infection, cells were incubated with 18.4 µm puromycin for 30 s at room temperature and then incubated with 18.4 µm puromycin and 208 µm emetine at room temperature for 1 min. cells were washed three times with room temperature 1× bes media, fixed and processed for immunofluorescence, as described above. anti-puromycin (sigma) was diluted 1:10000. to quantify immunofluorescence images, the puromycin signal in 100 cells was determined using imagej [38] . initially, the ability of ibv to induce sg during replication was assessed. vero cells were infected with ibv and at the indicated time points, cells were fixed and labelled with anti-dsrna to detect virus infection and with anti-g3bp1 to detect sg. at each time point, infected cells were present, with the number of infected cells increasing over time, as expected ( figure 1a ). in addition, at each of the time points tested, g3bp1 puncta were detected in a proportion of, but not all, infected cells, with diffuse g3bp1 found in the remaining infected and uninfected cells. subsequently, the number of infected cells with and without g3bp1 puncta was determined. the percentage of infected cells containing g3bp1 puncta was found to be between 10 and 25% ( figure 1b ) and this percentage remained unchanged over the course of infection, with no statistical difference between the percentages of cells containing puncta at any time point. therefore, ibv replication triggers the formation of g3bp1 puncta, but interestingly, only in 10%-25% of infected cells. following identification of sg in ibv infected cells, the requirement for active virus replication in induction of granules was assessed. cells were infected with wild type ibv or a bei-inactivated virus. after 24 hours, cells were fixed and labelled with anti-s2 and anti-g3bp1. while cells infected with wild type ibv contained sg as observed before, cells infected with the inactivated virus did not ( figure s1 ). in addition, none of the compounds used for virus inactivation had any effect on sg formation or inhibition. therefore, the induction of sg requires actively replicating virus and is not a response by the cell to the presence of the virus particle. some viruses inhibit the formation of sg by degradation of sg nucleating proteins, such as g3bp1. therefore, it was investigated whether g3bp1 is degraded during ibv infection. cells were infected with ibv and after 24 h, cells were lysed, proteins separated by sds-page and levels of g3bp1, ibv n and gapdh were measured by western blot ( figure 1c ). g3bp1 levels were unaltered compared to mock treated cells upon either ibv infection or sodium arsenite treatment. therefore, ibv infection does not result in the degradation of g3bp1. as ibv replication did not induce sg in every infected cell, it was hypothesised that ibv may be able to inhibit the formation of canonical sg. to test this, cells were infected with ibv for 24 h and prior to fixation, cells were treated with sodium arsenite for 1 hour or hydrogen peroxide for 2 h to induce sg formation. sodium arsenite induces eif2α-dependent sg by activating the eif2α kinase hri. hydrogen peroxide induces sg via hri and gcn2 [39] , but also in an eif2α independent process, by disrupting the eif4f complex. following fixation, cells were labelled with anti-dsrna to detect virus infected cells and anti-g3bp1 to visualize sg. in uninfected cells, treatment with either sodium arsenite or hydrogen peroxide resulted in the formation of sg (figure 2a ). however, in ibv infected cells, both sodium arsenite and h 2 o 2 induction of sg were blocked with g3bp1 in infected viruses 2020, 12, 536 5 of 20 cells, remaining largely diffuse ( figure 2a ). the percentage of cells containing g3bp1 foci was then determined ( figure 2b ). in the absence of chemical treatment, 17% of ibv infected cells contained sg. when mock infected cells were treated with sodium arsenite or hydrogen peroxide, 83% and 74% of cells were positive for sg, respectively. however, when ibv infected cells were sodium arsenite or hydrogen peroxide treated, only 18% and 9% infected cells contained sg, respectively. therefore, ibv infection inhibits both eif2α-dependent and independent sg induction. were detected using an anti-g3bp1 antibody (red) and ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). positive control cells were treated with sodium arsenite (naas) to induce eif2α-dependent sg. scale bar indicates 50 μm. (b) images in (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g3bp1 foci. a minimum of 100 cells were counted from three independent replicates. the mean and standard deviation is shown. asterisks indicate statistical significance, as measured by oneway anova, *** represents p < 0.005 and **** represents p < 0.0005, respectively. (c) vero cells were mock infected or infected with ibv. at 23 hpi, where indicated, cells were treated with naas. cells were lysed at 24 hpi and processed and labelled using anti-g3bp1, anti-gapdh or anti-ibv antibodies. mean g3bp1 band intensities for ibv and naas treatments were normalized relative to mock band. blot representative of 3 independent replicates. as ibv replication did not induce sg in every infected cell, it was hypothesised that ibv may be able to inhibit the formation of canonical sg. to test this, cells were infected with ibv for 24 h and prior to fixation, cells were treated with sodium arsenite for 1 hour or hydrogen peroxide for 2 h to were detected using an anti-g3bp1 antibody (red) and ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). positive control cells were treated with sodium arsenite (naas) to induce eif2α-dependent sg. scale bar indicates 50 µm. (b) images in (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g3bp1 foci. a minimum of 100 cells were counted from three independent replicates. the mean and standard deviation is shown. asterisks indicate statistical significance, as measured by one-way anova, *** represents p < 0.005 and **** represents p < 0.0005, respectively. (c) vero cells were mock infected or infected with ibv. at 23 hpi, where indicated, cells were treated with naas. cells were lysed at 24 hpi and processed and labelled using anti-g3bp1, anti-gapdh or anti-ibv antibodies. mean g3bp1 band intensities for ibv and naas treatments were normalized relative to mock band. blot representative of 3 independent replicates. viruses 2020, 12, 536 6 of 20 infected cells, both sodium arsenite and h2o2 induction of sg were blocked with g3bp1 in infected cells, remaining largely diffuse (figure 2a ). the percentage of cells containing g3bp1 foci was then determined ( figure 2b ). in the absence of chemical treatment, 17% of ibv infected cells contained sg. when mock infected cells were treated with sodium arsenite or hydrogen peroxide, 83% and 74% of cells were positive for sg, respectively. however, when ibv infected cells were sodium arsenite or hydrogen peroxide treated, only 18% and 9% infected cells contained sg, respectively. therefore, ibv infection inhibits both eif2α-dependent and independent sg induction. figure 2 . ibv inhibits eif2α-dependent and independent stress granule induction. (a) vero cells were mock infected or infected with ibv for 24 hours. prior to fixation, cells were treated for 1 hour with 500 μm sodium arsenite (naas) to activate the eif2α-dependent pathway or for 2 h with 2 µ m hydrogen peroxide (h2o2) to activate the eif2α-independent pathway. at 24 hpi, cells were fixed and stress granules (sg) were labelled with an anti-g3bp1 antibody (red). ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue) and scale bar indicates 50 μm. (b) images from (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g3bp1 foci. a minimum of 50 cells were counted. data from three independent replicates. asterisks indicate statistical significance as measured by one-way anova, **** p < 0.0001. several viruses have been shown to promote the formation of specific virus-induced cytoplasmic foci, by recruitment and relocalisation of many sg components, including g3bp1 and g3bp2 [22, 24, 25, 40] . therefore, following the identification of g3bp1 puncta in some ibv infected cells, the nature of these puncta was investigated, to determine whether they were canonical sg or virusspecific granules. canonical sg contain multiple sg markers such as g3bp1, translation initiation factors, ribosomal subunits and mrna. therefore, the presence of punctate translation initiation factors eif3η and eif4g in infected cells was investigated. cells were infected with ibv and after 24 h, cells were fixed and labelled with anti-dsrna and either anti-eif3η or anti-eif4g. as expected, eif3η and eif4g were diffuse within the cytoplasm in mock infected cells ( figure 3 ). similar to previous observations using g3bp1, in a proportion of virus infected cells, both eif3η and eif4g were figure 2 . ibv inhibits eif2α-dependent and independent stress granule induction. (a) vero cells were mock infected or infected with ibv for 24 hours. prior to fixation, cells were treated for 1 hour with 500 µm sodium arsenite (naas) to activate the eif2α-dependent pathway or for 2 h with 2 µm hydrogen peroxide (h 2 o 2 ) to activate the eif2α-independent pathway. at 24 hpi, cells were fixed and stress granules (sg) were labelled with an anti-g3bp1 antibody (red). ibv infection was detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue) and scale bar indicates 50 µm. (b) images from (a) were quantified by manual counting of sg positive cells, identified by counting infected or treated cells with g3bp1 foci. a minimum of 50 cells were counted. data from three independent replicates. asterisks indicate statistical significance as measured by one-way anova, **** p < 0.0001. several viruses have been shown to promote the formation of specific virus-induced cytoplasmic foci, by recruitment and relocalisation of many sg components, including g3bp1 and g3bp2 [22, 24, 25, 40] . therefore, following the identification of g3bp1 puncta in some ibv infected cells, the nature of these puncta was investigated, to determine whether they were canonical sg or virus-specific granules. canonical sg contain multiple sg markers such as g3bp1, translation initiation factors, ribosomal subunits and mrna. therefore, the presence of punctate translation initiation factors eif3η and eif4g in infected cells was investigated. cells were infected with ibv and after 24 h, cells were fixed and labelled with anti-dsrna and either anti-eif3η or anti-eif4g. as expected, eif3η and eif4g were diffuse within the cytoplasm in mock infected cells ( figure 3 ). similar to previous observations using g3bp1, in a proportion of virus infected cells, both eif3η and eif4g were found in cytoplasmic puncta, with the remaining infected cells containing diffuse eif3η or eif4g ( figure 3 ). therefore, ibv infection induces the formation of sg that contains multiple sg marker proteins. viruses 2018, 10, x for peer review 7 of 21 found in cytoplasmic puncta, with the remaining infected cells containing diffuse eif3η or eif4g ( figure 3 ). therefore, ibv infection induces the formation of sg that contains multiple sg marker proteins. in addition to containing multiple sg marker proteins, canonical sg are dissolved in the presence of cycloheximide. as mrnas are constantly shuttled between sg and ribosomes, cycloheximide binding to the ribosome, preventing release of mrna, inhibits recycling to sg. as a result, sg are dissolved. to further understand the nature of ibv induced sg, their susceptibility to cycloheximide treatment was determined. cells were infected with ibv for 24 h, and one hour prior to fixation, cells were treated with cycloheximide. cells were then labelled with anti-dsrna and anti-g3bp1. firstly, it was confirmed that sg induced with sodium arsenite in uninfected cells were dissolved by treatment with cycloheximide ( figure 4a ). when cycloheximide treatment was applied to ibv infected cells, a significant decrease in the number of cells containing sg was observed. the percentage of infected cells containing sg was quantified ( figure 4b ) and, interestingly, the number of ibv infected cycloheximide treated cells containing sg was reduced to a value not significantly different from mock cells. together, this shows that ibv infection induces sg that contain multiple sg markers and are susceptible to cycloheximide, indicating that they are likely to be canonical sg. in addition to containing multiple sg marker proteins, canonical sg are dissolved in the presence of cycloheximide. as mrnas are constantly shuttled between sg and ribosomes, cycloheximide binding to the ribosome, preventing release of mrna, inhibits recycling to sg. as a result, sg are dissolved. to further understand the nature of ibv induced sg, their susceptibility to cycloheximide treatment was determined. cells were infected with ibv for 24 h, and one hour prior to fixation, cells were treated with cycloheximide. cells were then labelled with anti-dsrna and anti-g3bp1. firstly, it was confirmed that sg induced with sodium arsenite in uninfected cells were dissolved by treatment with cycloheximide ( figure 4a ). when cycloheximide treatment was applied to ibv infected cells, a significant decrease in the number of cells containing sg was observed. the percentage of infected cells containing sg was quantified ( figure 4b ) and, interestingly, the number of ibv infected cycloheximide treated cells containing sg was reduced to a value not significantly different from mock cells. together, this shows that ibv infection induces sg that contain multiple sg markers and are susceptible to cycloheximide, indicating that they are likely to be canonical sg. it was previously found that during the replication of alphacoronavirus tgev, viral rna was targeted to virus-induced sg, and this was thought to be important for their anti-viral function [26] . therefore, to further understand ibv induced sg and to determine whether ibv rna is also targeted to sg, viral genomic rna was visualized using fish. cells were infected with ibv or mock infected. cells were then mock treated or treated with cycloheximide (chx) to dissolve sg. at 24 hpi, cells were fixed and labelled with anti-g3bp1 (red) to detect stress granules (sg) and ibv infected cells were detected with an anti-dsrna antibody (green). nuclei were stained with dapi (blue). scale bar indicates 20 µm. (b) images from (a) were quantified to determine the percentage of cells containing sg. a minimum of 50 cells were counted. mean and standard deviation of three independent replicates are shown. asterisks indicate statistical significance, as measured by one-way anova, **** p < 0.0001; ns, not significant. it was previously found that during the replication of alphacoronavirus tgev, viral rna was targeted to virus-induced sg, and this was thought to be important for their anti-viral function [26] . therefore, to further understand ibv induced sg and to determine whether ibv rna is also targeted to sg, viral genomic rna was visualized using fish. cells were infected with ibv or mock infected. after 24 hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g3bp1 ( figure 5 ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g3bp1 puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g3bp1 containing sg. therefore, ibv genomic rna is not targeted to sg. after 24 hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g3bp1 ( figure 5 ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g3bp1 puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g3bp1 containing sg. therefore, ibv genomic rna is not targeted to sg. figure 5 . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for 24 hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g3bp1 antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate 10 µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [41] [42] [43] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after 24 h, fixed and labelled with anti-g3bp1, as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp12, the viral rna-dependent rna polymerase or spike protein (anti-s2), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g3bp1 foci (figure 6 ). in addition, g3bp1 did not to co-localize with either nsp12 or spike. therefore, ibv does not direct sg markers to sites of virus replication. . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for 24 hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g3bp1 antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate 10 µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [41] [42] [43] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after 24 h, fixed and labelled with anti-g3bp1, as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp12, the viral rna-dependent rna polymerase or spike protein (anti-s2), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g3bp1 foci (figure 6 ). in addition, g3bp1 did not to co-localize with either nsp12 or spike. therefore, ibv does not direct sg markers to sites of virus replication. after 24 hours, cells were fixed and labelled with fish probes specific for ibv genomic rna and anti-g3bp1 ( figure 5 ). viral genomic rna was found to be located in foci within the cytoplasm. in addition, g3bp1 puncta were detected in a percentage of infected cells, as seen before. however, no co-localization was observed between viral genomic rna and g3bp1 containing sg. therefore, ibv genomic rna is not targeted to sg. figure 5 . ibv genomic rna is not diverted to stress granules during infection. vero cells were mock infected or infected with ibv for 24 hours. ibv genomic rna (red) was detected using fluorescent in situ hybridization (fish) probes and an anti-g3bp1 antibody was used to detect sg (green). nuclei were stained using dapi (blue). scale bars indicate 10 µm. images are representative of three independent replicates. during the replication of several other viruses including ebola virus, west nile virus, dengue virus and tick-borne encephalitis virus, sg markers are redirected to sites of virus replication [41] [42] [43] . to investigate whether ibv induced sg co-localize with sites of viral rna synthesis or virion assembly, cells were infected with ibv and after 24 h, fixed and labelled with anti-g3bp1, as well as antibodies specific for dsrna, thought to be an intermediate in viral rna synthesis, nsp12, the viral rna-dependent rna polymerase or spike protein (anti-s2), to label sites of progeny virus assembly. consistent with earlier experiments, dsrna did not co-localize with g3bp1 foci (figure 6 ). in addition, g3bp1 did not to co-localize with either nsp12 or spike. therefore, ibv does not direct sg markers to sites of virus replication. the formation of sg in cells is closely associated with an inhibition of translation. previous work has demonstrated that ibv replication is associated with a shut-off of host translation from around 12 hpi, with the translation of viral proteins also ceasing by 24 hpi [34] . however, translational activity on a single cell level has not been characterized. therefore, rpm was used to visualize actively translating ribosomes over a time course of infection [37] . cells were infected with ibv and nascent polypeptides labelled with puromycin at 12, 18 and 24 hpi, followed by the stalling of translating ribosomes with emetine. cells were then fixed and labelled with an anti-puromycin antibody to detect nascent polypeptides and an anti-ibv antibody to detect infected cells. in mock infected cells, active translation was detected, with a diffuse puromycin signal throughout the cytoplasm. this signal was absent without puromycin treatment or upon treatment of cells with sodium arsenite to inhibit translation ( figure 7a ). following ibv infection at all three time points studied, two phenotypes were observed. some cells contained the diffuse puromycin signal detected in mock infected cells. alternatively, a proportion of infected cells showed reduced puromycin signal ( figure 7b) . to enable the level of translational activity to be determined, puromycin signal was quantified in at least 50 infected cells and surrounding non-infected cells ( figure 7c ). this indicated that there was a shut-off of translation at all time points. furthermore, the degree of translational inhibition increased as infection progressed with a more pronounced shut-off at 18 and 24 than at 12 hpi. therefore, consistent with previous work [34] , this single cell rpm indicates that translational shut-off in infected cells is seen from 12 hpi and increases with the duration of infection. as previously demonstrated, ibv induces a translational shut-off that increases over the duration of infection, with two phenotypes showing variation in the degree of translational shut-off. in addition to this, ibv induces sg in 20% of infected cells. the translational status of infected cells containing g3bp1 foci was therefore assessed to determine whether the two different translational profiles could be attributed to the presence or absence of sg. cells were infected with ibv and then treated as before, using the rpm method. at 24 hpi cells were fixed and labelled using anti-puromycin, anti-g3bp1 and anti-ibv antibodies ( figure 8a ) and the intensity of puromycin label quantified as before ( figure 8b ). active translation was detected in mock infected cells, with a diffuse puromycin signal throughout the cytoplasm. upon stimulation with sodium arsenite, the accumulation of g3bp1 into sg correlated with a strong reduction in puromycin signal, confirming that sg formation is coupled to translational shut-off. the level of translational activity was then determined in infected cells, with and without sg. while infected cells containing sg exhibited a potent translational impairment with puromycin labelling levels similar to those detected in cells stimulated with sodium arsenite, infected cells that did not contain sg also displayed markedly reduced puromycin signal compared to uninfected cells. this demonstrates that the translational shut-off seen in ibv-infected cells cannot be solely attributed to sg formation and that translation inhibition and sg formation are uncoupled. viruses 2018, 10, x for peer review 11 of 21 asterisks indicate statistical significance, as measured by one-way anova, * p = 0.02; **** p < 0.0001. both sg formation and translational shut-off are usually associated with phosphorylation of eif2α. previous work by others has demonstrated that ibv infection results in eif2α phosphorylation at early time points, but that the virus inhibits this as infection progresses [32] . therefore, the phosphorylation status of eif2α was investigated. vero cells were infected with ibv and lysed at 6, 12, 18 and 24 hpi. proteins were separated by sds-page and transferred to nitrocellulose. blots were labelled using anti-eif2α to detect total eif2α, anti-eif2α-p to detect the phosphorylated eif2α, anti-ibv to detect virus and anti-gapdh as a loading control ( figure 9a ). total levels of eif2α remained unchanged throughout infection. although eif2α phosphorylation was achieved using sodium arsenite treatment, no eif2α phosphorylation was detected at any time point during ibv infection, with levels remaining comparable to that of mock infected cells. subsequently, to determine whether ibv infection actively inhibits phosphorylation of eif2α, vero cells were infected with ibv and then treated with sodium arsenite prior to cell lysis ( figure 9b ). as before, the sodium arsenite treatment of mock infected cells resulted in a significant increase in the level of phosphorylated eif2α. when ibv infected cells were treated with sodium arsenite, there was also a significant increase in the level of phosphorylated eif2α when compared to ibv infected untreated cells. significantly, the level of phosphorylated eif2α in these cells appeared comparable to that in mock infected sodium arsenite treated cells ( figure 9b ). together, this demonstrates that sg formation and translational shut-off observed during ibv replication both occur in the absence of detectable levels of eif2α phosphorylation, but that ibv infection does not actively inhibit eif2α phosphorylation. data presented is representative of three independent replicates. asterisks indicate statistical significance, as measured by one-way anova, *** p = 0.0004; **** p < 0.0001. both sg formation and translational shut-off are usually associated with phosphorylation of eif2α. previous work by others has demonstrated that ibv infection results in eif2α phosphorylation at early time points, but that the virus inhibits this as infection progresses [32] . therefore, the phosphorylation status of eif2α was investigated. vero cells were infected with ibv and lysed at 6, 12, 18 and 24 hpi. proteins were separated by sds-page and transferred to nitrocellulose. blots were labelled using anti-eif2α to detect total eif2α, anti-eif2α-p to detect the phosphorylated eif2α, anti-ibv to detect virus and anti-gapdh as a loading control ( figure 9a ). total levels of eif2α remained unchanged throughout infection. although eif2α phosphorylation was achieved using sodium arsenite treatment, no eif2α phosphorylation was detected at any time point during ibv infection, with levels remaining comparable to that of mock infected cells. subsequently, to determine whether ibv infection actively inhibits phosphorylation of eif2α, vero cells were infected with ibv and then treated with sodium arsenite prior to cell lysis ( figure 9b ). as before, the sodium arsenite treatment of mock infected cells resulted in a significant increase in the level of phosphorylated eif2α. when ibv infected cells were treated with sodium arsenite, there was also a significant increase in the level of phosphorylated eif2α when compared to ibv infected untreated cells. significantly, the level of phosphorylated eif2α in these cells appeared comparable to that in mock infected sodium arsenite treated cells ( figure 9b ). together, this demonstrates that sg formation and translational shut-off observed during ibv replication both occur in the absence of detectable levels of eif2α phosphorylation, but that ibv infection does not actively inhibit eif2α phosphorylation. hpi, cells were washed and lysed. samples were then separated by sds-page and transferred to nitrocellulose. total eif2α (eif2α-t) was detected with anti-eif2α, phosphorylated eif2α (eif2α-p) was detected with anti-eif2α-p. ibv proteins were detected using an anti-ibv antibody, with a band corresponding to the ibv nucleocapsid protein shown (ibv (n)) and an anti-gapdh antibody was used as a loading control. (b) vero cells were mock infected or infected with ibv. at 23 hpi, where indicated, cells were treated with naas. cells were lysed at 24 hpi and processed and labelled as in (a). blots are representative of three independent replicates. here, we present a study that furthers our understanding of how ibv regulates the important cellular pathways of the isr and translation. firstly, we have demonstrated that ibv infection inhibits both eif2α-dependent and independent sg formation. several other viruses, including kaposi's sarcoma-associated herpesvirus, zika virus, west nile virus and junin virus have been shown to inhibit sg signaling via regulation of the eif2α-dependent pathway [44] [45] [46] [47] . these viruses achieve this by inhibiting activation of pkr [45] , thereby preventing eif2α phosphorylation [47] or by total eif2α (eif2α-t) was detected with anti-eif2α, phosphorylated eif2α (eif2α-p) was detected with anti-eif2α-p. ibv proteins were detected using an anti-ibv antibody, with a band corresponding to the ibv nucleocapsid protein shown (ibv (n)) and an anti-gapdh antibody was used as a loading control. (b) vero cells were mock infected or infected with ibv. at 23 hpi, where indicated, cells were treated with naas. cells were lysed at 24 hpi and processed and labelled as in (a). blots are representative of three independent replicates. here, we present a study that furthers our understanding of how ibv regulates the important cellular pathways of the isr and translation. firstly, we have demonstrated that ibv infection inhibits both eif2α-dependent and independent sg formation. several other viruses, including kaposi's sarcoma-associated herpesvirus, zika virus, west nile virus and junin virus have been shown to inhibit sg signaling via regulation of the eif2α-dependent pathway [44] [45] [46] [47] . these viruses achieve this by inhibiting activation of pkr [45] , thereby preventing eif2α phosphorylation [47] or by dephosphorylating eif2α [44] . zika virus was found to upregulate growth arrest, and dna-damage-inducible 34 (gadd34), a component of the protein phosphatase 1 (pp1) complex, and subsequent dephosphorylation of eif2α [44] . interestingly, in the present study, ibv was also found to inhibit eif2α-independent sg signaling. flaviviruses and ebola virus also inhibit both eif2α-dependent and independent signaling. ebola virus achieves this via an interaction between vp35 and several sg components including; g3bp1, eif3 and eef2 [41, 48, 49] . whether any ibv proteins interact directly with sg components to inhibit sg formation remains to be determined. interestingly, hydrogen peroxide has also been found to inhibit sgs via the oxidation of tia-1. arimoto-matsuzaki et al. (2016) also concluded that when cells are exposed to both oxidative stress and er stress via protein misfolding, these cells cannot form sg [50] . it is therefore possible that the inhibition of hydrogen peroxide induced sgs presented here is achieved via er stress caused during ibv infection prior to hydrogen peroxide treatment [51] . despite ibv regulation of multiple sg signaling pathways, infection results in the formation of sg in approximately 20% of infected cells. numerous viruses such as poxviruses and reoviruses are known to divert sg components to sites of virus replication to benefit the virus [52, 53] . however, although not exhaustive, our analysis suggests that the sg formed in 20% of infected cells are canonical sg produced in response to virus replication. ibv induced sg were found to contain multiple sg marker proteins and were susceptible to cycloheximide treatment, hallmarks of canonical sg. in addition, the sg markers did not co-localize with markers for viral rna synthesis or particle assembly. therefore, they do not appear to resemble the virus-specific granules produced during the replication of other viruses. this then raises the interesting question of how sg form in this subset of cells. it is possible that the viral control of sg signaling may alter over the course of infection. however, eif2α was not phosphorylated, even at early time points post infection, and the sg positive subpopulation of cells remained constant at all time points tested. therefore, this would not appear to be the case. other viruses have also been shown previously to induce sg in only a proportion of infected cells. for example, semliki forest virus infection resulted in sg in 63% of infected cells at 4 hpi, with a further decrease after this point [54] . in chronic hepatitis c virus (hcv) infection, an oscillation of the stress response is seen, in which 40% of hcv infected cells treated with interferon-α had sg, but in a live time course, this was shown to oscillate in a cell-specific rhythm, with 97% of cells displaying sg at some point during infection. this appears to be a strategy by hcv to modulate the cellular stress response by pkr activated eif2α phosphorylation or conversely, dephosphorylation of eif2α by the upregulation of gadd34 in a balancing act to prolong cell survival with oscillating stalls in translation and cell division [55] . therefore, it is possible that in the ibv infected cells containing sg, viral regulation of eif2α phosphorylation or other sg signaling pathways is less efficient, or these cells perhaps represent a more complex balancing act. another possible explanation for this subset of sg displaying cells is a pre-priming of the cellular innate immunity via a paracrine signaling effect, as seen with interferon (ifn) signaling, in which paracrine ifn activates the jak/stat pathway and upregulates interferon stimulated genes (isgs). vero cells do not secrete ifn, and so are unlikely to use this specific paracrine signaling pathway, however it cannot be ruled out for other signaling molecules. analysis at the single cell level will likely be required to tease apart the mechanism of sg formation in the subset of ibv infected cells that contain them. other members of the cov family have been found to display markedly different relationships with sg and their regulation. transmissible gastroenteritis virus (tgev) infection was shown to induce specific antiviral sg. in contrast to observations seen here where ibv genomic rna did not co-localize with g3bp1, these tgev specific granules feature an interaction between polypyrimidine tract binding protein (ptb) and viral genomic and sub-genomic rna [26] . during mouse hepatitis virus (mhv) infection, translational shut-off and sg formation was also observed and mhv replication was enhanced upon infection of eif2αs51a −/− cells or tia −/− cells in which translational inhibition and sg formation are impaired, indicating an inhibitory role for sg during mhv replication [27] . similar to our observations here, mers-cov inhibits sg formation. this is achieved via an interaction between mers-cov accessory protein 4a and dsrna, preventing pkr activation [31] . therefore, taken together, and in agreement with our findings here that ibv inhibits multiple sg induction pathways, this suggests an antiviral function for sg during cov replication. sg usually form following translation inhibition, as a result of the aggregation of stalled mrnps, translation initiation factors and rna binding proteins. therefore, the translational activity of ibv infected cells was investigated. in agreement with previous work [34] , translation inhibition occurred during ibv replication from 12 hpi and the degree of inhibition increased as infection progressed. however, only 20% of infected cells contain sg and this remained constant across all time points studied. it was considered possible that cells with reduced translational activity are the 20% observed to contain sg, even though this would not account for all the cells found to have reduced levels of translation, particularly at later time points. therefore, the relationship between sg and translational inhibition in ibv infected cells was assessed using rpm and sg staining simultaneously. this confirmed that de novo protein synthesis in ibv infected cells without sg is significantly reduced, although it is reduced to a greater extent when sg are present. therefore, the translational inhibition seen during ibv infection cannot be solely attributed to sg formation and, interestingly, there is an uncoupling of translational arrest and sg formation. indeed, we observe both sg formation and translational inhibition in the absence of eif2α phosphorylation, indicating altered signaling for both pathways. this situation has also been observed during murine norovirus (mnv) replication, where cellular translation is inhibited and canonical sg assembly is blocked through the repurposing of g3bp1 independently from eif2α phosphorylation [56, 57] . mnv translational control is achieved via the phosphorylation of eif4e by mnk1, which in turn, is activated by the p38 kinase [58, 59] . in addition, several cov have been shown to activate the interferon-induced endoribonuclease rnase l [60, 61] . rnase l cleaves cellular and viral rnas producing ligands of pattern recognition receptors to amplify ifn production and the antiviral response [62] . interestingly, rnase l has recently been shown to reduce translation independently of pkr, phospho-eif2-alpha, and sgs [63] . moreover, rnase l activity has been linked with the assembly of specific membrane-less organelles [63, 64] . in cells treated with the pkr ligand poly(i:c), rnase l activation also resulted in the inhibition of chemically-induced sgs, similar to our observations with ibv, and the assembly rnase l-bodies (rlb) that are small sg-like punctate, that do not require g3bp1 for assembly and are uncoupled from translation inhibition via pkr [63] . interestingly, another study using the specific rnase l ligand 2-5a showed that rnase l stimulation resulted in the assembly of antiviral stress granules (avsgs) [64] . these avsgs are different from the rlbs in that they require g3bp1 for assembly, which interacts with pkr and rig-i in these granules, but also oas and rnase l. the assembly of avsgs further stimulates irf3-mediated ifn production, rather than ifn signaling, and may provide a platform for the interaction of rna ligands, with pattern recognition receptors to amplify the ifn production. despite these discrepancies, further investigation into the role of rnase l in ibv-mediated regulation of translation and sgs is warranted. the mechanism of ibv translational control is currently unknown. other covs have been shown to inhibit translation through the action of a viral non-structural protein, nsp1, which binds the 40s ribosomal subunit and cleaves host mrna [65] . however, ibv does not express nsp1. instead, ibv accessory protein 5b was found to be responsible for translational shut-off and the stability of some mrnas tested was actually increased upon ibv infection, suggesting a completely different mechanism for the control of cellular translation [34] . during this work, ibv replication did not result in phosphorylation of eif2α, at any of the time points tested. furthermore, infection was not able to limit sodium arsenite induction of eif2α phosphorylation, showing that ibv cannot actively inhibit eif2α phosphorylation. this is in contrast to previous findings that ibv nsp2 is a weak antagonist of pkr and that gadd34 is upregulated during ibv infection, resulting in decreased levels of phosphorylated eif2α [32] . however, recently gadd34 has been shown to promote sg disassembly and block further stress sensing and eif2a phosphorylation, but also to contribute to impairing sg assembly independently from the eif2a pathway [63, 66] . therefore, we do not exclude that gadd34 induction may contribute to the absence of sgs in ibv-infected cells. a subsequent study by the same laboratory found that ibv infection also induced the phosphorylation of perk, and the subsequent activation of atf4 and the proapoptotic gadd153, again resulting in dephosphorylation of eif2α [33] . the reason for the inconsistency between our current findings and the previous work is not clear, although one methodological difference in the current study is the use of sodium arsenite to induce eif2α phosphorylation, which acts via hri, whilst the previous studies showed the activation if eif2α phosphorylation in virus infected cells, via activation of either pkr or perk. notably however, in our work presented here ibv replication did not induce phosphorylation of eif2α at any time point, and it is therefore not necessarily surprising that mechanisms to dephosphorylate eif2α are also not activated. indeed, the signaling molecule required for activation of pkr, dsrna, is known to be concealed within virus induced vesicles during coronavirus replication [67] . furthermore, interferon signaling, which also relies upon the sensing of dsrna is not activated in ibv infected cells until very late time points, consistent with the shielding of dsrna from cellular detection [68] . therefore, the activation of these various cellular signaling pathways in response to ibv infection is likely to be prevented, consistent with our findings. in the present study, we have demonstrated that ibv replication effectively blocks both eif2α-dependent and eif2α-independent sg signaling pathways. in addition, ibv replication results in a shut-off of translation. however, interestingly, in a proportion of infected cells, canonical sg are formed that do not localize with sites of viral replication and do not contain viral rna. this raises the interesting future possibility of being able to study the composition and function of canonical cellular sg in virus infected cells. in addition to these findings, in ibv infected cells, both translational repression and sg formation were found to occur in the absence of eif2α phosphorylation, although ibv replication was not able to actively inhibit eif2α phosphorylation. therefore, the ibv infection of cells results in a dysregulation and uncoupling of several important cellular signaling pathways. the mechanism behind this dysregulation remains to be determined, but we have furthered our understanding of how ibv changes the cellular environment to make it favorable for virus replication. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/5/536/s1, figure s1 . active replication is required for ibv induced stress granules. translation inhibition and stress granules in the antiviral immune response tinkering with translation: protein synthesis in virus-infected cells stress granules: sites of mrna triage that regulate mrna stability and translatability the dsrna protein kinase pkr: virus and cell control protein translation and folding are coupled by an endoplasmicreticulum-resident kinase translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses activation of gcn2 in uv-irradiated cells inhibits translation herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perk and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein antiviral effect of the mammalian translation initiation factor 2alpha kinase gcn2 against rna viruses inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation inhibition of eukaryotic translation initiation by the marine natural product pateamine a stimulation of mammalian translation initiation factor eif4a activity by a small molecule inhibitor of eukaryotic translation functional characterization of ireses by an inhibitor of the rna helicase eif4a hydrogen peroxide induces stress granule formation independent of eif2alpha phosphorylation principles and properties of stress granules distinct stages in stress granule assembly and disassembly the role of stress granules and the nonsense-mediated mrna decay pathway in antiviral defence phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization formation and maturation of phase-separated liquid droplets by rna-binding proteins rna self-assembly contributes to stress granule formation and defining the stress granule transcriptome stress granules regulate double-stranded rna-dependent protein kinase activation through a complex containing g3bp1 and caprin1 valiente-echeverria, f. who regulates whom? an overview of rna granules and viral infections inhibition of cytoplasmic mrna stress granule formation by a viral proteinase chikungunya virus nsp3 blocks stress granule assembly by recruitment of g3bp into cytoplasmic foci analysis of subcellular g3bp redistribution during rubella virus infection the polypyrimidine tract-binding protein affects coronavirus rna accumulation levels and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies g3bp1 interacts directly with the fmdv ires and negatively regulates translation correction: g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses inhibition of protein kinase r activation and upregulation of 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stress granule formation during infection and sequesters stress granule proteins within viral inclusions the stress granule component tia-1 binds tick-borne encephalitis virus rna and is recruited to perinuclear sites of viral replication to inhibit viral translation interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly zika virus inhibits eif2alpha-dependent stress granule assembly kshv inhibits stress granule formation by viral orf57 blocking pkr activation arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in west nile virus-infected cells junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif2alpha phosphorylation ebola virus vp35 blocks stress granule assembly flavivirus infection uncouples translation suppression from cellular stress responses tia1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis the endoplasmic reticulum stress sensor ire1α protects cells from apoptosis induced by the coronavirus infectious bronchitis virus colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions virus-mediated compartmentalization of the host translational machinery sequestration of g3bp coupled with efficient translation inhibits stress granules in semliki forest virus infection dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection mouse norovirus infection arrests host cell translation uncoupled from the stress granule-pkr-eif2alpha axis norovirus infection results in eif2α independent host translation shut-off and remodels the g3bp1 interactome evading stress granule formation mitogen-activated protein kinases activate the serine/threonine kinases mnk1 and mnk2 murine norovirus 1 (mnv1) replication induces translational control of the host by regulating eif4e activity during infection middle east respiratory syndrome coronavirus ns4b protein inhibits host rnase l activation activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon a scientific journey through the 2-5a/rnase l system rnase l promotes the formation of unique ribonucleoprotein granules distinct from stress granules rnase l amplifies interferon signaling by inducing pkr-mediated antiviral stress granules coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression protein synthesis inhibition and gadd34 control ifn-β heterogeneous expression in response to dsrna sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum activation of the chicken type i interferon response by infectious bronchitis coronavirus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank ambi batra for providing bei-inactivated ibv. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-262940-eyejnexx authors: liu, genmei; lv, lishan; yin, lijuan; li, xiaoming; luo, dongu; liu, kang; xue, chunyi; cao, yongchang title: assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins date: 2013-11-12 journal: vaccine doi: 10.1016/j.vaccine.2013.09.024 sha: doc_id: 262940 cord_uid: eyejnexx infectious bronchitis virus (ibv) as an avian coronavirus is still posing a persistent and imminent threat to the poultry industry worldwide. here we report that transfection of sf9 cells with a single recombinant baculovirus encoding m and s proteins resulted in the assembly of ibv vlps; this is the first report that s protein plus m protein alone were able to be assembled into vlps for coronaviruses. we further showed that the generated ibv vlps could induce humoral immune responses in a level comparable to that of inactivated ibv vaccine, and more importantly the ibv vlps could elicit significantly higher cellular immune responses than the inactivated ibv vaccine. in summary, the assembly of ibv vlps with m and s proteins provided a simple strategy for generating vlps for coronaviruses, and the generated ibv vlps laid a feasible foundation for the development of an effective vaccine against infection of ibv in the future. avian infectious bronchitis virus (ibv) causes an acute and highly contagious viral disease of chickens, and leads to huge economic losses in poultry industry worldwide. ibv is an enveloped rna virus and its virus particle morphology is spherical with a 80-120 nm in diameter. ibv contains four viral structural proteins: membrane protein (m), spike protein (s), envelope protein (e), and nucleocapsid protein (n). the m protein contains a short amino-terminal ectodomain with 2 glycosylation sites, and is 23-35 kda with varying degrees of glycosylation. homotypic interactions among m proteins and interactions with the s protein and the other structural proteins are required for virus particle formation. thus, the m protein plays an important role in coronavirus assembly [1] [2] [3] [4] . ibv m protein is found in the cis-golgi network and cis-golgi complex when expressed alone [5] , and cannot be released into the supernatant unless ibv e is present [6] . in contrast, studies of other coronaviruses suggested that the m protein was observed in the golgi area and plasma membranes of a variety of cells when expressed alone [7] , and can form sedimentable particles and release from infected cells [8] . the s protein forms projections on the surface of the virion and generates two subunits, s1 and s2, through posttranslational cleavage. the s1 protein contains epitopes that can induce neutralization, hemagglutination inhibition (hi) and serotype-specific antibodies [9] [10] [11] [12] [13] [14] . obviously, the s protein is the main immunogenic protein and will be a target protein when designing new ibv vaccines. the s protein is transported to the plasma membrane when expressed alone [15] . incorporation of spikes into coronavirus particles is effected by interactions between the s protein and the m protein and governed by the carboxy-terminal domain of the s protein [16] . numerous vlps assemble in an in vitro expression system, contain the major structural viral proteins, and mimic the conformation and organization of authentic native viruses without the viral genome [17] . for ibv vlps, some researchers have reported that e proteins are sufficient for formation, but the efficiency is extremely low [6] . other researchers have described that interactions between the e and m proteins and the membrane bilayer probably played an important role in vlp formation and virus budding [18] . nowadays, ibv is controlled using live attenuated and inactivated vaccines, but ibv frequently outbreaks in endemic areas. thus, the development of new vaccines is urgent. in the present study, we assembled ibv vlps containing m and s proteins using a baculovirus expression system and we further evaluated the vlps immune responses in mice and chickens. spodoptera frugiperda (sf9) insect cells were grown adherent in t-flask in the complete grace's insect cell culture medium and incubated at 27 • c. human epithelial kidney cells (293t) were grown in the complete dulbecco's modified eagle's medium and incubated at 37 • c in 5% co 2 . h120 strain of ibv was propagated in 9-day-old chick embryos and inactivated by 0.1% formalin at 37 • c for 24 h. the inactivated h120 was purified by ultracentrifugation at 80,000 × g for 3 h at 4 • c on a discontinuous sucrose gradient of 20%, 30%, 40%, 50%, and 60% sucrose. ibv m and s genes were amplified from the total rna extracted from the allantoic fluid of h120-infected chick embryos using reverse transcriptase polymerase chain reaction (rt-pcr) and subcloned into plasmid pfastbac tm dual (pfdual) (invitrogen), either individually or simultaneously (fig. 1) . the recombinant plasmids were chemically transformed into competent dh10bac tm escherichia coli cells (invitrogen). the recombinant shuttle plasmids rbacmid-m, rbacmid-s, and rbacmid-s-m were obtained and identified by pcr using m13 primers. a total of 8 × 10 5 sf9 cells per well grown in 6-well culture plates were transfected with 5 g purified recombinant bacmid dna mixed with 6 l cellfectinr ii ® reagent (invitrogen) in 210 l in supplemented grace's medium. after incubating the transfected cells at 27 • c for 4 h, the transfection mixture was removed and replaced with complete growth medium, and the cells were incubated at 27 • c. the supernatant was collected through centrifugation when 90% of cells had cytopathogenic changes. the recombinant baculoviruses rb-m, rb-s, and rb-s-m harvested from the supernatant were propagated and purified 3 times using viral plaque in sf9 cells. at 72 h postinfection, supernatants from infected sf9 cells were collected, filtered, and centrifuged at 80,000 × g for 60 min at 4 • c. sediments were suspended in phosphate-buffered saline (pbs) plus 0.1 mm phenylmethylsulfonyl fluoride (pmsf). next, adherent cells were rinsed twice and collected in pbs plus 0.1 mm pmsf, sonicated, and microcentrifuged at 3500 × g for 15 min at 4 • c to remove cell debris. the samples were resolved through electrophoresis on 8%, 10%, and 12% sds-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (bio-rad). the expressed proteins were detected with chicken polyclonal sera raised against ibv virus at a 1:3000 dilution and horseradish peroxidase (hrp)conjugated anti-chicken secondary antibody at a 1:5000 dilution (ptglab, usa). at 48 h post-infection, the infected sf9 cells grew on glass cover slips were fixed in 100% ice-cold methanol at 4 • c and blocked with pbs-tween 3% bovine serum albumin plus 0.2% triton tm x-100. fixed cells were incubated with the primary antibody at a 1:200 dilution and with the secondary antibody at a 1:300 dilution. m proteins were detected with mouse polyclonal sera raised against m protein expression with 293t cells and secondary anti-mouse fluorescein isothiocyanate (fitc)-conjugated antibody (ptglab, usa). s proteins were detected with chicken polyclonal sera raised against s1 protein expression with 293t cells and secondary antichicken cy3 conjugated antibody (ptglab, usa). cell nuclei were stained with 4 ,6-diamidino-2-phenylindole. cover slips were visualized under a confocal laser scanning microscope (tcs sp5, leica). at 48-72 h post-infection, the culture media of infected sf9 cells was collected, filtered, and microcentrifuged at 3500 × g for 15 min at 4 • c to remove cell debris. the supernatant was ultracentrifuged at 80,000 × g for 60 min at 4 • c. vlps collected in the pellet were suspended in pbs. to further purify them, the vlps suspension was loaded on a discontinuous sucrose gradient of 20%, 30%, 40%, 50%, and 60% sucrose and ultracentrifuged at 80,000 × g for 3 h at 4 • c. vlps at the interface between 30% and 40% sucrose were collected and pelleted by ultracentrifugation at 80,000 × g for 1.5 h at 4 • c. vlp-containing pellets were resuspended in pbs and analyzed for the presence of ibv structural proteins using coomassie blue stained sds-page electrophoretogram and western blot. the purified vlps were placed onto carbon-coated, 200-mesh copper grids for 2 min. sample-containing grids were washed with water, dried with filter paper, and stained with 1% phosphotungstic acid (ph 6.5) for 1 min. excess staining solution was removed with filter paper. vlp-containing grids were viewed with a transmission electron microscope (jem-100cx, jeol ltd., japan). images were taken at 40,000× magnification. the total protein concentrations of vlp and inactivated ibv were determined using bradford protein assay kit (beyotime, china) with bovine serum albumin (bsa; takara, japan) as standard; and the s protein concentrations of vlp and inactivated ibv were determined using sds-page gel electrophoresis by genesnap and genetools from syngene software with bsa as standard. twenty-four 6-week-old specific-pathogen-free (spf) balb/c female mice (experimental animal center, sun yat-sen university) without ibv-specific antibody were randomly divided into 3 groups of 8 mice. the mice were housed in positive pressure and immunized 3 times (weeks 0, 2, and 4) with freund's adjuvant. group 1 mice were immunized with 2 g vlps (s protein) per mouse via subcutaneous injection. group 2 mice were injected with 2 g inactivated h120 (s protein) per mouse as a positive control. group 3 mice received pbs as a negative control. sera were collected from mice at days 14, 28, and 42 after initial immunization for ibv-specific antibody detection. sixty 10-day-old spf chickens without ibv-specific antibody were randomly divided into 3 groups of 20 chickens. the chickens were housed in individual isolators under positive pressure and immunized 2 times (weeks 0 and 2) with oil adjuvant. the immunization program and immunization dose of chickens were the same as the mice did. sera were collected from chickens at days 14 and 28 after primary immunization for ibv-specific antibody detection. the purified h120 virosomes were used as antigen to detect the ibv-specific antibodies in an indirect elisa. the h120 virosomes reconstitution with the detergent octaethylene glycol monododecyl ether (c12e8) and purification with discontinuous sucrose gradient centrifugation were carried out following the procedures as previously described [19, 20] . the secondary rabbit anti-mouse or donkey anti-chicken hrp-conjugated antibodies were used at a 1:5000 dilution. the optical density value was 450 nm. the neutralizing antibody titer was determined using a neutralization test performed on chick embryos following the procedures as previously described [21] . serum samples were incubated for 30 min at 56 • c to inactivate nonspecific inhibitors. treated sera were serially diluted twofold and incubated with an equal volume of h120 with 100 eid50 at the final concentration for 1 h at 37 • c. 100 l of the incubated sera-viral mixture was injected into 9-dayold spf chick embryos. the 100 eid50 of h120 without serum was used as a negative control. the injected eggs were incubated for 7 days at 37 • c. the neutralizing antibody titer was determined using the reciprocal of the highest dilution of serum that gave 50% neutralization of 100 eid50 of virus in chick embryos. spleens were collected from mice on day 42 after the primary immunization. the lymphocytes were isolated from the spleens using mouse 1× lymphocyte separation medium (dakewe, china). the number of ifn-r and il-4 secreting cells in the single-cell suspension of splenocytes was determined with a elispot kit (dakewe, china) following the manufacturer's protocol. the splenocytes were stimulated with purified h120 virosomes at a 10 g/ml concentration, and the spots were counted using the immunospot elispot reader (bioreader 4000, bio-sys, germany). the data between groups were statistically analyzed by using a student's two-tailed t test when only two groups were compared or by one-way analysis of variance (anova) when more than two groups were compared. p values less than 0.05 (p < 0.05) were considered statistically significant. the supernatant culture media and cell lysates from the recombinant baculovirus-infected sf9 cells were harvested and analyzed using western blot. the results showed that m and s proteins could be expressed in the sf9 cells (fig. 2) . interestingly, when m protein was expressed alone, it could be detected in the sedimentable particles of the culture media ((a), lane 2), but s protein could not be detected when it was expressed alone ((b), lane 2). more importantly, when s protein was expressed together with m protein, s protein could be detected in the sedimentable particles of the culture media ((c), lane 2). the expressions of m and s proteins were further confirmed by immunofluorescence staining analysis. sf9 cells were first infected with rb-m, rb-s or rb-s-m, respectively, and then stained with m-or s1-specific primary antibodies and fitc-or cy3-conjugated secondary antibodies. the results showed that both m and s proteins were mainly expressed near the plasma membranes, and m and s proteins were co-localized (fig. 3) . the detection of s protein in the sedimentable particles when it was co-expressed with m protein strongly suggested that ibv vlps could be generated using m and s proteins only. sf9 cells were infected with rb-s-m, and supernatant culture media were harvested and then the sedimentable particles were subjected to sucrose density gradient ultracentrifugation. fractions were collected and analyzed by western blot (data not shown). the fraction collected from the interface between 30% and 40% showed the coexpression of m and s proteins, where a distinct protein band of roughly 23-35 kda corresponding to the m protein and a 170 kda band corresponding to the s protein ( fig. 4a and b ). using em analysis, we could see a spherical morphology particle with a diameter of about 100 nm (fig. 4c ). we investigated the immunological characteristics of ibv vlps both in mice and chickens. ibv-specific antibody in the sera of immunized mice was detected using indirect elisa. the results indicated that, 2 weeks after the primary vaccination (day 14), serum igg titers could be detected in vlps and inactivated h120 groups, and the titers continued to increase following the second and third immunizations (days 28 and 42) (fig. 5) . the vlps and inactivated h120 groups had significantly higher igg titers (p < 0.01) than the pbs group. as for the vlps and h120 groups, the vlps group, after the first and second immunizations, had lower igg titers than the inactivated h120 group; and the vlps group, after the third immunization, had higher igg titers than the inactivated h120 group, but they were not statistically different (p > 0.05). we further researched the immunogenicity of ibv vlps in chickens. the results showed that, 2 weeks after the primary vaccination (day 14), both of vlps and inactivated h120 groups could not detected serum igg titers, and the differences between these and pbs groups were not statistically significant (p > 0.05); but following the second immunization (day 28), the igg titers of the vlps and inactivated h120 groups increased (fig. 6 ) and were significantly higher (p < 0.01) than the pbs group. the differences between vlps and inactivated h120 groups were not statistically significant (p > 0.05), although the vlps group had a slightly lower igg titers after the second immunization. antisera from vaccinated mice and chickens were analyzed using a neutralization assay to detect functional antibodies with neutralization activity against the h120 virus. the ibv-specific neutralization antibodies in mice were detected at 42 days after the primary immunization and in chickens the antibodies were detected at 28 days after the primary immunization. the results showed that vlps and inactivated h120 groups had statistically significantly higher neutralizing antibody titers (p < 0.01) than the pbs group (fig. 7) . the differences between vlps and inactivated h120 groups were not statistically significant (p > 0.05), although the vlp group had a slightly higher neutralizing antibody titer. 7 . neutralizing antibody titers of mice and chickens sera. y1 means neutralizing antibody titers of mice sera collected on day 42; y2 means neutralizing antibody titers of chickens sera collected on day 28. virus like particles (vlps) and inactivated h120 groups had significantly higher neutralizing antibody titers (p < 0.01) than the pbs group. the groups were not statistically different (p > 0.05), although the vlp group had a slightly higher neutralizing antibody titer. 3.5. ibv vlps induced significant higher cellular immunoresponses than inactivated h120 vaccine did in mice cellular immunoresponses were evaluated through detection of the number of cells secreting ifn-r and il-4 after h120 virosome stimulation of the single-cell suspension splenocytes from vaccinated mice using the elispot assay. the results revealed that the levels of ifn-r and il-4 in mice which inoculated with vlps were significantly higher than in those which inoculated with inactivated h120 and pbs (p < 0.01) (fig. 8 ). the molecules required for the formation of coronavirus vlps varied in different reports. ho et al. showed that m and e proteins were required for the assembly of human sars coronavirus-like particles in sf21 cells [22] ; but m, e and n proteins were required for the formation of sars-cov vlp as demonstrated by siu et al. in vero e6 cells [23] and nakauchi et al. in 293t cells [24] . liu et al. found that influenza m1 protein and sars s protein could assemble chimeric sars-cov vlps in sf9 cell [25] . for mouse hepatitis virus (mhv-a59), vennema et al. showed that transfection of ost7-1 cells with vaccinia virus encoding m and e proteins resulted in the vlp formation [26] . these reports strongly suggested that m and e were the minimal molecules required for the coronavirus-like particles regardless of virus types and cell lines used in the studies. in contrast, our results showed for the first time that ibv m and s proteins were efficient assembled into vlps in sf9 cells with a single recombinant baculovirus encoding m and s proteins. this might not be a complete surprise. as for ibv, corse and machamer reported that while the presence of e protein could enhance the release of m proteins in the sedimentable particles from the culture supernatants, m protein alone was capable of being released in the sedimentable particles from the culture supernatants in ost7-1cells with a vaccinia-t7 polymerase expression system [18, particularly fig. 6 ]. in addition, the native envelope trimeric s glycoprotein is not necessary for particle formation but is incorporated into vlps when present [22] ; and incorporation of spikes into coronavirus particles is mediated by the s protein's carboxy-terminal domain and effected by interactions between the s protein and the m protein [16] . the showing of ibv vlps formed by m and s proteins demonstrated a simple strategy for production of coronavirus-like particles for coronaviruses. s protein is a main antigen for coronaviruses [9, 12] . in line with previous literatures, our results showed that ibv vlps induced humoral antibody responses (i.e., serum ibv-specific igg antibodies and ibv-specific neutralization antibodies) in a level comparable to that of the inactivated ibv vaccines in mice and chickens, and both ibv vlps and inactivated ibv vaccine induced significantly higher humoral antibody responses (p < 0.01). for the cellular immune responses, the ifn-r and il-4 were detected using the elispot assay. the results revealed that ibv vlps elicited high levels of ifn-r and il-4, but the inactivated h120 vaccine only induced il-4 at a significantly lower level (p < 0.01). the results demonstrated that ibv vlps could elicit both th1-and th2-type cellular immune responses, but inactivated h120 only stimulated th2-type cellular immune response. previous studies showed that vlps could elicit more effective antibody responses than proteins in their nonnative forms [27, 28] . the reason for the results that the inactivated h120 vaccine used in this study had slightly lower neutralizing antibody titers and a significantly lower level of cellular immune responses than vlps might be that a portion of s molecules lost their native forms during inactivation with formalin. our results demonstrated that the ibv vlps mimic the conformation and organization of authentic native viruses, and their immunogenicities were reserved. in summary, we have successfully generated ibv vlps carrying m and s proteins and shown that the generated ibv vlps could effectively elicit humoral immune responses both in mice and chickens, and more importantly, ibv vlps could induce significantly higher cellular immune responses than inactivated ibv vaccine in mice. as the results taken together demonstrated a simple strategy for generating coronavirus-like particles and provided a candidate vaccine against the infection of ibv for future development. assembly of the coronavirus envelope: homotypic interactions between the m proteins coronavirus structural proteins and virus assembly the molecular biology of coronaviruses protein interactions during coronavirus assembly the e1 glycoprotein of an avian coronavirus is targeted to the cis golgi complex infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles self-assembly of severe acute respiratory syndrome coronavirus membrane protein generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection structural proteins of avian infectious bronchitis virus: role in immunity and protection the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens immune responses to structural proteins of avian infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction reconstitution of the fusogenic activity of vesicular stomatitis virus functional reconstitution of influenza virus envelopes animal virology assembly of human severe acute respiratory syndrome coronavirus-like particles the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles analysis of severe acute respiratory syndrome coronavirus structural proteins in virus-like particle assembly chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin membrane embedded hiv-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes this study was supported by the grants from chinese national high-tech r&d program (863 program, 2011aa10a209) and cooperation project in industry, education and research of guangdong province and ministry of education of people's republic of china (grant no. 2010b090301019). we thank to dr. george d. liu for critical review and revision of the manuscript. key: cord-273661-egpyvqrw authors: mo, mei-lan; li, meng; huang, bai-cheng; fan, wen-sheng; wei, ping; wei, tian-chao; cheng, qiu-ying; wei, zheng-ji; lang, ya-hui title: molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china date: 2013-12-04 journal: viruses doi: 10.3390/v5123007 sha: doc_id: 273661 cord_uid: egpyvqrw to gain comprehensive genetic information of circulating avian coronavirus infectious bronchitis virus (ibv) isolates in china, analysis of the phylogenetic tree, entropy of the amino acid sequences, and the positive selection as well as computational recombinations of s1, m and n genes of 23 ibv isolates was conducted in the present study. the phylogenetic trees based on the s1, m and n genes exhibited considerably different topology and the ck/ch/lsc/99i-type isolates were the predominant ibvs based on the phylogenetic analysis of s1 gene. results of entropy of amino acid sequences revealed that the s1 gene had the largest variation; the m gene had less variation than the n gene. positive selections were detected in not only s1 but also m and n gene proteins. in addition, five s1 gene recombinants between vaccine strain 4/91 and ck/ch/lsc/99i-type field isolate were confirmed. in conclusion, multiple ibv genotypes co-circulated; genetic diversity and positive selections existed in s1, m and n genes; 4/91 vaccine recombinants emerged in china. our results show that field ibvs in china are continuing to evolve and vaccine strains may have an important role in the appearance of new ibv strains via recombination. in addition, the present study indicates that ibv evolution is driven by both generations of genetic diversity and selection. infectious bronchitis (ib) is an acute, highly infectious and contagious disease of domestic chickens worldwide caused by avian infectious bronchitis virus (ibv), a member of genus gammacoronavirus, subfamily coronavirinae, family coronaviridae [1] . ib affects chickens of all ages and ibv replicates primarily in the respiratory tract, and also in some epithelial cells of the kidney, gut and oviduct, resulting in reduced performance, reduced egg quality and quantity, increased susceptibility to infections with other pathogens, and condemnations at processing [2] . multiple ibv serotypes or genotypes have been identified worldwide and different serotypes of ibvs confer little or no cross-protection against the others. ibv genome consists of a linear, single-stranded, positive-sense rna of 27.6 kb, which encodes four major structural proteins, the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) protein and the envelope or small membrane (e) protein [3] . the s glycoprotein is post-translationally cleaved into s1 and s2 subunits and s1 is the most divergent region, which carries conformationally-dependent virus-neutralizing and serotype-specific epitopes [4, 5] . the n protein located in the capsid of the virion is involved in rna replication, assembly and carries group-specific antigenic determinants [6] and has high immunogenicity, readily inducing antibodies and cytotoxic t-lymphocyte immunity in chickens [7] . s1 and n genes have been used most frequently to determine the relatedness of emerging strains of ibv [5, 8] . the m protein is a structural membrane protein and plays an important role in the viral assembly process and particularly is indispensable for many biological functions including viral core stability. interactions of m and e proteins are important for virus budding and formation of virus-like particles, which are involved in mucosal immunity [9] . the genetic diversity and viral evolution of ibv are mainly monitored by analysis of the s1 gene because of its high variability and close serotype correlation [10] , but viruses within the same serotype can have a high degree of genetic variability outside of the spike gene [11] . pathogenicity of ibv is associated with the spike gene as well as genes outside of the spike gene [12] . the m protein is associated with virus assembly and change this protein will affect the efficiency of virus particles formation and subsequent transmission of the virus [3] . the n protein plays an important role in viral replication, assembly, and immunity. in addition to s1 glycoprotein, the n protein could represent an important target in the prevention of ib outbreaks [13] . recent evidence revealed that there are significant variations in the n and m genes between strains [13, 14] . therefore, it is necessary to analyze multiple genes especially to analyze the genetic variation of s1, m and n genes considering their importance as structural proteins. the major challenge for the prevention and control of ib is the increasing number of new serotypes or variants of ibv, which was caused by frequent gene mutation and recombination [15] [16] [17] [18] . recombination is thought to be a contributing factor in the emergence and evolution of ibv or even the emergence of new coronaviruses and new diseases [3] . the studies of ibv recombination are very important for ibv control, because they will further our understanding of the diversity and evolution mechanisms of these viruses and thus enable the development of better control methods [3, 18] . ibv strains within a geographic region are unique and distinct [19] although many countries share some common antigenic types. therefore, it is extremely critical to identify the prevalence of ibvs and genetic characteristics of circulating strains in a region or a country in order to develop effective vaccines for the control of the disease. outbreaks of ib have been occurring frequently in china in spite of intensive vaccinations for many years [15, 17, [20] [21] [22] . ib is still a major problem in guangxi province [15, 17] , which is located in southern china and produces a total of 700 million birds per year and ranks third in china [23] . it is very important to know the genetic characteristics of prevalent strains of ibvs in this region. we previously reported the genotype diversity of guangxi ibv isolates based on the hypervariable region i (hvr i) of s1 gene [15] , but the available comprehensive genetic information of circulating ibv strains in this region was limited. therefore, in the present study we performed the analysis of the phylogenetic tree, of the entropy of the amino acid sequences, of positive selection as well as of computational recombination based on the sequencing results of the viral structural protein genes s1, m and n in order to provide molecular epidemiology information of ibv and to lay a good foundation for the control of ib in the field. the nucleotide and deduced amino acid (aa) sequence identities of the s1, m and n genes among the 23 isolates were 76.1%-99.9% (aa: 74.7%-99.8%), 88.1%-100.0% (aa: 89.1%-100.0%) and 86%-100.0% (aa: 90.4%-100.0%), respectively. the identities of nucleotide and deduced amino acid sequences of s1, m and n genes between the 23 isolates and all the reference strains were 57.9%-99.9% (aa: 47.2%-99.8%), 78.5%-100.0% (aa: 66.2%-100.0%) and 85.6%-99.9% (aa: 83.4%-99.7%), respectively. compared with the most popularly used vaccine strain h120, all the isolates had lower nucleotide sequence identities (s1: 76.2%-83%; m: 87.9%-90.2%; n: 85.7%-87.9%) except for gx-nn1 and gx-nn2 (99.5% and 99.7) in the s1 gene, gx-nn1, gx-nn2 and gx-nn5 (99.8%, 99.7% and 99.8%) in the m gene, gx-nn5 (99.1%) in the n gene (supplementary table s1 ). both the phylogenetic trees constructed with the neighbor-joining and maximum-likelihood method had very similar topography, so only the neighbor-joining trees are shown ( figure 1 ). the phylogenetic trees based on s1 gene amino acid sequences showed that all ibv isolates except gx-c were divided into five distinct groups (figure 1a ). eleven out of 23 isolates were grouped into the ck/ch/lsc/99i-type with china ibv reference strains ck/ch/lsc/99i, saibk and a2, which were isolated during 2004-2008. isolates gx-nn7, gx-nn11, gx-nn9, gx-nn10, gx-nn8, gx-yl7 and reference vaccine strain 4/91 were classified into the 4/91-type, but the latter five isolates occupied another offshoot. gx-yl6 and reference strains ldt3, partridge/gd/s14/2003 and korea strain km91 were grouped as the tl/ch/ldt3/03-type. amazingly, gx-g and gx-xd isolated in 1988 were grouped with taiwan reference strains tw2296/95, tw2575/98 as taiwan-type. isolates gx-nn1 and gx-nn2 showed a close relationship with commonly used vaccine strains h120, h52, ma5, m41 and other china vaccine strains w93, h94, d41, ibn, hk and grouped as mass-type. gx-c, isolated in 1985, showed considerable low homology with the above five genotypes and belonged to a separate group. (a) s1 the phylogenetic trees of m and n genes showed that the 23 isolates were segregated into 4 distinct groups, which exhibited considerably different topology than that of the s1 gene (figure 1b,c) . in the phylogenetic trees of the m gene, 12, 2, 3 and 6 isolates were designated as ck/ch/lsc/99i-type, lx4-type, mass-type and bj-type respectively. in the phylogenetic trees of the n gene, 9, 4, 9 and 1 isolates were designated as lx4-type, new-type, ck/ch/lsc/99i-type and mass-type respectively. the results of the codon-based tests of positive selection (z-test, mega5) for analyzing the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) on the s1, m and n proteins were displayed as supplementary material (supplementary figures s1). no significant evidence for positive selection of s1 protein of taiwan-type and mass-type groups was observed (p > 0.05). however the result of analysis of entropy of s1, m and n genes on amino acid sequences was shown in figure 2 . the higher the peak is, the greater the entropy is, indicating the higher variation frequency of amino acid sites. numerous high entropy amino acid sites were distributed throughout the entire s1 gene; only a few high entropy amino acid sites were scattered within the m gene. the number of high entropy amino acid sites within the n gene is less than that of the s1 gene but more than that of the m gene (supplementary material). an entropy value bigger than 0.4 indicated the corresponding amino acid site was not conserved. the percentages of entropy bigger than 0.4 in amino acids sequences of s1, m and n gene were 29.87% (164/549), 6.61% (15/227) and 11.46% (47/410), respectively. the descending average entropy order were s1 (0.2651) > n (0.0953) > m (0.0831). therefore, the s1 gene amino acid sequences had the largest variation; the m gene had less variation than the n gene. recombinant events were detected in the s1 gene of isolates gx-nn8, gx-nn9, gx-nn10, gx-nn11 and gx-yl7 by all recombination detection methods implemented in the rdp4.14 software. these five isolates were found to be recombinants between the vaccine strain 4/91 and the ck/ch/lsc/99i-type field strain gx-yl2 (figure 3) one of the major problems caused by ibv in the field is the frequent emergence of new variants. ibv strains within a geographic region are unique and distinct [19] . outbreaks of ib still occurred in guangxi [15, 17] although vaccines have been applied and the molecular epidemiology information available was limited. hence, we investigated the genetic characteristics of s1, m and n genes of ibvs circulating in this region. this is the first report on the analysis of entropy of amino acid sequence and the positive selection of s1, m and n genes of ibvs. some investigators reported the genetic typing based on hvr i of the s1 gene is representative of the grouping based on the whole s1 gene [8, 24] , but another study disagreed with these findings [25] . the present results and our previous report from hvr i [15] also indicated that genotypes based on hvr i are not representative of that based on the whole s1 gene. the reason was that mutations in non-hvr i of the s1 gene were also detected [25] . in addition, in addition, our results showed that genotypes based on s1 gene exhibited considerably difference from m and n genes. the discordance of topology in the s1-based tree and other gene-based trees were also described by other investigators [26, 27] . the co-circulation of multiple ibv types and the ongoing emergence of ibv variants are the epidemiological challenges in china. nine genotypes including lx4-type, ck/ch/lsc/99i-type, tl/ch/ldt3/03-type, ck/ch/ldl/97i-type, bj-type, ck/ch/lhlj/95i-type, mass-type, 4/91-type, and n1/62 associated circulated in china and lx4-type was the dominant genotype [20] . recently, the taiwan ii-type was firstly reported in china [22] . in our study, ck/ch/lsc/99i-type, tl/ch/ldt3/03-type, 4/91-type, taiwan-type and mass-type were identified, which suggested that multiple genotypes of ibvs were co-circulating in guangxi province. eleven out of 23 isolates sharing 76.8-77.9% s1 gene amino acid sequence similarity with the vaccine strain h120 belonged to ck/ch/lsc/99i-type. however, the prevalent genotype in this region was ck/ch/lsc/99i-type not the lx4-type, which was different from other reports that lx4-type was the dominant genotype in china [20, 22] , indicating ibv strains within this region are unique and distinct. ibv has been diagnosed in china since the early 1980s [20] . surprisingly, the gx-g and gx-xd isolated in 1988 showed closed relationship with taiwan ii-type strain tw2296/95 and no taiwan-type strain occurred in recent year in our study. recently other investigators reported that taiwan ii-type strains of ibv occurred in mainland china [22] . whether the taiwan-type ibvs entered china by long distances migration of wild birds or importing of poultry products or improper use of vaccines？it is unclear. identification of tai-wan and china-like recombinant ibvs in taiwan was reported [28] . so it is very important to monitor the vaccine, birds and poultry products in china. natural selection generally causes a reduction in deleterious mutations while promoting advantageous mutations. a gene which undergoes positive selection promoted by natural selection usually has highly important functions [29] . some investigators reported positive selection wasn't detected in the spike protein of ibvs although they differed markedly in the sequence of the spike protein [26, 30] . however, other investigators showed different results. positive selection was detected in the spike protein of ibv california-type viruses, for which no vaccine exists but was not detected in massachusetts-and connecticut-types where attenuated live vaccines are routinely used [11] . another report showed that positive selection was found in the s1 protein of variants isolated from layer-type birds but was not found in variants isolated from broilers, even though a high number of mutations was significantly associated with broiler-type chickens [25] . positively selected sites in the nucleocapsid protein of the taiwan ibv and their effects on rna-binding activity were reported recently [31] . previous reports on sars-cov indicated that positive selection on s protein was changeable in different epidemic groups and positive selection on replicase of sars-cov was detected only in human patients, not in any proteins of bat sars-like-cov [29] . we found positive selection was observed on s1 protein of 4/91-type and ck/ch/lsc/99i-type strains, m protein of ck/ch/lsc/99i-type and bj-type strains, n protein of ck/ch/lsc/99i-type and lx4-type strains. thus, not only s1 protein but also m and n proteins experienced positive selection during the ibv epidemics. the variation of positive selection of s, m and n proteins among different groups may explain why these field variants escape immune pressure and may provide valuable evidence that these three structural proteins may be critical for virus evolution. it is the first time to analyze the positive selection of s1, m and n genes of ibvs. the entropy is one useful quantification of diversity in a single position of amino acid sequences [32] . high scoring amino acid positions may correlate with structurally or functionally important residues [33] . the greater the entropy is, the higher variation frequency of amino acid sites is. an entropy value bigger than 0.4 indicated the corresponding amino acid site was not conserved [34] . a shannon entropy analysis of immunoglobulin and t cell receptor revealed that the t cell receptor is significantly more diverse than immunoglobulin-suggesting t cell receptor has new complementarity determining regions, which represent a larger antigen combining site, additional combining sites, or an evolutionary strategy to avoid inappropriate interaction with other molecules [35] . a recent study used the shannon entropy and relative entropy to measure the diversity of amino acid site of h3 ha between the 1992-1993 season and the 2009-2010 season and showed that the rate of evolution increases with the virus diversity in the current season and the shannon entropy of the sequence in the current season predicts relative entropy between sequences in the current season and those in the next season [32] . according to our results, the average entropy of amino acid sequences, the percentages of entropy bigger than 0.4 and the number of amino acid sites with high entropy of s1 gene are biggest, and those of n gene were bigger than m gene. thus, these observations revealed that amino acid sequences of s1 gene had the largest variation; the m gene had less variation than the n gene. to our knowledge, it is the first time to analyze the entropy of s1, m and n gene amino acid sequences. the variation of amino acids will have an important effect on the biological function and evolution of viruses. hence, observing the biological function of the amino acid residues with higher entropy and identifying the positively selected sites among ibvs will be further studied. recombination is involved in the emergence and evolution of ibv or can even directly lead to the emergence of new coronaviruses and related diseases [36] recombination can occur between field isolates or between field and vaccine viruses [36] [37] [38] . in our study, convincing evidence showed five s1-gene recombinants gx-nn8, gx-nn9, gx-nn10, gx-nn11 and gx-yl7, with their putative parental strains of vaccine strain 4/91 and ck/ch/lsc/99i-type field strain gx-yl2, and their crossover regions were at nucleotide position 7-677 or 7-678. a recently report showed a recombinant (ck/ch/lzj/111113 strain) came from a chinese field isolate (ck/ch/ldl/091022 strain, lx4-type) and a 4/91-like strain, with switches at 3 sites, namely upstream of s, the n gene and the 3' utr [39] . besides the mass-type vaccine, 4/91-type live vaccines are also commonly used in china including during the breeding period [39] , even without official authorization. our finding provides another evidence that 4/91 vaccine strains are contributing to the emergence of variants in the field in china. therefore, it is necessary to strengthen the vaccine licensing system before introduction of exotic ibv strains. we should continued 49/1-type recombinants surveillance in china. the pathogenicity of 4/91-derived recombinants should be assessed in further studies. twenty-three ibv strains, isolated as previously described [15] were analyzed in the present study. the ibv field isolates were propagated in 9 to 11-day-old specific pathogen free embryonated chicken eggs via the allantoic cavity route. allantoic fluids were harvested at 48 h post-inoculation, frozen, and stored at −70 °c until used. for each ibv strain, the entire s1, m and n genes were amplified. the s1 primers were designed according to the previous report and the anticipated amplification segment is about 1760 bp encompassing the entire s1 gene including the protease cleavage motif [40] . the m gene sense primer was:5'-cgagtttcctaagaacggttggaa-3', and the anti-sense primer was: 5'-cccctctctacacgcacacatttat-3'. the n gene sense primer was: 5'-ccatggcaagcggtaaagcar-3', and the anti-sense primer was: 5'-ccactcaaagttcattctctcc-3'. the anticipated amplification segments for m and n genes are 750 bp and 1236 bp respectively. viral rna was extracted from the infectious allantic fluid by the trizol reagents (invitrogen, usa) according to the manufacturer's instruction. the first cdna strand was synthesized in 25 µl mixture consisting of 9 µl of rna extract, 1µl of 50 µm/µl random 9 mers, 5µl of 5 ×reverse transcriptase first strand buffer, 1 µl of 40 u/µl rnase inhibitor (takara, japan), 1 µl of 200 u/µl amv reverse transcriptase (takara, japan) and 8µl of 2.5mmol/l dntpmix (takara, japan). the mixture was incubated at 42 °c for 1 h, and then inactivated at 99 °c for 5 min. for the following pcr assays, a total of 25 µl reaction mixture consisted of 2 µl of the cdna, 2.5 µl of 10× pcr buffer, 2 µl of 2.5 mmol/l dntpmix (takara, japan), 1 µl of 25 µmol/l of each of the two primers and 0.25 µl of 5 u/µl taq dna polymerase (takara, japan). the pcr conditions for the s1 gene amplification were 94 °c for 6 min, 35 cycles of 94 °c for 45 s, 55 °c for 45 s, and 72 °c for 2 min, followed by 72 °c for 10 min; that for the m gene were 94 °c for 5 min, 35 cycles of 94 °c for 1min, 50 °c for 1 min, and 72 °c for 1 min, followed by 72 °c for 10 min; and that for the n gene were 94 °c for 5 min, 35 cycles of 94 °c for 1 min, 50 °c for 1 min, and 72 °c for 2 min, followed by 72 °c for 10 min. the pcr products were analyzed on 1.0% agarose-gel electrophoresis. the pcr products were purified, cloned and then sequenced by sangon bio-company (shanghai, china). for each gene, three independent clones were selected randomly and sequenced twice from both directions. the open reading frames of s1, m and n gene were determined using the dnastar version (dnastar, madison, wi). the nucleotide sequences of s1, m and n genes have been submitted to genbank database and assigned accession numbers (supplementary table s2 ). the nucleotide and the deduced amino acid sequences alignments were generated using the clustalw multiple alignment method of bioedit version 7.0.9.0 and compared with those of 42 reference ibv strains retrieved from the genbank database with the accession numbers listed in in supplementary material (supplementary table s3 ). phylogenetic trees were constructed based on the amino acid sequences of s1, m and n genes with the neighbor-joining method (jones-taylor-thornton (jtt) model) and maximum-likelihood method (jtt model) using mega 5.05 version. the bootstrap values were determined from 1000 replicates of the original data. the entropy is one useful quantification of diversity in a single position of amino acid sequences. a large entropy means the amino acid in the given position is prone to be substituted. in order to understand the variation degree of s1, m and n genes, the entropy of aligned amino acid sequences within these genes of the isolates was calculated by bioedit version 7.0.9.0. in addition, codon-based tests of positive selection (z-test, mega5) were used to estimate the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) within the s1, m and n proteins in order to understand whether these proteins are submit to positive selection.4.6. computational recombination analysis. aligned nucleotide sequences of s1, m and n genes were analyzed with the recombination detection program (rdp4, version 4.14) to detect potential within-gene recombination events. the window size was adjusted to 40 bp from the default setting 30 bp because ibv has a high mutation rate, which can mask recombination signals. the highest acceptable p value was 0.05 and the detection of recombination events was applied between sequences sharing 0 and 100% identity. seven algorithms in rdp 4.14, including rdp, geneconv, bootscan, maxchi, chimaera, siscan and 3seq were used to confirm the recombination events. two phylogenetic trees, which were constructed from the portion of the alignment between the inferred breakpoints and the remainder of the alignment were made and compared to assess recombination events further. recombination events and recombination breakpoints were further confirmed by similarity plot and bootscan analyses using the simplot program (version 3.5.1.). in conclusion, multiple ibv genotypes co-circulated; genetic diversity and positive selections existed in s1, m and n genes; 4/91 vaccine recombinants emerged in china. our results show that field ibvs in china are continuing to evolve and vaccine strains may have an important role in the appearance of new ibv strains via recombination. in addition, the present study indicates that ibv evolution is driven by both generations of genetic diversity and selection. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus recombination in avian gamma-coronavirus infectious bronchitis virus location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions immune responses to structural proteins of avian infectious bronchitis virus rapid detection and identification of avian infectious bronchitis virus typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s1 gene the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction 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infectious bronchitis viruses and emergence of new recombinants in korea molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates a 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china identification of the avian infectious bronchitis coronaviruses with mutations in gene 3 genetic diversity of avian infectious bronchitis coronavirus in recent years in china molecular epidemiological investigation of marek's disease virus from guangxi relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus genetic diversity of avian infectious bronchitis virus california variants isolated between 1988 and 2001 based on the s1 subunit of the spike glycoprotein recombinational histories of avian infectious bronchitis virus and turkey coronavirus complete nucleotide sequences of s1 and n genes of infectious bronchitis virus isolated in japan and taiwan identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan differential stepwise evolution of sars coronavirus functional proteins in different host species identification of intertypic recombinant infectious bronchitis viruses from slaughtered chickens evolution of infectious bronchitis virus in taiwan: positively selected sites in the nucleocapsid protein and their effects on rna-binding activity quantifying selection and diversity in viruses by entropy methods, with application to the haemagglutinin of h3n2 influenza incorporating background frequency improves entropy-based residue conservation measures crystal structure of the polymerase pa(c)-pb1(n) complex from an avian influenza h5n1 virus a shannon entropy analysis of immunoglobulin and tcell receptor molecular characterization of infectious bronchitis virus isolates from russia and neighbouring countries: identification of intertypic recombination in the s1 gene complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h120 characterization of a recombinant coronavirus infectious bronchitis virus with distinct s1 subunits of spike and nucleocapsid genes and a 3' untranslated region isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during this work was supported by grants from the national natural science foundation of china (31360611, 31160516), guangxi natural science foundation (2013gxnsfca019010), and the guangxi provincial programs for science and technology development (0993009-2). and the manuscript was kindly reviewed by richard roberts, aurora, co 80014, usa. the authors declare no conflict of interest. key: cord-261388-d56ci0hl authors: tibbles, k.w; cavanagh, d; brown, t.d.k title: activity of a purified his-tagged 3c-like proteinase from the coronavirus infectious bronchitis virus date: 1999-05-28 journal: virus res doi: 10.1016/s0168-1702(99)00011-8 sha: doc_id: 261388 cord_uid: d56ci0hl previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (ibv) large open reading frame (orf1), confirmed the activity of a predicted 3c-like proteinase (3clp) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kda protein, 3clpro, capable of further cleavages in trans. in order to identify such cleavages within the orf1 polyprotein mediated by 3clpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3clp domain as targets. the proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3clpro. a histidine (his(6)) tag was introduced close to the c-terminus of the proteinase to aid purification. processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. from these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. this tentatively completes the processing map for the orf1 region with respect to 3clpro. the coronavirus infectious bronchitis virus (ibv) encodes the major portion of its non-struc-tural proteins within two large open reading frames, orfs 1a and 1b, situated at the 5' end and occupying the major portion of the 27.6 kb single-stranded positive-sense rna genome . the orfs overlap slightly with orf1b accessed via programmed ribosomal frameshifting (brierley et al., 1989) resulting in orf1a and orf1a/1b (a fusion) trans-lation products. the large size of the orfs suggests that the translation products are polyproteins that are processed to generate a set of mature non-structural proteins. in common with all other coronaviruses so far examined, ibv encodes a 3c-like proteinase domain which is located towards the c-terminus of the orf1a polyprotein and approximately centrally in the orf1a/1b fusion product. the 3clp domain was first identified as a possible component of ibv polyproteins (gorbalenya et al., 1989) due to sequence similarity to picornavirus 3c proteinases (reviewed in ryan and flint, 1997) . in the same study, predictions were made as to likely target dipeptide cleavage sites for a 3c-like proteinase within the orf1 polyproteins, the large number suggested implicating this proteinase in a pivotal role in the maturation of the polyproteins. similar domains were subsequently identified in mouse hepatitis virus (mhv) (lee et al., 1991) and human coronavirus (hcv 229e) (herold et al., 1993) . there are no cellular counterparts for the activities of the 3c-like proteinases, therefore they are essential to the successful maturation of their respective polyproteins and subsequent replication events. this, and the comparatively high degree of conservation (at functional level), makes the 3clike proteinases attractive targets for antiviral intervention. given the essential role played by the 3c-like proteinase of the coronaviruses in maturation of the 'polymerase' polyproteins, there has been considerable interest in characterising the enzyme, with initial emphasis on defining the processing events involving the proteinase. such studies are already at an advanced stage for both ibv (liu et al., 1994 and hcv 229e orf1b regions (grotzinger et al., 1996; heusipp et al., 1997a,b) . 3c-like proteinase activities have been confirmed recently for three representatives of the coronavirus group, mhv (lu et al., 1995) , hcv 229e (ziebuhr et al., 1995) and ibv (tibbles et al., 1996) and the protein has been identified in vivo during viral infection in the case of hcv (ziebuhr et al., 1995) and mhv (lu et al., 1996) . in addition, the proteinases from these two viruses have been purified and partially characterised (seybert et al., 1997; ziebuhr et al., 1997) . the identification of the ibv 3clpro was first realised in vitro when a cloned fragment of the polyprotein was expressed in reticulocyte lysate (tibbles et al., 1996) . in the presence of membranes, upon which activity was vitally dependent, processing of the region containing the protease domain was observed with the release of a 35 kda protein (p35). this product was capable of trans processing, but was inefficient and of limited use for mapping studies. we undertook the identification of the remaining ibv 3clpro cleavage targets within the orf1 polyprotein using bacterial expression to obtain significant quantities of 3clpro. the bacterial expression system described utilises the minimum processing unit identified in our previous studies which established that, in addition to the 3clp domain, mp2 protein sequence between the q/s 4 cleavage target and a point delimited by an ncoi restriction site (ntd position 10118) was that minimally required for processing. a bamhi fragment released from pkt205 (see below) was introduced into bamhi digested pet3xa (novagen) to yield pet205 ( fig. 1 ). this construct would allow expression to be monitored through the synthesis of the strongly expressed t7 gene 10 protein, which composes the major insoluble fraction component upon extraction. expression beyond the ncoi site was restricted by digestion of pet205 with ncoi followed by end repair and religation. this manipulation introduces a frame-shift in the sequence which results in a translation stop signal being encountered. this plasmid was then used to create an internal deletion between the vector initiator atg triplet and the ibv sequence by digestion with nhei and bamhi. the newly generated plasmid, petdn205dc nco , was modified to include a his-6 tag approximately 35 aminoacid residues from the c-terminus of 3clpro to aid in purification. a mutation which replaced the amino-acid sequence vknsqw (single letter code) with six histidine residues near the c-terminus of the 3clp domain in pkt205 (ntds 9679fig. 1 . schematic representation of the ibv orf1a/1b polyprotein with positions of predicted cleavage dipeptide targets shown as solid vertical bars, those with arrowheads have been experimentally confirmed. predicted catalytic domains are also indicated (mp1/2; membrane proteins, 3clp; 3c-like proteinase, pol; polymerase, zn; zinc finger, hel; helicase). the derivation of the eukaryotic and bacterial expression clones and the principal cloning vectors used are depicted. the principal restriction sites used in cloning manipulations (adjacent solid bars), promoters (t7, sp6 and ptac) and translation (tln) start sites for the plasmids are also shown. 9696) was made, giving rise to pkt205his. the positioning of this tag sequence was determined by consideration of the possible amino-acid sequences involved in cleavage at the q/s 4 site and potential for interaction with the affinity matrix based on predicted hydrophilicity (kyte and doolittle, 1982) of the proteinase sequence. this modified sequence was transferred to the 205bearing pet plasmid via replacement of a nsii fragment. the processing properties of the modified proteinase were tested extensively, using a variety of systems, and were found to be indistinguishable from wild-type (data not shown). therefore the appropriate pet plasmids (studier et al., 1990) were introduced into e.coli bl21 plyss cells (studier, 1991) . cultures were grown for approximately 3 h until the od 600 was between 0.6 and 0.9 and expression induced by the introduction of iptg (at 100 mg/ml) into the growth medium. the cells were harvested after a further 3 h. cells were lysed, clarified and the supernatant applied to a chelating agarose column charged with cobalt chloride. the use of cobalt instead of the usual nickel resulted in lower binding of background material (data not shown). alternatively, cultures were induced after 1.5 -2 h (od 600 0.3 approx.) and rifampicin was added (to 200 mg/ml) after a further hour and incubation continued for 3 h. this resulted in a substantial inhibition of cellular protein expression without affecting ongoing t7-dependent expression. although this resulted in a lower overall protein yield, there was a higher proportion of 3clpro and background binding for the metal chelate column selection was significantly reduced. typical results are shown in fig. 2 where the appearance of a novel 35 kda protein was observed after induction (compare lanes 1 and 2, panel a). clarification of the crude lysate (lane 3) by centrifugation revealed that the major portion of the protein of interest (i.e. 35 kda) remained in the supernatant (lane 5, compare to lane 4, pellet fraction). comparison of the total applied protein and the unbound fraction (lanes 5 and 6 respectively) revealed that over 90% of the novel band seen after induction was retained on the column. this putative 3clproh6 bound with relatively low affinity and was seen to elute at 60mm imidazole (lane 8), conditions normally associated with removing background binding. no observable protein remained to be eluted with 1m imidazole (lane 9). the background contaminating proteins could be significantly reduced by expression in the presence of rifampicin. while this resulted in a reduction in the total protein yield of some 80%, the proportion that was 3clproh6 was greater and resulted in less background in the purified product (panel b, compare products under normal conditions, lane 1, with those obtained in the presence of rifampicin, lane 2, after single step purification on a cobalt column). only fractions seen to contain the putative 3clproh6 were found to cleave target polypeptide targets in a trans assay which supports the identity of the induced product as 3clproh6 (data not shown). from comparison with known concentration protein standards (bsa) we estimated the yield to be approximately 2-4 mg/l of culture (0.5 -1 mg/l in the presence of rifampicin), which compares favourably with that obtained by ziebuhr et al., 1997 . different regions of the orf1a/1b polyprotein were expressed in reticulocyte lysate (tnt, promega) or bacterial cell extract (s30, promega) in the presence of [ 35 s] methionine using the following expression plasmids (see fig. 1 ). construction of the pkt205 and pktbp5 plasmids (encoding ibv sequence from nucleotides (ntd) 8693 -10927 and 10752 -12600 respectively) has been described elsewhere (liu et al., 1994) . these encode predicted cleavage sites q/s 3 to q/g 6 and q/s 7 to q/s 9 , respectively, within the c-terminal region of orf1a. ibv sequence between ntd 16785 and 23060 (boursnell et al., 1987) , amplified by rt-pcr using a first-strand cdna synthesis kit (pharmacia) and pfu polymerase (stratagene), was cloned into pcrscript (stratagene). this plasmid, pcrf4, was digested with espi and psti (following end repair of espi site with dna polymerase) and the fragment introduced into ecorv-psti digested pkt0/ns1 vector to produce pkt1bc (encoding nucleotides 16841 -20926). the polyprotein fragment encoded contains the three predicted 3clpro cleavage sites q/g 11 , q/s 12 and q/s 13 from the c-terminal region of orf1b. cleavage dipeptides were mutated (to g/s or a/s) by oligonucleotide-directed mutagenesis as described previously (tibbles et al., 1996) . this region was transferred as a spei fragment to pgex4t2 (pharmacia) and deletions made to express portions containing each target dipeptide individually. the n-terminal half of the orf1a region (up to nucleotide position 6500 approx.) is encoded by the plasmid pkt1a3, constructed as described previously (liu et al., 1995) . analysis of the q/g 5 site was made using a kpni deletion of the pet205 plasmid, pet205d kpn . a hincii fragment (ntd 10438-10902) bearing the q/g 6 target from pkt205 was introduced into pgex4t2 to yield pgexmp2c (resulting in the production of a fusion with glutathione-s-transferrase (gst), smith and johnson, 1988) . the region encoding the mp1 region and q/g 2 was derived from pcr amplification and a fragment (ntds 6919-8693) cloned upstream of the 205 sequence in pkt205/ bp5. purified proteinase was added in varying amounts to the radiolabelled target fragments and incubated for up to 16 h at room temperature. cleavage patterns were analysed following polyacrylamide gel electrophoresis and an example of these analyses is shown in fig. 3 . complete digestion conditions for the fragment corresponding to the bp5 region of the polyprotein revealed a cleavage pattern that was consistent with the pattern expected from the cleavage predictions made by gorbalenya et al., 1989 . this target contained the predicted cleavage sites of q/s 7 , q/s 8 and q/s 9 , all of which, along with an additional unpredicted q/n site, have been experimentally confirmed (liu et al., 1997; ng and liu, 1998) . cleavage within bp5 would be expected to yield processed products of approximate sizes of 6, 24, 10, 17 and 3 kda (as they lie on the polyprotein) assuming cleavage at q/s 7 , q/n, q/s 8 and q/s 9 . in fig. 3 the effect of processing by the addition of 3clproh6 can be seen. the uncleaved target (lane 1) consisted of two species (of approximately 60 and 70 kda) because the sequence contained the frame-shift signal, followed by approximately 300 nucleotides from orf 1b. only the two largest cleavage products were resolved in this experiment (lane 2) which correspond to cleavage reinforced by comparing them with those produced after incorporation of [ 35 s] cysteine. the relative intensity of the two bands is reversed reflecting the inverse ratio of the two amino acids in the 24 and 17 kda products (data not shown). no cleavage was apparent in the absence of 3clproh6 (data not shown). cleavage at the predicted q/s 7 site was tested by mutation of the cleavage dipeptide to an uncleavable form. failure to cleave at the mutated q/s 7 site should result in the extension of the n-terminus of the 24 kda product by 6 kda resulting in the generation of a new cleavage product of approximately 30 kda. comparison of the cleaved wild-type 24 kda product (lane 2) and the product from the mutated polyprotein fragment (lane 3) revealed a decrease in the mobility of the more intensively labelled product (equating to approximately 30 kda), consistent with failure to cleave at the mutated q/s 7 site. polyprotein fragments constituting the remaining areas of the orf1 region were synthesised and similarly analysed for cleavage by purified 3clproh6. the results of all of these trans assay analyses are summarised in table 1 . we found that all so far identified target dipeptides are utilised by the 3clproh6 enzyme in vitro. it was at q/s 7 and q/n for the largest product of 24 kda and at q/s 8 and q/s 9 for the smaller of 17 kda. the identities of these two products were noted that under conditions expected to result in complete cleavage, not all target sites were digested to completion. these tended to be in orf1b. only three of the predicted sites fail to be cleaved, namely q/g 2 , q/g 5 and q/g 6 and no novel target sites were detected. the first of the uncleaved sites (q/g 2 ) lies upstream of both the 3clp and mp1 domains while the latter pair fall within the mp2 region. either these sites are not utilised by 3clpro or the conformation of the target fragments bearing them in our assays do not facilitate their recognition by the proteinase. however in the case of the q/g 6 -bearing gst fusion, cleavage consistent with use of the thrombin site within the fusion junction region was observed (data not shown), which suggests that no gross conformational abnormality exists that might preclude proteolysis. we have used bacterial expression of viral protein, comprising the ibv orf1a polyprotein fragment that gives rise to 3clpro via autocatalytic processing in vitro (tibbles et al., 1996) , to obtain significant amounts of highly purified active proteinase for trans processing studies. our rationale for modifying the starting pet plasmid containing the 205 sequence was as follows. expression of hydrophobic proteins from picornaviruses with deleterious effects on bacterial cells has been reported (lama and carrasco, 1996) . therefore termination of expression at the ncoi site within mp2 would still allow processing, according to our in vitro data (tibbles et al., 1996) , while at the same time minimising the possible toxicity of the highly hydrophobic mp2 region. the gene 10 sequence was deleted in order to promote the production of a soluble polyprotein fragment, equivalent to that which is able to mature in vitro, and so ensure processing would proceed. evidence suggests that in bacteria folding is post-translational (netzer and hartl, 1997) and so there was potential for the proteinase to become inactive due to the characteristic sequestration of pet fusion proteins into inclusion bodies. in the event this latter measure appeared unnecessary as soluble processed 3clpro was produced by the cells expressing fusion proteins (data not shown). this suggests that folding and processing were completed if not before completion of translation then before the sequestration into inclusion bodies. thus by whatever means the proteinase matures in its usual eukaryotic context seems to be facilitated in the bacterial cell, though not necessarily by analogous interactions. the precise nature of the interaction between mp2 and cellular components (membranes) necessary for maturation of 3clpro and its role in the mechanism of 3clpro processing (maturation or release) remain to be determined. we do not know as yet whether the membrane requirement for maturation of 3clpro also reflects a need for other associated cellular components such as chaperone functions. the fact that processing is able to proceed in an heterologous environment offers a possible means of investigating this further. in order to simplify purification of 3clpro, we introduced a histidine (his 6 ) tag which facilitates separation on a suitable metal chelation matrix. the tag could not be placed at the extreme n or c-termini of the protein as is usual since the cleavage dipeptide and surrounding sequences are crucial for correct processing (ryan and flint, 1997) . we introduced the tag into the nearest region of predicted hydrophilicity to the c-terminal processing site q/s4 so as to minimise interference with the sequences involved in processing and allow interaction of the exposed histidine residues with the purification matrix. the tagged proteinase (3clproh6) appears to be indistinguishable from wild-type and is readily purified to high purity. however, the affinity between modified proteinase and matrix is low and similar to background proteins suggesting that the tag may not be fully accessible. our observation seems to conflict with the interpretation of a mutational analysis of hcv 229e 3clpro by ziebuhr et al., 1997 which concluded that the c-terminus of 3clpro was important to function and intolerant of mutation. the his 6 -tag mutation here (a substitution) was well tolerated and would therefore appear to indicate a so called 'permissive site' in the c-terminus of the protein. we have shown by trans cleavage analysis that processing occurs at the majority of the predicted 3clpro cleavage dipeptides within the ibv orf1 polyprotein and that no further cleavages are apparent. our data reinforce recently reported in vivo studies and also partially overcome a potential shortcoming of these approaches where some products may not be consistently detectable, due to poor reactivity with antisera or short half life for example. this analysis tentatively completes the processing map of the orf1 region with respect to 3clpro. there appear to be no target sites for 3clpro in the n-terminal half of the orf1a polyprotein (covered by the 1a3 clone) so that processing of this region and possibly regions upstream is probably achieved through the plp domain (liu et al., 1995) , alone or in combination with other as yet unidentified proteinase activities. those target dipeptides that were not cleaved in our assays are associated with the hydrophobic domains. cleavage of the mp1 region downstream of 3clpro may be carried out by the plp domain or an alternative activity, although failure for 3clpro to cleave q/g 2 may reflect a shortcoming in the presentation of the targets. the data presented here also suggest that there is no processing between q/s 3 (the c-terminus of the 3clp domain) and q/s 7 so that the mp2 product containing uncleaved q/g 5 and q/g 6 dipeptides would be expected to be approximately 42 kda. notably these three hydrophobic region targets are not represented in either mhv or hcv 229e, although, along with ibv itself, these viruses bear a potential target at a position equivalent to just downstream of the ibv q/g 6 site. these possibilities and the assembly of processed products into polymerase complexes may be addressed using specific antisera raised to amino or carboxyl terminal regions of processing products indicated by the present studies. completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot the coronavirus non-structural proteins coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis characterization of a 105-kda polypeptide encoded in gene 1 of the human coronavirus hcv 229e nucleotide sequence of the human coronavirus 229e rna polymerase locus identification and subcellular localization of a 41 kda, polyprotein 1ab processing product in human coronavirus 229e-infected cells identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e a simple method for displaying the hydropathic character of a protein screening for membrane-permeabilizing mutants of the poliovirus protein 3ab the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products identification, expression, and processing of an 87-kda polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites intracellular and in vitro-translated 27-kda proteins contain the 3c-like proteinase activity of the coronavirus mhv-a59 identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 recombination of protein domains facilitated by co-translational folding in eukaryotes identification of a 24-kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites virus-encoded proteinases of the picornavirus super-group expression and characterization of a recombinant murine coronavirus 3c-like proteinase single-step purification of polypeptides expressed in escherichia coli as fusions with glutathione s-transferase use of bacteriophage t7 lysozyme to improve an inducible t7 expression system use of t7 rna polymerase to direct expression of cloned genes characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus a region of the infectious bronchitis virus 1a polyprotein encoding the 3c-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate characterization of a human coronavirus (strain 229e) 3c-like proteinase activity biosynthesis, purification, and characterization of the human coronavirus 229e 3c-like proteinase we thank ian brierley for critical reading of the manuscript. this work was supported by the wellcome trust, the biotechnology and biological sciences research council and the ministry of agriculture, fisheries and food, uk. key: cord-288309-6pw7t512 authors: kusters, j. g.; jager, e. j.; niesters, h.g.m.; van der zeijst, b.a.m. title: sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus date: 1990-12-31 journal: vaccine doi: 10.1016/0264-410x(90)90018-h sha: doc_id: 288309 cord_uid: 6pw7t512 abstract under laboratory conditions coronaviruses were shown to have a high frequency of recombination. in the netherlands, vaccination against infectious bronchitis virus (ibv) is performed with vaccines that contain several life-attenuated virus strains. these highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. this paper addresses the question of the frequency of recombination of avian coronavirus ibv in the field. a method was sought to detect and quantify recombination from sequence data. nucleotide sequences of eight ibv isolates in a region of the genome suspected to contain recombination, were aligned and compared. phylogenetic trees were constructed for different sections of this region. differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo rna recombinant had occurred. infectious bronchitis virus (ibv) causes considerable damage in the poultry industry by infections of the respiratory tract and the reproductive organs 1. ibv is the prototype strain of the coronaviridae, a family of positive stranded rna viruses with a genome of about 28kb. this genome contains the information for three structural proteins and a number of enzymes involved in virus replication (figure 2 ). although vaccines offer protection, they are often made ineffective by the continuous emergence of new serotypes 2"3. serotypespecific protective immunity is thought to be mediated by antibodies to a protein on the surface of the virion, the so-called peplomer protein *--7. the peplomer protein is synthesized as a precursor glycoprotein, which is cleaved into the subunits s1 and $2, derived from its n-and c-terminal half, respectively a-~°. point mutations are obviously involved in the generation of new serotypes. however, previous studies 1~ suggested that rna recombination is also involved in the generation of new antigenic variants. to address the question of the frequency of recombination in the generation of new field isolates the nucleotide sequences in five windows of homologous sequences of the genomes of eight ibv strains have been compared. to make this comparison the sequences of the $2 genes of d207 and d1466 were newly determined. both strains are dutch field isolates and are included in vaccines currently used in the netherlands. the alignment of the sequences suggests that the dutch field strain d20712, the british field strain 6/8213 and the japanese field strain kb8523 t4 result from rna recombination events. the origin of the ibv strains as well as the procedures used for the cloning and sequencing of the peplomer genes have been published previously 3'11'15. sequence data were processed using the programs of microgenic (beckman instruments, ref. 16) . phylogenetic trees were derived from nucleotide difference-matrices using the program of dr j. felsenstein (university of seattle) distributed as part of the phylip package 2.617. the $2 sequences the newly determined d207 and d 1466 sequences are based on data from two or more independent cdna clones. with strain d1466, two nucleotide differences between cdna clones were observed. neither of these differences resulted in an amino acid replacement. from the nucleotide sequences (submitted to the embl genbank and ddbj nucleotide sequence databases), amino acid sequences have been deduced. in figure 1 the amino acid sequences are listed together with those of all other known $2 sequences. assuming the conserved arg-arg-x-arg-arg-ser sequence to be the cleavage site between s1 and $28, the (table i) . however, 19 out of 20 cysteine residues and most glycosylation sites are completely conserved. s k v v n rk l s k v t v --r v z e n m v v -----f e k v y s v -p asg e k v h s v --p asg fq kq s s h i gla qdna miq r d nsf -'f--a d ~ i••dllft••e••glptddaykn•ta•plgflkdla•areyngll•l•pi•taem•tlyt••l•a•mafggitaagaipfa n f a -a -v ics k f i s dk l g s qk s v v qk am i s m41u,~y~da~ana~drl~tgrls~ls~la~ak~aeh~r~s~relat~k~ne~vk~ry~f~gngr/~ltip~napng f y l y t s d i yy s d pi yy k le l yak t ke t g g m ibis m41u,h m42h '\ m428 kb8523 6/82 d207 d1466 96q 97q 980 99q 1000 1010 1020 1030 ivfihfsytpd~fvnvt---a~vgf~vkpana-~s~yai~pangrgifi~vng--ssyyitardmympra~tagdi~tlts~anyvs v t e ~ ~ v ~ h t e n d t e -n ~ ~ d l m l t e y ----v 8d-'d'te gl vt ve ---t s n g v k q m41u,h m42h,s kb8523 6/82 d207 d1466 m41u m41h m42s,h kb8523 6•82 d207 d1466 104q 1050 106q 107q 1080 1090 ll0o iii0 vnkt____.vittfvdnddfdfndel skwwndt___~elpdfdkfnyt___vp ildid s eidr iqgviqglnds lidlekls ilktyikw e d i'e--y 0 --i v n tyd k ee k -t -d -e-g t i-'rr. ~ --rsrdf n q ~i v n__~sn ~. ---the nucleotide and amino acid sequences were compared for possible recombination points. from this comparison we conclude that the japanese field strain kb8523 is probably a recombinant. the $2 protein of this strain is almost identical to $2 of m41, except for the region corresponding to amino acids 929 to 1102 ( figure i) where the sequence is remarkably similar to d1466. this suggests a recombination event with two crossovers in this region. the alignment of the $2 genes indicates a second potential recombinant: the british field isolate 6/82. its s1 gene and most of the $2 gene are almost identical to those of strain d207. at the 3' end of the $2 sequence (at the lys-1105 codon) the percentage of identical nucleotides switches from 99% upstream to 57% downstream. at exactly the same position the nucleotide sequence of 6/82 becomes highly similar to the d1466 sequence: from 73% identity upstream to 97% downstream. the nucleotide identity between d207 and figure 1 d1466 is about the same on both sides of this putative crossover site: 73 and 67%, respectively. the above comparisons are both time consuming and subjective. a method was sought to test and quantify these results more rigorously. this was achieved by comparing phylogenetic trees for each of five sections (windows) of the $2 gene and its flanking sequences (see figure 2a ). the $2 gene was divided into three sections. vaccine, vol. 8, december 1990 607 in vivo recombination of ibv: j.g. kusters et al. the first section (ii in figure 2b ) contained the 5'-part of the $2 gene up to the putative kb8523 crossover site (in the codon for ile-929 in figure 1) . a second $2 tree (iii in figure 2b ) was constructed for the part of the $2 gene between the kb8523 and 6/82 crossover sites (from ile-929 to lys-1105). the 3'-border of this region did not exactly coincide with the second kb8523 crossover site (in the ser-ll02 instead of the lys-ll05 codon) but changing this border had no significant effect on the topology. the third $2 tree (iv in figure 2b ) was calculated for the region from the 6/82 crossover site (in the lys-ll05 codon) until the stop codon on position 1182. sequences upstream and downstream from the $2 gene were also used to construct phylogenetic trees. the tree based on s1 gene sequences (i in figure 2b ) has the same topology as an $2 tree based on region ii in $2. kb8523 has shifted to the d1466 branch in a tree for region iii of $2. region iv of $2 yields a tree in which 6/82 has shifted from the d207 to the d1466 branch, while kb8523 is again near m41 and m42. finally a tree (v) constructed from the e1 gene sequences shows that, compared to tree iv, also strain d207 has shifted towards the d1466 branch, and therefore may also be a recombinant. with the murine coronavirus mhv a high frequency of recombination was found both in vitro and in mouse brain after infection with a ts mutant of mhv strain a59 and wild type jhm-virus 18'19 phylogenetic trees constructed from five different windows of the genomes of eight ibv strains were used to detect crossover events. a clear advantage of this method is that, due to its simplicity, large numbers of homologous genes can easily be screened. the data presented in this paper suggest that genomic recombination not only occurs under laboratory conditions but also plays an important role in the generation of new virulent strains; at least three ibv strains, all isolated from outbreaks, are potential recombinants. pre,)ious data ~1 already indicated that the dutch field isolate v1397 is also the product of a recombination. considering the limited number of sequences analysed so far, in vivo recombination of ibv seems to occur at a rather high frequency. this has implications for the use of ibv vaccines consisting of several serotypes of live attenuated ibv strains. these vaccines offer the best protection, but they may prove dangerous in the long term by the induction of new variants, not only by point mutations but also through recombination. in this respect vaccination protocols that combine ibv with other live attenuated viruses are equally suspect. they may create ideal conditions for non-homologous recombination which may result in the emergence of recombinant virus species with unwanted properties. the biology and pathogenesis of coronaviruses the classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in britain between 1981 and 1983 molecular epidemiology of infectious bronchitis virus in the netherlands induction of humoral neutralising and haemagglutination-inhibiting antibody by the spike protein of avian infectious bronchitis virus coronavirus ibv; partial amino terminal sequencing of spike polypeptide $2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor of ibv strains beaudette and m41 antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies monoclonal antibodies to the $1 spike and membrane proteins of avian infectious bronchitis virus strain massachusetts m4t coronavirus ibv: virus retaining spike glycopolypeptide s2 but not $1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection eyidence for a coiled-coil structure in the spike protein of coronaviruses coronavirus proteins: biogenesis of avian bronchitis virus virion proteins phylogeny of antigenic variants of avian coronavirus ibv occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6•82 with that of ibv beaudette cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the peplomer protein sequence of the m41 strain of coronavirus ibv and its comparison with beaudette strains a comprehensive sequence analysis program for the ibm personal computer confidence limits on phylogenies: an approach using the bootstrap highfrequency rna recombination of murine coronaviruses in vivo rna-rna recombination of coronavirus in mouse brain cloning and sequencing of the gene encoding the spike protein of coronavirus ibv evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes the authors are grateful to ms g.a.w.m. kremers and ms k.a. zwaagstra for experimental contributions and to dr j.a. lenstra for advice and help with computer programs. the use of the caos/camm computer key: cord-293081-40pa5g89 authors: li, jun; shen, wei; liao, ming; bartlam, mark title: preliminary crystallographic analysis of avian infectious bronchitis virus main protease date: 2006-12-16 journal: acta crystallographica section f structural biology and crystallization communications doi: 10.1107/s1744309106052341 sha: doc_id: 293081 cord_uid: 40pa5g89 infectious bronchitis virus (ibv) is the prototype of the genus coronavirus. it causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. the coronavirus (cov) main protease (m(pro)), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. in this study, ibv m(pro) was overexpressed in escherichia coli. crystals suitable for x-ray crystallography have been obtained using microseeding techniques and belong to space group p6(1)22. x-ray diffraction data were collected in-house to 2.7 å resolution from a single crystal. the unit-cell parameters were a = b = 119.1, c = 270.7 å, α = β = 90, γ = 120°. three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function. infectious bronchitis virus (ibv) is the prototype of the genus coronavirus (spaan & cavanagh, 2004; lai & holmes, 2001) , the members of which can infect humans and multiple species of animals, causing a variety of highly prevalent and severe diseases (spaan & cavanagh, 2004; ziebuhr, 2005; pereira, 1989; lai & holmes, 2001) . for instance, a previously unknown human coronavirus called severe acute respiratory syndrome coronavirus (sars-cov), the emergence of which was most likely to have been the result of animalhuman transmission (lau et al., 2005; li et al., 2005) , was identified as the aetiological agent of a global outbreak of a life-threatening form of pneumonia called severe acute respiratory syndrome (peiris et al., 2003; kuiken et al., 2003; ksiazek et al., 2003; drosten et al., 2003) . from their genome sequences, coronaviruses can be classified into three distinct groups (spaan & cavanagh, 2004) . ibv belongs to group iii (lai & holmes, 2001) , which only infects avian species, and leads to a highly contagious disease affecting the respiratory, reproductive, neurological and renal systems of chickens, resulting in a drop in egg production in adult birds and damaging the developing reproductive system in young birds. ibv infection predisposes chickens to infection with secondary pathogens. the incidence of ibv-related nephritis can result in high mortality rates (siddell et al., 2005; cavanagh, 2005; ignjatovic & sapats, 2000) . ibv infection is prevalent in all countries with an intensive poultry industry, with the incidence of infection approaching 100% in most locations, causing great economic loss worldwide (ignjatovic & sapats, 2000) . at the present time, control of ibv relies almost exclusively on vaccination and management (holmes, 2001; siddell et al., 2005) . however, there are two major problems associated with the use of vaccines. firstly, there are many serotypes of ibv; secondly, there is at least circumstantial evidence to suggest that new serotypes may emerge by recombination, possibly involving the live vaccine virus itself (lee & jackwood, 2000; cavanagh, 2003) . the coronavirus main protease (m pro ) is a chymotrypsin-like cysteine protease (anand et al., 2002; yang et al., 2003) . it not only processes at its own flanking sites within the replicase polyproteins involved in viral rna synthesis, but also directs the processing of all downstream domains of the replicase polyproteins via at least 11 conserved cleavage sites, mediating coronavirus replication and transcription (anand et al., 2005; ziebuhr et al., 2000; ziebuhr, 2005) . the functional importance and the absence of cellular homologues identifies it as an attractive target for anti-coronavrius drug design anand et al., 2005) . to date, several crystal structures of coronavirus m pro s have been reported for group i and ii coronaviruses, which mostly infect mammals (anand et al., 2002 yang et al., 2003) . however, there are no structures available for group iii coronaviruses, which are distinct from those belonging to groups i and ii. in this report, we describe the crystallization and preliminary crystallographic analysis of ibv m pro as a prelude to the elucidation of its three-dimensional structure in order to provide a basis for rational drug design. the cdna encoding ibv m pro (m41 strain) was a gift from professor ming liao (south china agricultural university, people's republic of china). the gene sequence encoding the ibv m pro was amplified using the pcr method with forward primer 5 0 -cgg-gatcctctggttttaagaaa-3 0 and reverse primer 5 0 -ccg-ctcgagtcattgtaatctaacacc-3 0 and then inserted into the bamhi and xhoi sites of the pgex-4t-1 plasmid (pharmacia, new york, usa). the resulting plasmid was then used to transform escherichia coli bl21 (de3) cells. the sequence of the insert was verified by dideoxynucleotide sequencing. the positive clones harbouring the recombinant plasmid were grown to an od 600 of 0.6 at 310 k with shaking in 1 l lb medium containing 0.1 mg ml à1 ampicillin. the gst-fusion protein, gst-ibv m pro , was then expressed by introducing 0.5 mm iptg and incubating at 289 k for 10 h. cells were harvested by centrifugation, resuspended in lysis buffer (20 mm tris-hcl ph 7.3, 140 mm nacl, 2.7 mm kcl) and sonicated at 277 k. the cell lysate was centrifuged, the supernatant was collected and the fusion protein was purified by gst-glutathione affinity chromatography. the gst tag of the fusion protein was cleaved with gst-rhinovirus 3c protease. the recombinant ibv m pro was further purified using anion-exchange chromatography. the purified and concentrated ibv m pro (25 mg ml à1 ) was stored in 10 mm tris-hcl ph 7.5, 1 mm dtt for crystallization. the purified protein was concentrated to 25 mg ml à1 using a 5k filtron ultrafiltration membrane. protein concentrations were determined by absorbance at 280 nm. hampton research crystal screen kits were used to screen crystallization conditions and positive hits were then optimized. each drop was formed by mixing equal volumes (1.5 ml) of protein solution and reservoir solution and was allowed to equilibrate via vapour diffusion over 400 ml reservoir solution at 291 k. after one month, we obtained a cluster of small crystals. some small crystals were transferred into a microcentrifuge tube containing 20 ml stabilizing solution including 5% peg 4000, 12% 2-propanol, 0.1 m sodium cacodylate ph 6.5. the crystals were then crushed with a 3 mm diameter ptfe bead by vortexing (20 â 1 s cycles on a vortex mixer) and 180 ml of stabilizing solution was added to the suspension, making up the seed stock. the seeds in the seed stock were then transferred using a fine whisker to new drops that had been pre-equilibrated for 4-5 d. one week later, several single crystals appeared in the drops (fig. 1) . diffraction data were collected at the beijing synchrotron radiation facility (bsrf, institute of high energy physics, beijing, people's republic of china) at a wavelength of $1.0 å with a mar 345 (mar research, hamburg) image-plate detector at 100 k. the crystal was mounted on a nylon loop and flash-cooled in a cold nitrogen-gas stream at 100 k using an oxford cryosystems cryostream. the cryoprotectant solution contained 20% glycerol, 2% peg 4000, 9.6% 2-propanol, 0.08 m sodium cacodylate ph 6.5. a total of 120 frames of data were collected with an oscillation angle of 0.6 and an exposure time of 180 s for each image (fig. 2) . all intensity data were indexed, integrated and scaled with the hkl-2000 programs denzo and scalepack (otwinowski & minor, 1997) . initial microcrystals were obtained from condition no. 41 of crystal screen i (hampton research) containing 20% peg 4000, 10% 2-propanol, 0.1 m na hepes ph 7.5. optimization of the conditions yielded the best crystals from a solution containing 2.5% peg 4000, 12% 2-propanol, 0.1 m sodium cacodylate ph 6.5, but the crystals were found to be unsuitable for x-ray diffraction. crystal growth was further optimized by the microseeding technique and ibv m pro crystals subsequently diffracted to 2.7 å resolution. the crystals belong to space group p6 1 22, with unit-cell parameters a = b = 119.1, c = 270.7 å , = = 90, = 120 (see table 1 typical crystals of ibv m pro grown by the hanging-drop method in 2.5% peg 4000, 12% 2-propanol, 0.1 m sodium cacodylate ph 6.5. typical crystal dimensions are 0.3 â 0.06 â 0.05 mm. computational project, number 4, 1994) , the solvent content is estimated to be 55% and the matthews coefficient (v m ; matthews, 1968) 2.7 å 3 da à1 . selected data statistics are given in table 1 . the structural and functional analysis of ibv m pro will be published elsewhere. fields virology fields virology proc. natl acad. sci. usa andrewes' viruses of vertebrates topley and wilson's microbiology and microbia infections: virology virus taxonomy. viiith report of the ictv proc. natl acad. sci. usa we thank professor zihe rao for generous support and xiaoyu xue, qi zhao and haitao yang for technical assistance. key: cord-291718-cz1bi0ym authors: yu, liping; zhang, xiaorong; wu, tianqi; wang, yuyang; meng, jie; liu, qian; niu, xiaosai; wu, yantao title: the papain-like protease of avian infectious bronchitis virus has deubiquitinating activity date: 2017-03-18 journal: arch virol doi: 10.1007/s00705-017-3328-y sha: doc_id: 291718 cord_uid: cz1bi0ym coronavirus papain-like proteases (plps) can act as proteases that process virus-encoded large replicase polyproteins and also as deubiquitinating (dub) enzymes. like the plps of other coronaviruses (covs), the avian infectious bronchitis virus (ibv) plp catalyzes proteolysis of gly-gly dipeptide bonds to release mature cleavage products. however, the other functions of the ibv plp are not well understood. in this study, we found that ibv exhibits strong global dub activity with significant reductions of the levels of ubiquitin (ub)-, k48-, and k63-conjugated proteins. the dub activity exhibited a clear time dependence, with stronger dub activity in the early stage of viral infection. furthermore, the ibv replicase-encoded plp, including the downstream transmembrane (tm) domain, is a dub enzyme and dramatically reduced the level of ub-conjugated proteins, while processing both k48and k63-linked polyubiquitin chains. by contrast, plp did not cause any reduction of haemagglutinin (ha)-ub-conjugated proteins. in addition, mutations of the catalytic residues of plp-tm, cys1274ser and his1437lys, reduced dub activity against ub-, k48and k63conjugated proteins, indicating that the dub activity of the plp-tm wild-type protein is not completely dependent on its catalytic activity. overall, these results demonstrate that the ibv-encoded plp-tm functions as a dub enzyme and suggest that ibv may interfere with the activation of host antiviral signaling pathway by degrading polyubiquitin-associated proteins. avian infectious bronchitis virus (ibv) is the prototype of gamma coronaviruses (cov), a family of enveloped viruses that possess a large continuous positive-stranded rna genome [4] . the genomic rna is 27.6 kb in length, and approximately two thirds of the nucleotide sequence encodes orf 1, which includes orf 1a and orf 1b [11, [22] [23] [24] [25] . a papain-like protease (plp) is encoded by the region from nucleotides 4243 to 5553 of orf 1a [21] , comprising the catalytic domain of nonstructural protein 3 (nsp3). proteolysis at the gly673-gly674 and gly2265-gly2266 dipeptide bonds leads to release of the 87-kda and 195-kda n-terminal mature proteins and the c-terminal 41-kda cleavage product [20, 21, 23] . site-directed mutagenesis studies have confirmed that the cys1274 and his1437 residues of plp are essential for proteinase activity [20] . covs such as the human coronavirus nl63 (hcov-nl63) [6] and murine hepatitis virus-a59 (mhv-a59) [2] encode two functional papain-like proteases (plp), termed plp1 and plp2. at least two covs, severe acute respiratory syndrome coronavirus (sars-cov) [1] and ibv [21] encode only one functional papain-like protease, termed plp. the proteolytic processing mediated by the plp encoded by ibv [20] is similar to that of plp2 encoded by other covs [3, 6] . plps of covs also perform deubiquitinating (dub) and interferon (ifn) antagonism activities. for instance, plp2 of mhv-a59 [29] can bind to irf3, cause its deubiquitination and prevent its nuclear translocation, thus inhibiting cellular irf3-mediated type i ifn production and promoting viral infection. the sars-cov plp pro-catalytic core also has dub activity and can deubiquitinate irf3, thereby stalling its migration to the nucleus and preventing the host antiviral response [7, 10, 26] . similarly, purified plp2 of hcov-nl63 can hydrolyze k48linked hexa-ubiquitin (k48-ub6) to produce monoubiquitin [6, 7] , and therefore negatively regulate antiviral defenses by disrupting the sting-mediated ifn induction [26] . structural and enzymatic studies have revealed that covs plps can act as both a protease, to process virusencoded large replicase polyproteins, and a dub enzyme, to cleave the isopeptide bonds found in polyubiquitin chains [7, 20] . here, we demonstrate that ibv has dub activity, and like other covs, ibv can recognize and process both k48-and k63-linked polyubiquitin chains. we also demonstrate that the core domain of ibv plp-tm is a coronaviral dub enzyme. we also evaluated the role of plp catalytic activity in dub activity and found that plp-tm does not require catalytic activity to cleave polyubiquitin-linked proteins. this study represents a first step in elucidating the role of plp-tm in ibv pathogenesis and provides new insights on how ibv escapes host antiviral immune mechanisms. chicken embryonic kidney (cek) cells were aseptically generated from 20-day-old specific pathogen-free (spf) chicken embryos (beijing merial vital laboratory animal technology company). the cell suspension was obtained by trypsinization of kidneys for 30 min at 37°c and subsequent filtration through a 100-lm mesh. the cells were then cultured in m199 medium (hyclone) supplemented with 3% fetal bovine serum (fbs). the df1 chicken fibroblast cell line was used for all transfection-based assays. the cells were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 10% fbs. ibv (js/2010/12 strain, genbank accession no. jq900122.1) was cultured in 10-day spf chicken embryos or cek cells. the initial ibv stock was inoculated in chicken embryos for six passages (p6). this study was conducted based on this p6 stock of ibv. the 50% tissue culture infective dose (tcid 50 ) of the ibv p6 stock was determined by identifying the cytopathic effect (cpe) of the virus in cek cells. the plasmid pcdna-5' flag was a kind gift from dr. meng (dalian medical university). prk5-ha-ub and the mutant derivatives prk5-ha-ub-k48 and prk5-ha-ub-k63 were obtained from addgene (plasmids #17608, #17605 and #17606) [19] . attachment of ubiquitin (ub) modifiers is a reversible post-translational modification that regulates the fate and function of proteins. the ubiquitin molecule contains a total of seven lysine residues at positions 6, 11, 27, 29, 33, 48 (k48), and 63 (k63). these lysine residues potentially mediate ubiquitin chain elongation. the two most common types of polyubiquitin chains are linked through ubiquitin lysine 48 (k48) and lysine 63 (k63). plp is encoded by ibv orf 1a at the region of nsp3 between nucleotides 4243 and 5553 [22] . biological analysis software (tmhmm server v. 2.0) was used to analyze the amino acid sequence of nsp3 to predict the transmembrane (tm) domain near plp [9] . in figure 2b , the red area indicates the transmembrane domain; the extracellular protein and short cytoplasmic domain are indicated by pink and blue, respectively (fig. 2b) . plp and plp-tm constructs were generated using specific primers (table 1 ) to amplify the designated a cysteine residue (cys 1274) and histidine residue (his 1437) of plp are the catalytic residues of the proteinase activity [20] . either of these two sites could play an important role in the dub activity of plp-tm. to explore these possibilities, substitution mutations of the cys 1274 residue with ser and the his 1437 residue with lys were constructed to obtain the mutant constructs plp-tm cys1274ser (plp-tm c1274s) and plp-tm his1437lys (plp-tm h1437k) [20] . to generate specific mutations of the catalytic residues, mutagenic primers (table 1) were incorporated into newly synthesized dna using the fast mutagenesis system protocol (trans, fast mutagenesis system, fm111) according to the manufacturer's instructions. the transfections were performed using lipofectamine 2000 (invitrogen) according to the manufacturer's instructions. this method resulted in a transfection rate of at least 70%. briefly, the plasmid (2 lg for 6-well) was diluted into opti-mem, and lipofectamine 2000 (5 ll for 6-well) was diluted into opti-mem. the diluted dna was added to diluted lipofectamine 2000 (1:1 ratio), incubated for 5 min, and then added to the cell cultures. df1 cells were co-transfected with prk5-ha-ub, prk5-ha-ub-k48 or prk5-ha-ub-k63 plasmids and the indicated amounts of ibv plp, plp-tm and specific catalytic mutants. the cells were then lysed in ripa lysis buffer (beyotime institute of biotechnology, china). the cell lysates were analyzed for ha-conjugated proteins using western blotting with monoclonal anti-ha antibody (1:5000, sigma). an anti-n antibody was used to detect the viral replication level. to confirm the expression levels of plp and the mutants, anti-flag antibody (1:5000, sigma) was used to detect the flag-tagged proteins. actin was detected using b-actin antibody (1:5000, sigma) as a protein loading control. the effect of ibv on ubiquitinated proteins in cells was assessed as follows. df1 cells were infected with ibv at a multiplicity of infection (moi) of 10 or mock-infected. the cells were then transfected with prk5-ha-ub, prk5-ha-ub-k48 or prk5-ha-ub-k63 for an additional 24 h. to investigate the relationship between ibv replication and dub activity, df1 cells were infected with ibv at an moi of 10 or mock-infected and then transfected with prk5-ha-ub at 12 h, 18 h or 24 h post-infection for an additional 24 h. cell lysates were then prepared and the extent of protein ubiquitination was assessed by western blot as described. assays for the dub activity of plp, plp-tm and the catalytic mutants the effects of plp, plp-tm and the catalytic mutants on ubiquitinated proteins in cultured cells was assessed as follows. df1 cells were co-transfected with prk5-ha-ub/ prk5-ha-ub-k48/prk5-ha-ub-k63 and the indicated amounts of the constructs encoding plp-tm or the corresponding catalytic mutants. the pcdna-5' flag vector was used to standardize the quantity of dna. the total cells were harvested, and ubiquitinated proteins were assessed as described previously 24 h post infection. to determine if ibv exhibits dub activity against host cellular substrates after infection, df1 cells were transfected with prk5-ha-ub, prk5-ha-ub-k48 or prk5-ha-ub-k63 after infection with ibv or mock infection. the levels of ub(fig. 1a, lane 3) , k48(fig. 1a , lane 5) and k63(fig. 1a, lane 7) conjugated proteins were reduced dramatically in df1 cells after infection with ibv. this suggests that ibv exhibits strong dub activity that recognizes and processes both k48-and k63-linked polyubiquitin chains. to investigate the relationship between ibv replication and dub activity, df1 cells were transfected with prk5-ha-ub at different times post-infection. western blot analysis revealed that ibv decreased the levels of ub-conjugated proteins at different times, and the levels of ub-conjugated proteins were lower at 12 h than at 18 h and 24 h. thus, ibv exhibited stronger dub activity in the early stage of infection (fig. 1b, lanes 3, 4 and 5; fig. 1c ). overall, ibv has global dub activity against ubiquitinated proteins, and the dub activity exhibits a clear time dependence in cultured cells. plp is the catalytic domain of ibv, encoded by the genomic region from nucleotides 4243 to 5553, and the tm domain is encoded by the region from nucleotides 5781 to 6252, downstream of plp (fig. 2a, b) . to determine if plp and plp-tm have dub activity, plp and plp-tm were separately co-transfected with prk5-ha-ub into df1 cells. we found that expression of plp-tm resulted in a dramatic reduction in the level of ub-conjugated proteins (fig. 2c, lane 3) . by contrast, expression of plp did not result in any significant reduction of ha-ub conjugates (fig. 2c, lane 4) . this result indicates that ibv plp-tm has effective dub activity that can remove ub conjugates from many cellular substrate proteins. the two most common types of ubiquitinated proteins are linked through ubiquitin k48 and k63. to determine if plp-tm of ibv has selectivity for ubiquitinated substrates with k48 and k63 linkages, df1 cells were transfected with plp-tm with or without prk5-ha-ub (fig. 2d, lanes 2 and 3) , prk5-ha-ub-k48 (fig. 2d, lanes 4 and 5) or prk5-ha-ub-k63 (fig. 2d, lanes 6 and 7) . the extent of ubiquitinated products was assessed by western blot. consistent with the processing of ub-linked proteins by plp-tm, plp-tm dramatically reduced the levels of both k48-and k63linked ubiquitin (fig. 2b, lanes 3, 5 and 7) . this result shows that both major forms of polyubiquitinated proteins can be recognized and degraded by plp-tm. the dub activity of plp-tm does not completely depend on its catalytic activity plp contained within the nsp3 has been shown to be responsible for the cleavage of orf 1a at the two gly-gly dipeptide bonds to release mature protein, and the catalytic dyad for this activity was cys1274 and his1437 [20] . either of the two catalytic residue sites may play an important role in the dub activity of plp-tm. to explore these possibilities, df1 cells were transfected with plp-tm, plp-tm c1274s or plp-tm h1437k, together with prk5-ha-ub (fig. 3a) , prk5-ha-ub-k48 (fig. 3b) , or prk5-ha-ub-k63 (fig. 3c) , and the extent of ubiquitinated products was assessed by western blot. we found that the catalytic mutants (c1274s and h1437k) of plp-tm exhibited reduced dub activity against ub-, k48-and k63-linked proteins (fig. 3a-c) . these results demonstrate that the dub activity of plp-tm is not completely dependent on its catalytic activity. in this study, we characterized the dub activity of ibv plp in df1 cells and the core domain of the dub activity. we found that ibv exhibits strong global dub activity during infection of df1 cells, indicating that ibv infection disrupts polyubiquitin modification in host cells or encodes a protein with dub activity [14] . further experiments indicated that the proteinase plp-tm plays an important role in ibv dub activity and can process both k48-and k63-linked polyubiquitin chains. moreover, the dub activity of plp-tm was not completely dependent on its catalytic activity. deubiquitinating function of an avian ibv papain-like protease 1947 similar to the cov porcine epidemic diarrhea virus (pedv) [28] , the dub activity of ibv also dramatically reduced ub-conjugated protein levels in virus-infected cells, and the ibv plp plays an important role in this dub activity. plp2 of pedv has potent dub activity that is dependent on its catalytic activity. in notable contrast to pedv plp2, the dub activity of ibv plp is not dependent on its protease activity. another group reported that ibv plp can degrade k48-and k63-linked polyubiquitin chains to monoubiquitin but cannot degrade linear polyubiquitin [18] . hcov-nl63 plp2 has a dub activity that is dependent on its protease activity [7] . by contrast, both the plp2-tm and catalytic mutants of plp2-tm [26] had dub activity, although the dub activity of the plp2-tm catalytic mutants was lower than that of plp2-tm, suggesting that the tm domain plays a role in plp2 dub activity. similarly, our study confirmed that the tm domain downstream of ibv plp is essential for plp dub activity. the ibv plp cleaves the orf 1a to generate nonstructural proteins that associate with endoplasmic reticulum membranes to generate convoluted membranes and double membrane vesicles (dmvs), which are the site of viral replication. the plps are tethered to the dmvs by a tm domain [12, 17] . many reports have demonstrated that the tm domain plays an important role in covs dub activity, possibly by facilitating correct protein folding or interactions with cellular proteins. additional studies are needed to identify the exact functions of the tm domain during ibv replication and the interaction between the host innate immune system and ibv infection. this study raises important questions concerning the role of viral dub activity in cov replication and pathogenesis. coronaviral dub activity may target the ubproteasome pathway to facilitate virus replication and damage host defense mechanisms, including innate immunity [5, 8] . some covs may utilize dub activity to escape the host innate antiviral response [13, 27] . for example, plp2 of mhv-a59 can bind to irf3, cause its deubiquitination and prevent its nuclear translocation. coexpression of plp2 inhibits cardif-, tbk1-and irf3mediated ifn-b reporter activities [29] . mhv-a59 may use dub activity to reduce ifn induction, to promote viral growth and to escape from host innate antiviral responses [29] . pedv infection suppresses the production of ifn-b and plp2 acts as a viral dub to interfere with rig-i-and sting-mediated signaling pathways [28] . however, some covs plp-mediated interferon antagonism is independent on protease and dub activity. the plp2 of nl63 has dub activity and antagonizes the induction of type i ifn, whereas plp-mediated ifn antagonism is independent of dub activity [6] . these data show that covs plps target the activity of type i ifn through dub activity and inhibit the activation of the innate immune system. joeri kint et al. showed that ibv inhibits the synthesis of host proteins, including type iifn, a key component of the antiviral response [15, 16] . our study also indicates that ibv plp-tm process both k48-and k63-linked ubiquitin. k48-and k63-linked ubiquitin are the most common types of modified polyubiquitin and play key roles in protein degradation and in response to changes in the innate and adaptive immunity systems. further investigations are necessary to determine if ibv plp dub activity inhibits the host antiviral response. overall, the results of our study show that ibv has dub activity and confirm that plp-tm is not only a classic papain-like protease encoded by ibv but is also a multifunctional protein that plays important roles in the regulation of interactions between ibv and host antiviral innate immune response proteins. ubiquitin modifications play key regulatory roles in protein degradation and in innate and adaptive immunity signaling pathways. ibv plp-tm may prevent the activation of host antiviral signaling pathways by degrading polyubiquitin chains associated with ubiquitin proteins. the interactions between ibv and the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity characterization of the leader papain-like proteinase of mhv-a59: identification of a new in vitro cleavage site characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain a59 nidovirales: a new order comprising coronaviridae and arteriviridae viral hijacking of cellular ubiquitination pathways as an anti-innate immunity strategy proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl63 deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3c-like proteinase severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis rna replication of mouse hepatitis virus takes place at double-membrane vesicles ubiquitin and ubiquitin-like proteins in protein regulation modification of proteins by ubiquitin and ubiquitin-like proteins infectious bronchitis coronavirus inhibits stat1 signaling and requires accessory proteins for resistance to type i interferon activity infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b sarscoronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum structural view and substrate specificity of papain-like protease from avian infectious bronchitis virus parkin mediates nonclassical, proteasomalindependent ubiquitination of synphilin-1: implications for lewy body formation characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87-kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products characterisation and mutational analysis of an orf 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling ubiquitin and ubiquitin-like proteins as multifunctional signals deubiquitinating function of an avian ibv papain-like protease 1949 the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production key: cord-271359-dpa8zzc3 authors: sapats, s. i.; ashton, f.; wright, p. j.; ignjatovic, j. title: novel variation in the n protein of avian infectious bronchitis virus date: 1996-12-15 journal: virology doi: 10.1006/viro.1996.0670 sha: doc_id: 271359 cord_uid: dpa8zzc3 abstract the nucleocapsid protein of coronaviruses has been considered highly conserved, showing greater than 94% conservation within strains of a given species. we determined the nucleotide sequence of the n gene and the 3′ untranslated region (utr) of eight naturally occurring strains of ibv which differed in pathogenicity and tissue tropism. in pairwise comparisons, the deduced amino acid sequences of n of five strains vic s, n1/62, n9/74, n2/75, and v5/90 (group i) shared 92.3–98.8% identity. the three strains n1/88, q3/88, and v18/91 (group ii) shared 85.8–89.2% identity with each other, but only 60.0–63.3% identity with viruses of group i. amino acid substitutions, deletions, and insertions occurred throughout the n protein and involved regions previously identified as being conserved. despite the considerable variation observed between the two virus groups, all n proteins contained a high proportion of basic residues, 80% of which were conserved in position. in addition, all strains contained approximately 30 serine residues of which 10 were conserved, the majority occurring between positions 168 and 194. as for all other coronaviruses, the region between positions 92 and 103 was highly conserved. hence, a large number of amino acid changes can be tolerated within the n protein without affecting its integrity or functioning. the 3′ utr immediately downstream from the n gene was highly heterogeneous with extensive deletions occurring in the group ii strains. infectious bronchitis virus (ibv), a member of the family be highly conserved differing by only 2 to 6% at the amino acid level (4, 5) . the high level of conservation in the n coronaviridae, causes an acute highly contagious disprotein has resulted in the widely held view that the s1 ease of chickens resulting in significant economic losses glycoprotein, which may show up to 49% variation (6) , to poultry industries throughout the world. the ibv geis the only relevant structural element in assessing the nome consists of a single strand of positive sense rna genetic diversity and evolutionary direction of ibv. measuring 27.6 kb in length excluding the poly(a) tail (1) . immediately downstream of the n gene is the 3 un-the genes encoding the three major structural proteins translated region (utr) which is presumably important are situated within an 8-kb region located at the 3 end in the initiation of negative-strand rna synthesis. the of the genome. these proteins are the spike glycoprotein organization of the 3 utr differs among the coronavi-(s), the membrane glycoprotein (m), and the phosphoryruses. in porcine, canine, and feline coronaviruses this lated nucleocapsid protein (n). the n protein plays a region is conserved among strains within a species and role in viral replication, assembly, and immunity. it intercontains at least one open reading frame (orf) (7, 8) . acts with leader rna sequences facilitating viral mrna to the present time, functional orfs have not been desynthesis and also binds to the viral rna forming a helitected in the 3 utr of ibv. however, the virulent m41 cal nucleocapsid (2). the n protein of all coronaviruses virus lacks a sequence of 184-196 nucleotides that has is overall very basic and in ibv contains 409 amino acids been detected in five other ibv strains. this sequence with a predicted m r of approximately 50,000 (3); it also has been termed a hypervariable region (hvr) and was contains a high proportion of serine residues which act proposed to be exogenous in origin (3, 9) . the region as sites for phosphorylation (2). located downstream of the hvr (315 nucleotides ending the n protein of 27 strains of ibv isolated over a period at the poly(a) tail) is highly conserved in strains of ibv, of 60 years from diverse locations such as the u.s.a., the probably indicative of its role in the synthesis of negative-uk, holland, saudi arabia, and japan has been shown to strand rna. recently we reported the isolation of three ibv strains, other australian ibv strains. based on s1 sequences, the ibv strains and methods used for their propagation have been described (10, 11) . vic s is a commercial these strains formed a distant and novel genetic group of ibv (10) . in addition, the n protein of these strains vaccine (arthur webster pty ltd, castle hill, australia). strain n1/62 was isolated from unvaccinated chicks in failed to react with five monoclonal antibodies directed against different epitopes on the n protein. these epi-1962, whereas n9/74, n2/75, n1/88, q3/88, v5/90, and v18/91 were isolated from vaccinated commercial chicks topes are conserved in other australian strains of ibv, indicating unusual changes in the n genes of n1/88, q3/ between 1974 and 1991. strains were cloned either in chicken embryo kidney cells (by plaque assay) or tra-88, and v18/91 (11) . we have now sequenced the n gene and 3 utr of eight australian ibv strains isolated over cheal organ cultures (by limiting dilutions) and passaged 1-2 times in embryonated chicken eggs. all strains repli-the period of thirty years from both vaccinated and unvaccinated flocks. the results demonstrated less conserva-cated in the trachea and strains vic s, v5/90, n1/62, n9/ 74, and n2/75 also replicated in the kidneys, the latter tion in the n protein and 3 utr of ibv than previously detected. three causing 32 to 96% mortality (10). fig. 1. sequence alignment of the n protein of australian strains of ibv. the complete deduced amino acid sequence of n of vic s is shown. gaps (dashes) were introduced to align the sequences. dots indicate residues identical to vic s. asterisks indicate residues conserved in all strains. the longest region of complete conservation is boxed. the serine-rich region is underlined; the t cell epitope is double underlined. clusters of basic residues are shaded. the clustal v program was used for all sequence alignments (17) . of these five strains were also similar to those of geographically distant strains isolated in the u.s.a., europe, and japan with which they shared 91.4-92.9% identity at the amino acid level (results not shown). this confirmed the previous observation of the tendency for conservation of the n protein over a long time (ç30 years) irrespective of geographical distances and immunological pressures. contrary to this, however, the n protein of three other strains n1/88, q3/88, and v18/91 (group ii) shared only 60.0-63.3% amino acid identity with the n proteins of strains in the first group, while sharing 85.8-89.2% amino acid identity with each other. this lack of conservation of the n protein has not been reported before for any other coronavirus. an alignment of the deduced amino acid sequence of the n gene of vic s with sequences of the other australian strains is shown in fig. 1 n (3, 4, 18) . the tree was constructed using tion 337. the group ii strains also contained a number the neighbor-joining method (19) . of insertions and deletions relative to group i strains, the majority of which were at the amino and carboxy termini between positions 7 and 23 and 339 and 401, respec-viral rna was purified using methods described (10, tively. overall, only 54% (221/411) of the residues were 12). vic s, n1/88, and q3/88 cdna was synthesized using conserved in all strains, the longest region of complete random primers, and sequences obtained from the conservation occurring between positions 92 and 103, cloned cdna were used to design primers for amplificacorresponding to the part of the n protein previously tion of the n gene of all strains by reverse transcription found to have the highest degree of conservation in ibv and polymerase chain reaction. all cdnas were cloned and all other coronaviruses (13). published sequences into puc series plasmids. for each virus two or more for the n proteins of ibv also contain a region of complete independent cdna clones were sequenced using the conservation between positions 242 and 296 (4). as evi-pharmacia t7 sequencing kit. pairwise comparisons of dent from fig. 1 , the corresponding region is not conthe nucleotide and deduced amino acid sequences of served among the australian strains. the precise locathe n genes of the eight strains vic s, v5/90, n1/62, n9/ tion and role of functional domains within the n protein 74, n2/75, n1/88, q3/88, and v18/91 (excluding the first are not well understood (2, 4, 14) . however, a t cell 17 nucleotides of v5/90, n1/62, n9/74, and n2/75) are epitope has been identified in ibv corresponding to posishown in table 1 . the strains formed two distinct genotions 74-81 in fig. 1 (15) . examination of these setypic groups based on the level of nucleotide and amino quences reveals that they are completely conserved in acid identities. the first five strains in table 1 2, 3, 14) . (i) all strains possessed a high proportion of basic residues (17.2-19.6%). the majority of revealed values of identity ranging from 22.2 to 100%. within group i the identity was 92.1-100%; within group these (80%) were conserved in position and generally clustered in three regions between positions 66 and 88, ii it was 22.2-100% identity (results not shown). the hvr of group i viruses (i) contained a high u content which 181 and 234, and 334 and 373 (fig. 1) . this basic character probably facilitates protein-rna interactions. (ii) the was evenly distributed, and (ii) was similar to that of beaudette, ark99, and gray viruses (9) . little similarity to carboxy terminus contained a clustering of acidic residues located between positions 375 and 415, a region kb8523 and holl52 viruses was observed (results not shown). which otherwise showed very little conservation. however, the acidic residues were less conserved (66%) in this hvr downstream of the n gene was originally identified in the vaccine strain beaudette by comparison position than the basic residues. (iii) all strains contained approximately 30 serine residues, their precise locations with the virulent m41 strain and considered to be an insert of exogenous origin acquired through recombina-varying considerably. only 10 were totally conserved in position. the majority of these conserved serine residues tion during adaptation in eggs (3, 9) . however, the presence of the long hvr (153 to 216) in group i strains, occurred between positions 168 and 194. serine residues are potential sites for phosphorylation (2). hence it which received a low number of passages in eggs, suggests that the hvr region may have been present in appears that the n protein is able to tolerate some variability in the distribution of basic, acidic, and serine resi-an ancestral strain and subsequently deleted in some strains. in addition, in contrast with the beaudette and dues, although the basic residues were the most conserved consistent with the function of the protein as a m41 comparison, the shorter forms (10 to 38) of the hvr in the australian strains n1/88, q3/88, and v18/91 (group ribonucleocapsid protein. these residues are expected to directly influence protein charge, state of phosphoryla-ii) are associated with a decrease in virulence. thus there is no clear association between the length of the tion, and secondary structure. the phylogenetic relationship between australian and hvr and virulence. the remaining 294 nucleotides downstream of the hvr (from position 220 in fig. 3b ) showed other strains of ibv based upon the published nucleotide sequences for the n gene is shown in fig. 2 . the group considerable conservation with values of identity ranging from 84.6-100.0% in pairwise comparisons. within group i and group ii australian strains formed two clusters distinct from previously published strains. the five group i i strains, the identity was greater than 97.3%. an orf was detected within the 3 utr of n1/62 and strains formed one cluster with vic s and v5/90 being the most closely related and showing approximately the n9/74 with the potential to encode hydrophobic proteins of 8793 m r and 8711 m r , respectively. a similar orf has same degree of relatedness to n1/62 and n2/75, while n9/74 appeared to be the most divergent. the group ii been identified in four other ibv strains (9) . although the initiation codons of these orfs fit the kozak consensus strains formed another cluster having diverged markedly from all other clusters. the phylogenetic relationships sequence (16) with an a at position 03 and a g at position /4, there are no ibv mrna transcription motifs (ctt/ among the australian ibv strains based on the n nucleotide sequences were similar to those based on s1 (10). gaacaa) directly upstream. other coronaviruses such as porcine transmissible gastroenteritis virus also con-this suggests that the s1 and n genes of strains in group ii have evolved in parallel. interestingly, a comparison tain an orf within the 3 utr. the latter virus directs the synthesis of a hydrophobic and membrane-associated of the dutch strain d1466 with other european strains showed that the d1466 s1 protein had significantly di-protein of 9101 m r (7) . at present it is unknown whether the corresponding protein for ibv is synthesized in in-verged from the s1 of the other strains, similar to the distance between the australian group i and ii strains. fected cells and what its function in virus replication may be. more experiments are required to detect the protein however, this divergence was not matched by the n protein, which was conserved between d1466 and the and to examine possible mechanisms for the initiation of its translation. other european strains (5) . the 3 utr, located immediately downstream of the n gene, was sequenced for vic s and the other australian acknowledgment strains (figs. 3a and 3b) . a hvr was identified, ranging fig. 3b ) than previously reported (9) . in the references group i strains n1/62, n9/74, and n2/75 the hvr was tom-214, 214, and 216 nucleotides, respectively, while for vic ley the coronaviridae virus 12 key: cord-285330-td4vr0zv authors: mohammadi, ali; asasi, keramat; boroomand, zahra; namazi, fatemeh; hosseinian, seyedeh alemeh title: viral quantity and pathological changes in broilers experimentally infected by irfibv32 isolate of infectious bronchitis virus date: 2015-11-12 journal: virusdisease doi: 10.1007/s13337-015-0286-4 sha: doc_id: 285330 cord_uid: td4vr0zv an iranian isolate of avian infectious bronchitis virus irfibv32 was quantified in experimentally infected broilers using real-time reverse transcriptase polymerase chain reaction and histopathological changes was investigated. thirty-six 3-week-old commercial broilers were inoculated by 10(5) eld50/0.1 ml of the virus. on the various days post inoculation (dpi) different tissues were collected. the virus strongly started the replication in trachea at 1 dpi and reached to the maximum titer at 3 dpi. the highest ibv rna level was shown in this organ. in lung, the virus was replicated with the titer lower than that of the trachea, but it rose up more at 5 dpi. the kidneys were the tissues with the least viral genome copy number, although the duration of the virus presence was considerable. the virus replicated in testes sooner than ovaries also disappeared sooner but the maximum viral yield in the ovaries was more. the virus titer in the studied tissues had an interesting fluctuation especially in caecal tonsils. testes and ovaries were the organs that the virus could reactivate without using any chemical. the most severe lesions were observed in tracheae but they appeared in the lungs later. lymphocyte infiltration in the kidneys was noted from 5 dpi even sooner than the lungs. there were no lesions in the caecal tonsils, testes and ovaries in spite of the virus replication in a high titer. infectious bronchitis (ib) is an acute, highly contagious upper respiratory disease of chickens. ib is characterized by tracheal rales, sneezing and coughing, increased oculo-nasal secretion and excess mucus in the trachea accompanied by decreases in both weight gain and feed efficiency. economic consequences to the poultry industry comprise growth retardation and high morbidity and mortality rates in meattype birds. in addition, reduced egg production, decreased internal, external egg quality, hatchability and fertility have been documented in layers and breeders [8] . this disease is caused by infectious bronchitis virus (ibv), a member of the family coronaviridae (order nidovirales) and genus coronavirus [6] . ibv has been shown to replicate in many respiratory tissues including trachea, lungs, and air sacs, causing respiratory infection; in some urogenital tissues such as kidney, testes and oviduct, causing minor or major nephritis; and in many parts of the alimentary tract as well [12] . live attenuated and inactivated vaccines have been developed to control ib. despite extensive vaccination, ib has been a perpetual problem in the iranian poultry industry since the disease was first reported in 1994 [2, 18] . a new isolate of ib virus (irfibv32) from broiler chicken in fars province, southern iran, is isolated and identified by boroomand et al. [6] . furthermore, the tissue distribution and clinical signs of experimentally infected chicks by this isolate are also studied [5] . currently, real time reverse transcription polymerase chain reaction (rrt pcr) technique has been developed and used widely for rapid, sensitive and accurate diagnosis of different diseases. a rrt pcr test has been developed using a taqman probe for the detection of american strains of ibv from tracheal swabs [7] and a locked nucleic acid (lna) probe-based rrt pcr assay for the detection of the australian ibv strains [10] . jackwood et al. [14] used a rrt pcr diagnostic test with the use of fluorescence resonance energy transfer (fret) technology to detect several common strains of ibv. in this study, we identified ibv load in different tissues of experimentally infected broilers to clarify the replication strength of irfibv32 isolate at intervals post challenge. this was achieved using taqman ò -labeled probe two-step rrt pcr assay targeting the n gene of the virus. the n gene was chosen since it is highly conserved among ibv strains. we believe that the quantitation of the virus in different organs of the infected bird at different times post virus inoculation accompanied with pathological study may further help us to investigate the virus pathogenesis. the virus isolate used in this study was irfibv32 (genbank: hq123359.1). some characteristics of the isolate reported previously [6] . virus propagation was performed in 10-day-old embryonated chicken eggs, as described previously [17] . the embryo lethal dose (eld 50 ) of infected allantoic fluid was calculated according to the reed and muench [17] method. in the present study, allantoic fluid containing 10 5 eld 50 /0.1 ml of the virus was used to induce the infection. fifty-nine apparently healthy 1-day-old ross broiler chicks were divided randomly into two experimental groups comprising group 1 (challenged group: n = 41) and group 2 (control group: n = 18). two groups of chicks were reared separately in the animal research unit of the veterinary school of shiraz university and received feed and water ad libitum during the experiment. all experiments were conducted after institutional approval of the animal use committee of shiraz university. prior to challenge, five birds from the experimental group (group 1) were killed and their organs were investigated for ibv detection. at 20 days old, all birds in group 1 was challenged intranasally with 100 ll allantoic fluid containing 10 5 eld50 of the virus. the chicks in group 2 kept as unchallenged control. all the chicks were monitored daily for 20 days for clinical signs, and mortality. on 1, 2, 3, 5, 7, 11, 13, 15 and 20 days post inoculation (dpi) four birds from the challenged group and two birds from the control group were randomly selected. then, they were bled for plasma separation and sacrificed for collecting tissue samples. gross lesions were recorded and their trachea, lungs, kidneys, caecal tonsils, testes and ovaries were aseptically collected separately for the virus detection, titration and histopathological evaluations. all tissue samples were immediately frozen and stored at -70°c until needed for the rna extraction. the samples for histopathological evaluations were fixed in 10 % neutral buffered formalin, embedded in paraffin and the sections were stained with haematoxylin and eosin and studied by an ordinary light microscope. rna extraction from clinical samples was performed with the accuzol tm (bio-neer corporation, south korea) according to the manufacturer's protocol. appropriate amount of plasma (250 ll) or homogenized tissues (50-100 mg) were used for the extraction. isolated rnas were solved in a final volume of 50 ll distilled water (dw) and was quantified by spectrophotometry. appropriate rnase inhibitor and dnase (fermentas) were added and stored at -70°c until assayed. for standard preparation, 100 ll infected allantoic fluid was used for rna extraction and cdna synthesis was carried out using bioneer rt-premix kit according to the manufacturer's instructions. pcr was performed to amplify 276 bp fragment of n protein gene of ibv that included in the rrt pcr assay amplicons [1] . five microliter of total rna and 10 pmol of antisense primer were used for cdna preparation. three microliter of cdna were added to 22 ll pcr master mix containing: 2.5 ll 10x pcr buffer, 0.75 ll mgcl 2 (50 mm), 0.5 ll dntps (10 mm), 10 pmol of each primer, 15.75 ll dnase/ rnase free dw and 0.5 ll taq dna polymerase (5 iu/ ll). the program in ependorf thermal cycler was 95°c for 3 min and 35 cycles including: 95°c for 45 s, 55.6°c for 45 s, 72°c for 50 s and a final extension step at 72°c for 7 min. the pcr products were separated in a 1 % (w/v) agarose gel and visualized under ultraviolet light after staining with ethidium bromide. pcr products (276 bp) was cloned into a t-vector (ptz57r/t) based on fermentas i nstaclonetmpcr cloning kit instructions. the plasmid dna was amplified in e. coli strain gm2163 and purified using bioneer accuprep plasmid extraction kit. the recombinant plasmid was verified by dna sequencing. the concentration of the plasmid dna was calculated using spectrophotometery. recombinant plasmids were serially diluted tenfold as a standard in the rrt pcr. the cdna synthesis from normalized rna of the samples was carried out and then rrt pcr was performed using a mixture of 20 pmol random hexamer and 20 pmol of reverse primer [1] . the rrt pcr primers and taqman probe used in this study were described previously by chousalkar et al. [10] . the assays were performed in the bio-rad miniopticontm system. the reaction mixture contained 5 ll of target cdna, 1 ll of each primer and probe at concentration of 10 pmol/ll, 10 ll of 2x master mixes, 0.2 ll of ung (uracil-n-glycosylase), and 1.8 ll of dw in a final volume of 20 ll. ung treatment and two step cycles program were as previous [10] . viral rna copy numbers (expressed as copies per 1 lg of total rna) were quantified by comparison with a tenfold serially diluted recombinant plasmid standard of known concentration. the quantity of the virus rna in various organs of the challenged birds at the different dpi was determined by rrt pcr. all organs of the five birds killed at 0 dpi (prior to challenge) were negative for viral rna. the results showed that the plasma samples of the challenged chickens were negative for the virus in any times post inoculation. the load of the virus in various tissues after challenge was shown in table 1 . the virus had its highest load in trachea on 3 dpi meanwhile lungs had the maximum level of the virus on 5 dpi. the virus existed at both organs up to 13 dpi. it was interesting that the titers on 13 dpi were more than those on 11 dpi. although the viral titer in kidneys was lower than the other tissues, the duration of virus replication was considerable (up to 15 dpi). the maximum viral load in this organ was on 7 dpi. there was no any evidence for virus replication in the ovary up to 3 dpi but it was appeared on 5 dpi with the highest level. although the virus was absent on 15 dpi, it was seen on 20 dpi again. testes were the organs that the virus replicated very soon and it was continued till 11 dpi with the highest level on 3 dpi. caecal tonsils were the tissues with the highest duration of the virus replication and a considerable amount of the viral rna. the maximum yield was seen on 5 dpi. it was the unique tissue that not only viral replication extended in all days but also there was an interesting fluctuation in the viral titer in the different days. the quantity of the viral rna was remarkable even in the last day of the assay (table 1) . no virus was detected in samples from unchallenged birds, indicating that biosecurity precautions were adequate. histopathological examination of the tracheal sections showed loss of cilia, and degenerative changes in the epithelial cells, decreased goblet cells and mucus glands with infiltration of plasma cells and lymphocytes into the lamina propria from 1 to 9 dpi. on 11, 15 and 20 dpi, no changes were recorded in sections of trachea. infiltration of mononuclear cells and interstitial pneumonia was evident in these chicks on 7 and 9 dpi. the kidney lesions consisted of infiltration of lymphocytes in the interstitial space. the lesions were seen from 5 to 20 dpi. there were no lesions in the caecal tonsils, testes and oviducts. also no lesions were found in the tissue sections of the control animals. ibv infection in chickens is known to be initiated by replication in the upper respiratory tract, which is usually restricted to the ciliated and mucus secreting cells of the nostrils, harderian gland, trachea, lungs, and air sacs. different type of ibv shows different tissue tropisms [5] . the maximum amount of viral rna in the trachea was observed on 3 dpi, while that for lung was at 5 dpi. the viral rna in trachea was not detected at 15 dpi therefore; the tracheal epithelium can support a finite amount of the virus. it is generally believed that upper respiratory tract is the primary site of ibv infection, although ibv can also replicate in the reproductive, renal, and digestive systems [8] . callison et al. [7] detected the maximal load of viral rna in the trachea at 5 dpi. mahdavi et al. [15] detected increasingly viral antigen in the lung and tracheal epithelium cells from 2 to 5 dpi. grgi´c et al. [13] found that histological lesions were very prominent in the trachea during the first 6-7 days after exposure to ibv while, in the present study, tracheal lesions were seen from 1 to 7 dpi. in histopathological examination, tissue lesions including loss of cilia and degenerative changes in the epithelial cells, decreased mucus glands and mononuclear cells infiltration in sections of trachea, interstitial pneumonia and interstitial nephritis are consistent with previous studies [10] . as the isolate irfibv32 was able to replicate in trachea for a longer duration, it may be a reason for severity of its virulence. in this study urinary tract showed marked infection from 1 to 15 dpi and the maximal level of the virus was found on 7 dpi. all kidneys sampled on 20 dpi lacked the viral rna but the kidney lesions were seen until 20 dpi. comparison of presence pattern of the virus in kidneys, to trachea and lung showed that presence of the virus more prolonged in the kidneys. these data indicate that irfibv32 has nephrotropic properties as many strains of ibv [15] . the results showed that the ovary can support active replication of the irfibv32 isolate of ibv between 5 and 13 dpi and virus load appears to peak at 5 dpi. chousalkar et al. [9] detected t strain of ibv in oviduct from 6 dpi and the viral load appeared to peak between 10 and 14 dpi. ibv rnas were detected in testes between 1 and 11 dpi. although the viral load in the ovaries was more than testes, the virus replication in the ovaries was overdue in comparison to the testes. detection of ibv rna in the testes corroborates findings by others that ibv replicates in the male testicles [11] . in spite of the virus replication in male and female reproductive system, there were no lesions in these organs. ibv infection of enteric tissues does usually not manifest clinically but persists for long periods and results in fecal virus shedding. we detected the viral rna in the caecal tonsils of infected chicken from 1 to 20 dpi and the maximal loads of the virus were on 5 dpi. comparison of detection of the virus from the caecal tonsils and the other organs showed that the irfibv32 isolate can replicate and be detected in the caecal tonsils for a longer time. naqi et al. [16] isolated massachusetts serotype vaccine virus from cloacal swabs at 63 dpi. mahdavi et al. [15] detected the viral antigens in intestine and kidney epithelial cells from 3 to 5 dpi. the viral rna was not detected in the blood of inoculated chickens in this study. no virus was detected in any tissue of the unchallenged group. the broad distribution of 2, 3-linked sialic acid in different organs explains the pan-tropic nature of ibv in chicken, but it cannot be the only determinant of the pathogenicity because such neuraminic acid is also present on cells that are not infected by ibv. it may be that the sialic acid binding is an initial step requiring further contacts to be made perhaps involving a more specific and less generally distributed secondary receptor [3] . extended and intermittent shedding of ibv from caecal tonsils is evidence for the potential risk of flock to flock transmission via contamination of personnel or equipment. the probability of continuous excretion of the virus at levels usually below the detection limit of the diagnostic tests, or reactivation in sites such as caecal tonsils or kidneys have been already shown [4] . in conclusions, the results of this study clearly corroborate that irfibv32 isolate is capable of distribution and proliferation in different tissues with the longest in the intestine of the experimentally infected chicks. it also is a virulent virus because of its relative long replication period in trachea. the virus titer in the studied tissues had an interesting fluctuation especially in caecal tonsils. testes and ovaries were the organs that the virus could reactivate without using any chemical. in the last days of the existence in the most tissues the viral titer increased after decreasing and then reached to the zero. it seems that the high titer of the virus is not the only reason for the lesions in the tissues because lesions were present even after disappearing of the virus in the kidneys. in addition, there was no any lesion in the ovaries and testes in spite of the high titer of the virus. more studies are needed to explain more the pathogenesis of the disease. molecular analysis of the 793/b serotype of infectious bronchitis virus in great britain studies on avian viral infections in iran immunohistochemistry for detection of avian infectious bronchitis virus strain m41 in the proventriculus and nervous system of experimentally infected chicken embryos re-excretion of infectious bronchitis virus in chickens induced by cyclosporin pathogenesis and tissue distribution of avian infectious bronchitis virus isolate irfibv32 (793/b serotype) in experimentally infected broiler chickens isolation and identification of a new isolate of avian infectious bronchitis virus irfibv32 and a study of its pathogenicity development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens infectious bronchitis detection of infectious bronchitis virus strain n1/88 from the oviduct and feces of experimentally infected vaccinated and unvaccinated hens lna probe-based real-time rt-pcr for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens detection the 4/91 strain of infectious bronchitis virus in testicular tissue from experimentally infected rooster by reverse transcription-polymerase chain reaction virulent avian infectious bronchitis virus, people's republic of china pathogenicity of infectious bronchitis virus isolates from ontario chickens detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the beaudette strain the immunohistochemistry study of lesions due to avian infectious bronchitis (serotype 4/91) on different tissues in specific pathogen free chicks establishment of persistent avian infectious bronchitis virus infection in antibodyfree and antibody-positive chickens a simple method of estimating fifty percent endpoint isolation and identification of infectious bronchitis viruses in chickens in iran viral quantity and pathological changes in broilers experimentally infected by irfibv32 isolate… 323 acknowledgments the authors extend thanks to the school of veterinary medicine, shiraz university, for providing the necessary facilities and financial support to carry out this study. key: cord-259480-1tqfoecc authors: li, huixin; wang, yulong; han, zongxi; wang, yu; liang, shulin; jiang, lu; hu, yonghao; kong, xiangang; liu, shengwang title: recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens date: 2016-03-02 journal: antiviral res doi: 10.1016/j.antiviral.2016.03.003 sha: doc_id: 259480 cord_uid: 1tqfoecc to design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rdevs) expressing the n, s, or s1 protein of infectious bronchitis virus (ibv) were constructed using conventional homologous recombination methods, and were designated as rdev-n, rdev-s, and rdev-s1, respectively. chickens were divided into five vaccinated groups, which were each immunized with one of the rdevs, covalent vaccination with rdev-n & rdev-s, or covalent vaccination with rdev-n & rdev-s1, and a control group. an antibody response against ibv was detectable and the ratio of cd4(+)/cd8(+) t-lymphocytes decreased at 7 days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as 7 days post-immunization. after challenge with a homologous virulent ibv strain at 21 days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p < 0.01), as the two covalent-vaccination groups and the rdev-s group provided better protection than the rdev-nor rdev-s1-vaccinated group. there was less viral shedding in the rdev-n & rdev-s(2/10) and rdev-n & rdev-s1(2/10) vaccinated groups than the other three vaccinated groups. based on the clinical signs, viral shedding, and mortality rates, rdev-n & rdev-s1 covalent vaccination conferred better protection than use of any of the single rdevs. infectious bronchitis (ib) is a highly contagious viral disease of the upper respiratory and urogenital tracts of chickens that is caused by the infectious bronchitis virus (ibv). the disease is prevalent in nearly all countries with an intensive poultry industry, causing respiratory and renal diseases in chickens of all ages. it also reduces the quality and quantity of eggs produced by mature hens, causing heavy economic losses to the poultry industry. in addition, high mortality often occurs in young chickens infected with nephropathogenic strains as a result of renal pathology (cavanagh and gelb, 2008) . the ibv genome consists of a linear, single-stranded, positivesense rna, which encodes four major structural proteins, which include the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope or small membrane (e) protein. the n phosphoprotein is conserved among different ibv serotypes and can induce high titers of cross-reactive antibodies and cell-mediated immunity that protects chickens from acute infection, thus it is used as a target protein in designing vaccines against ib (williams et al., 1992; collisson et al., 2000; seo et al., 1997) . the s glycoprotein is responsible for receptor binding and membrane fusion (hofmann et al., 2004) , and consists of the nterminal s1 and c-terminal s2 subunits (bosch et al., 2003) . most of the conformation-dependent, neutralizing antigenic, and serotypespecific determinants in ibv have been mapped to s1, while other immunodominant regions are located in the n-terminal regions of s2 (koch et al., 1990; kusters et al., 1989; lenstra et al., 1989) . in addition, interactions between the s1 and s2 subunits might affect the conformation of the s1 subunit, thereby accounting for differences in serologic protection (callison et al., 1999) . the m glycoprotein of coronaviruses gives the virion envelope its shape. it has been reported that the s glycoprotein interacts with the transmembrane region of the m glycoprotein and the cytoplasmic tail of the ibv e protein is responsible for its interaction with the ibv m glycoprotein (cavanagh, 2007) . measures to control ib in poultry rely primarily on vaccination. multiple live attenuated ibv vaccines are most often required because of poor cross protection between vaccines produced from different ibv serotypes (liu et al., 2009 (liu et al., , 2014 . live attenuated ibv vaccines do not provide adequate protection throughout the lifetime of layers or breeders, whereas inactivated vaccines convey certain advantages, such as slow antigen release and long-lasting immunity throughout the laying period. unfortunately, inactivated ibv vaccines are not effective when used alone, as birds require one or a series of vaccinations with live-attenuated ibv vaccines (live priming) prior to administration of an inactivated vaccine (cook et al., 2012) . conventional live ibv vaccines are attenuated by multiple serial passages in embryonated eggs (gelb and cloud, 1983; jackwood et al., 2003; bijlenga et al., 2004; huang and wang, 2006) , although this is a time-consuming process. genetically engineered vaccines present an alternative to inactivated and attenuated vaccines. in previous studies, a multivalent dna vaccine expressing s1, n, and m conferred 85% protection , while a multivalent dna vaccine combined with an inactivated vaccine booster conferred complete protection (yan et al., 2013) . in addition, a recombinant newcastle disease virus expressing the s2 protein of ibv was shown to provide broad protection against ibv challenge (toro et al., 2014) . duck enteritis virus (dev) causes duck plague, an acute, contagious, and lethal disease that affects birds of all ages of the order anseriformes (davison et al., 1993) . dev is a member of the family herpesviridae with a genome approximately 158 kb in size . because certain dev genes are not essential for viral replication in vitro (wang and osterrieder, 2011; liu et al., 2011) , dev has been used as a replicating vaccine vector in chickens to provide rapid protection against the h5n1 influenza virus (liu et al., 2013a,b) . in our current study, we used dev as a viral vector to construct three recombinant viruses expressing the n, s, and s1 proteins of ibv, and evaluated their protective efficacy in chickens against virulent ibv challenge. the nephropathogenic ibv strain ck/ch/ldl/091022 is an lx4type (qx-like) strain that was first isolated in china in 2009 (sun et al., 2011) . the dev clone-03 was isolated from a commercial vaccine by plaque assay (li et al., 2006; liu et al., 2007) . primary chicken embryo fibroblasts (cefs) were used for dev propagation (li et al., 2006) . specific pathogen-free (spf) white leghorn chickens, chicken embryo eggs and duck embryo eggs were obtained from harbin veterinary research institute (hvri; harbin, china). the birds were maintained in isolators under negative pressure and provided with food and water ad libitum. all experiments were performed in strict accordance with the recommendations of the guide for the care and use of laboratory animals of the ministry of science and technology of the people's republic of china, and the study protocols were approved by the committee of the ethics of animal experiments of the hvri. dev genomic dna was extracted as previously described (prigge et al., 2004) . the left and right homologous arms of the transfer vector were amplified by polymerase chain reaction (pcr) using primers vl1, vl2, vr1, and vr2. the enhanced green fluorescent protein (egfp) cassette was amplified by primers r1 and r2 (table 1 ) from the pegfp-n1 plasmid and cloned into the pmd18 tsimple plasmid to produce pt-egfp. the left and right arm pcr products were inserted through the clai and blni restriction sites and the mlui and avaiii restriction sites, respectively, of the pt-egfp plasmid to produce pus10-egfp. complementary dna (cdna) of the n, s, and s1 genes of the virulent ibv strain ck/ch/ldl/091022 was synthesized from the viral genomic rna by reverse transcription (rt)-pcr (sun et al., 2011) . the pus10-n, pus10-s, and pus10-s1 plasmids were produced by inserting the n, s, and s1 pcr products, respectively, between the xhoi and noti restriction sites flanking the egfp open reading frame (orf) in the pus10-egfp plasmid. the strategy for the construction of the recombinant devs (rdevs) is depicted in fig. 1a . briefly, the genomic dna of dev and the pus10-egfp transfer vector were cotransfected into cefs using turbofect transfection reagent (thermo fisher scientific, inc., waltham, ma, usa). rdev containing egfp (rdev-egfp) with deletion of the complete us10 gene was selected by plaque assay and used as the parental virus for constructing rdevs expressing the n, s, and s1 proteins of ibv. the selection of rdev-n, rdev-s, and rdev-s1 was conducted using fluorescence microscopy, and plaques without green fluorescence were purified by plaque assay. n1f, n1r, sf, sr, s1f, and s1r gene-specific primers (table 1) were used to confirm the identity of rdev-n, rdev-s, and rdev-s1 by pcr. in addition, the primer pair df and dr, corresponding to the flanking sequence of the dev us10 gene (table 1) , were used to differentiate wild-type (wt) dev from the rdevs. western blotting was performed to detect the expression of ibv proteins from the rdevs (han et al., 2013) . rabbit anti-gfp igg (sigmaealdrich corporation, st. louis, mo, usa), mouse anti-ibv n protein monoclonal antibody (4f10) (han et al., 2013) , and chicken anti-ibv serum were used as primary antibodies for detection of egfp, n, s, and s1 expressed from rdev-egfp, rdev-n, rdev-s, and rdev-s1, respectively. in addition, an anti-chicken b-actin monoclonal antibody of mouse origin (sigmaealdrich corporation) and mouse anti-dev gl serum (prepared in our laboratory) were used to detect reference proteins and the efficacious replication of dev or rdevs in cefs. horseradish peroxidase-conjugated anti-rabbit igg, anti-mouse igg, or anti-chicken igg (sigmaealdrich corporation) were used as secondary antibodies. an indirect immunofluorescence assay was performed to detect protein expression in infected cefs. briefly, cefs were infected with rdevs at multiplicity of infection (moi) of 0.001 and then fixed with 4% paraformaldehyde at 48 h postinfection. the antibodies described in section 2.5 were used as primary antibodies, while fluorescein isothiocyanate (fitc)-conjugated anti-mouse or anti-chicken igg (sigmaealdrich corporation) was used as the secondary antibody. expression of foreign proteins in recombinant viruses was observed by fluorescent microscopy. table 1 primers used in this study. primer sequence template egfp, enhanced green fluorescence protein; ibv, infectious bronchitis virus. schematic of the insertion of the n, s, and s1 genes of ibv into the dev genome. rdev-egfp was used as the parental virus for construction of rdev-n, rdev-s, and rdev-s1. (b) pcr identification by specific primers and differentiated primers. primer pairs nf and nr, sf and sr, and s1f and s1r were used to amplify specific genes from corresponding rdevs. the differentiated primer pair df and dr, corresponding to flanking regions of the us10 orf of dev, was used to analyze the purity and genetic stability of rdevs. there were differences in the fragment lengths amplified from the wt dev and each rdevs (dev clone-03, 637 bp; rdev-egfp, 1727 bp; rdev-n, 2217 bp; rdev-s, 4502 bp; and rdev-s1, 2610 bp). (c) western blot analysis of the expression of the n, s, and s1 proteins of ibv from the rdevs. the expression of chicken b-actin and the gl protein of dev were used as internal references of cefs infected with the rdevs. (d) immunofluorescence detection of the n, s, and s1 proteins of ibv in rdev-infected cefs. for detection of the n, s, and s1 proteins, rdev-infected cefs were fixed using 4% paraformaldehyde at 48 h postinfection. dapi was used to stain the nuclei and uninfected cefs were used as a control. to examine the growth kinetics of cefs infected with rdev-egfp, rdev-n, rdev-s, rdev-s1, or dev clone-03 at an moi of 0.001, the infected cefs and supernatants were harvested at 12, 24, 48, 72 and 96 h postinfection, and then titrated according to the method of reed and muench (1938) . to evaluate genetic stability, rdevs were passaged 20 times in cefs. after passages 5, 10, 15 and 20, the identity of each rdev was confirmed by pcr as described in section 2.5. a total of 150 four-week-old spf chickens were divided into six groups of 25 birds each. five groups of 25 chickens were inoculated intramuscularly with 10 6 pfu of rdev-n, rdev-s, rdev-s1, or received covalent vaccinations with rdev-n & rdev-s or rdev-n & rdev-s1. the remaining 25 chickens were immunized with dulbecco's modified eagle's medium, as a negative control. oropharyngeal and cloacal swabs were collected on post-vaccination days 3 and 6 for detection of the replication of the rdevs in chickens. peripheral blood samples from five vaccinated chickens were collected in sodium heparin-coated tubes to analyze the cellular immune responses to the rdevs on post-vaccination days 3, 7, 14, and 21, and on post-challenge day 5. on post-vaccination day 21, 10 chickens, including five that were stable for cellular immune analysis, were selected from each group and challenged with 10 6 50% egg infectious dose (eid 50 ) of the virulent ibv strain ck/ch/ldl/ 091022 by the oculonasal route. on post-challenge day 5, oropharyngeal swabs were collected from all chickens in each group to evaluate shedding of ibv. serum samples were collected from the remaining 15 chickens in each group to monitor levels of anti-ibv antibodies at 4 and 5 weeks post-vaccination. chickens were monitored daily for clinical signs of infection. snicks (abnormal respiratory sounds) made by each of the birds were counted by three individuals over a 2-min period. birds were checked individually for tracheal rales, nasal discharge, watery eyes, and wheezing. the percentage of birds that died or exhibited clinical signs was recorded daily for 20 days following ibv challenge. to determine whether the rdevs had the ability to replicate in chickens, cefs and 9-day-old spf embryonated chicken and duck eggs were used to re-isolate the viruses from the oropharyngeal and cloacal swabs of the vaccinated chickens. swabs maintained in phosphate-buffered saline (pbs) were centrifuged instantaneously at 8000 â g and the supernatants were filtered using a 0.45-mm syringe filter (millipore corporation, billerica, ma, usa) and inoculated in cefs and chicken or duck embryos through the chorioallantoic membrane, respectively. three blind passages were conducted for each swab sample. samples cultured for 5 days were detected by pcr using primers p1 and p2 (li et al., 2006) . serum samples were analyzed for the presence of anti-ibv antibodies using an enzyme-linked immunosorbent assay (elisa). briefly, 96-well plates were coated with homologous ibv strain ck/ ch/ldl/091022, which was concentrated and purified by differential centrifugation and sucrose gradient ultracentrifugation. standard positive and negative sera, secondary antibodies, substrates, and stop solution were provided with the ibv antibody elisa kit (idexx corporation, westbrook, me, usa). titers were automatically calculated using a microplate reader at an optical density of 605 nm. each serum titer was detected in triplicate, calculated based on the mean serum-to-positive (s/p) ratios (de wit et al., 1998; liu et al., 2006) , the cp value was determined as 0.25 and an s/p ratio ! 0.25 was considered positive. 2.10. analysis of cd4 þ , cd8 þ , and cd3 þ t-lymphocytes lymphocytes were isolated from peripheral blood by ficollehypaque density gradient centrifugation using the chicken lymphocyte separation kit (beijing solarbio science & technology co., ltd., beijing, china), according to the manufacturer's instructions. peripheral blood mononuclear cells were isolated from each blood sample, adjusted to a concentration of 1 â 10 6 cells/ 100 ml, and co-stained with mouse anti-chicken cd3-sprd, mouse anti-chicken cd8-pe, and mouse anti-chicken cd4-fitc (south-ernbiotech, birmingham, al, usa) antibodies for 1 h at room temperature. flow cytometry was performed using the bd facsaria cell sorter (bd biosciences, franklin lake, nj, usa) to calculate the percentages of cd4 þ , cd8 þ , and cd3 þ t-lymphocytes. viral shedding was quantified based on ibv rna levels in the oropharyngeal secretions of chickens challenged with strain ck/ch/ ldl/091022. the oropharyngeal swabs contained in pbs were treated and detected as described previously (jones et al., 2011; cao et al., 2011 cao et al., , 2012 ). all samples were tested in triplicate and the data were analyzed using the lightcycler 480 software, version 1.5 (roche diagnostics, basel, switzerland). the antibody titers of vaccinated chickens, the ratio of cd4 þ / cd8 þ t-lymphocytes, the growth kinetics of recombinant viruses, percentages of birds showing clinical signs, and mortality rates were statistical analyzed using multiple t-tests (graphpad prism6; graphpad software, inc., la jolla, ca, usa). all data are presented as means ± standard deviations. plaques formed by rdev-egfp exhibited green fluorescence, whereas those formed by rdev-n, rdev-s, and rdev-s1 did not (data not shown). the pcr screening and dna sequencing data confirmed that the n, s, and s1 cdnas from ibv were properly inserted into the dev genome with the deletion of the entire us10 gene. the primer pair df and dr, corresponding to the flanking regions of the us10 gene orf, were used to differentiate the wt from the rdevs. single fragments of varying lengths were amplified from the wt dev clone-03, rdev-egfp, rdev-n, rdev-s, or rdev-s1, indicating that the rdevs had been purified (fig. 1b) . western blotting analysis of the rdev-egfp-infected cells revealed a band of approximately 27 kda, which corresponded to the size of egfp. specific bands corresponding to the n (50 kda), s (128 kda), and s1 (71 kda) proteins of ibv were detected in the cells infected with rdev-n, rdev-s, and rdev-s1, respectively. the gl protein of dev was detected in cells infected with wt dev clone-03 or rdevs, indicating efficient dev replication as a viral vector in cefs (fig. 1c) . the immunofluorescence analysis showed that the n, s, and s1 proteins were expressed in cefs infected with rdev-n, rdev-s, and rdev-s1, respectively, whereas the mock-infected cefs exhibited no response to the anti-ibv or anti-ibv-n monoclonal antibodies (fig. 1d) . generally, the growth trends of rdev-egfp, rdev-n, rdev-s, and rdev-s1 were consistent with that of wt dev clone-03, which showed that the viruses reached the highest titer at 72 h and decreased at 96 h postinfection ( fig. 2a) . however, statistical analysis demonstrated that the titers of the rdevs at each time point significantly differed from that of the wt dev clone-03 (*p < 0.05, **p < 0.01). the primer pair df and dr was used to confirm the identity of three rdevs at passages 5, 10, 15, and 20. however, no 637-bp or 1727-bp fragment (existed in wt dev or rdev-egfp, respectively) was detected, demonstrating that three rdevs expressing ibv proteins were genetically stable without reversion (fig. 2b) . oropharyngeal and cloacal swabs were collected postvaccination for the detection of rdevs in chickens. pcr detection showed that viral isolation was negative from the cefs, as well as the chicken and duck embryo cultures, suggesting that the vaccinated chickens do not shed rdevs from the respiratory and gastrointestinal tracts. chickens were challenged with 10 6 eid 50 of the virulent ibv strain ck/ch/ldl/091022 at 21 days post-vaccination. the percentage of chickens that showed clinical signs in the rdev-n, rdev-s, rdev-s1, rdev-n & rdev-s, rdev-n & rdev-s1, and control groups was 30%, 20%, 40%, 20%, 20%, and 80% (p < 0.01, very significant difference, each group compared with the control group only), respectively (table 2) . birds began to exhibit ruffled feathers and snicks at 5 days after challenge with virulent ibv, and developed depression and huddle, dark combs, and death as time went on. mortality rates in the rdev-n, rdev-s, rdev-s1, rdev-n & rdev-s, rdev-n & rdev-s1, and control groups were 30%, 10%, 30%, 20%, 10% and 40%, respectively (p > 0.05, no significant difference) ( table 2) . gross lesions were confined primarily to the kidneys. the renal parenchyma of the affected birds was pale, swollen, and mottled, while the renal tubules and urethras were distended due to accumulation of uric acid crystals. we evaluated viral shedding using qrt-pcr to quantify the ibv rna in oropharyngeal swabs. in the control group, 90% of the chickens shed viruses, while challenged chickens in the rdev-n, rdev-s, rdev-s1, rdev-n & rdev-s, and rdev-n & rdev-s1 groups showed relatively lower viral shedding rates of only 20%, 30%, 40%, 20%, and 20%, respectively (p < 0.01, each group compared to the control group only). the antibody titers of anti-n, -s, and -s1 of vaccinated chickens were measured weekly using an elisa with plates coated with the homologous virus ck/ch/ldl/091022. in general, all groups showed relatively high antibody titers and seroconversion rates at week 1 post-vaccination, which then decreased gradually. at week 1 postvaccination, 84% (21/25) of the chickens vaccinated with rdev-n were seropositive, although seropositivity gradually decreased from week 2 (76%) to week 5 (46.7%). in the rdev-s vaccinated group, 92% (23/25) of the chickens exhibited seroconversion at week 1, but this percentage decreased rapidly afterward to 52% at week 2 and 16% at week 3 post-vaccination. in the rdev-s1 group, the rdevs with the us10 deletion exhibited statistically significant lower viral titers (**p < 0.01, *p < 0.05). viral titers are shown in tcid 50 /100 ml. (b) the genetic stability of the rdevs expressing the major structural proteins of infectious bronchitis virus (ibv). the identity of the rdevs was confirmed by pcr using the differentiated primer pair df and dr following the 5th, 10th, 15th, and 20th passages. no reversion occurred during the passage from the rdevs to the wt dev clone-03 (dev clone-03, 637 bp; rdev-n, 2217 bp; rdev-s, 4502 bp; rdev-s1, 2610 bp). 92% (23/25) of the chickens were seropositive, but this rate decreased sharply to 28% at week 2 and to 8% at week 3. the antibody levels and seroconversion rates of the rdev-n & rdev-s covalent-vaccination group were similar to those of the rdev-n and rdev-s group. the antibody titers of the rdev-n & rdev-s1 covalent-vaccination group were higher than all other groups and all of the chickens in this group were ibv antibody-positive at weeks 1 (100%) and 2 (100%), although these rates then slowly decreased from week 3 post-vaccination (84%). none of the chickens in the control group exhibited an antibody response (table 2) . there were significant differences (**p < 0.01, *p < 0.05) in antibody titers of each single vaccinated rdev group, as compared to the corresponding covalent-vaccination group (fig. 3) , suggesting that the covalent rdev-n & rdev-s1 vaccine enhanced the humoral response. cd4 þ and cd8 þ t-lymphocytes are important parameters of cell-mediated immune responses in vaccinated chickens. to evaluate the cellular response induced by rdevs in vaccinated chickens, the percentages of cd4 þ cd3 þ and cd8 þ cd3 þ t-lymphocytes, respectively, in peripheral blood were analyzed by flow cytometry, which showed that the ratio of cd4 þ and cd8 þ t-lymphocytes decreased at day 7 post-vaccination in each vaccinated group, and slightly increased from day 14 post-vaccination, as compared with that of birds in the control group (table 3) . there was a significant difference (p < 0.05) in the cd4 þ /cd8 þ ratio of the rdev-n group at day 7 post-vaccination, as compared to the control group. there was no significant difference among the three single-vaccination groups and the two covalent-vaccination groups (p > 0.05) ( table 3) . the ibv genome codes for four structural proteins, which play different roles in immune protection of vaccinated chickens. the n protein is an immunodominant antigen that induces high titers of cross-reactive antibodies. the m glycoprotein elicits low titers of antibodies with limited cross-reactivity, whereas the s1 glycoprotein induces production of serotype-specific and cross-reactive antibodies (ignjatovic and galli, 1993) . the s1 glycoprotein induces virus neutralizing and cross-reactive antibodies and cellmediated immune responses galli, 1994, 1995) . moreover, ignjatovic and galli (1993) reported that immunization of chickens with purified m glycoproteins did not induce protection against virulent ibv challenge, whereas immunization with the s1 glycoprotein prevented replication of nephropathogenic ibv in the kidneys, but not the tracheas, of immunized chickens. hence, the n, s, and s1 proteins of ibv were chosen as the target proteins to construct rdevs in order to evaluate the immune protection of these proteins against the virulent ibv strain ck/ch/ldl/091022. in the current study, the us10 gene of dev was replaced with the n, s, or s1 gene of ibv, respectively, to produce rdevs that expressed the n, s, and s1 proteins of ibv, respectively. virus titers of rdevs decreased 10-fold at 72 h postinfection compared to that of the wt dev clone-03. however, it was reported that virus titers of the ul44 gene deletion rdev decreased by 50e100-fold, as compared to the parental virus (wang and osterrieder, 2011) , suggesting that gene deletion of dev does affect viral replication, while the influence depends on the deletion site in the viral genome. the antibody levels of the chickens in the rdev-n & rdev-s1 covalent-vaccination groups were higher than those of all other vaccinated groups. at week 3 post-vaccination, the antibody levels in the rdev-s and rdev-s1 groups decreased sharply, whereas the covalent-vaccination groups had higher antibody levels. single rdev-n and single rdev-s1 vaccination invoked weak antibody responses, while covalent vaccination with these two rdevs induced strong antibody responses. the high antibody titers were related to the lower mortality in the rdev-n & rdev-s1 covalentvaccination group, indicating that covalent vaccination with the ibv structural proteins n and s1 provided better protection against challenge with virulent ibv strains. the ibv s glycoprotein consists (0) a number of chickens shedding ibv on day 5 post-challenge. b clinical signs (%) are presented as the percentage of birds showed clinical signs out of the total number of vaccinated chickens in the group after virulent ibv challenge. c p < 0.01, very significant difference, each group was only compared to the control group. fig. 3 . antibody responses of chickens vaccinated with recombinant duck enteritis viruses expressing the n, s or s1 gene of infectious bronchitis virus (ibv). sera from chickens immunized with rdev-n, rdev-s, rdev-s1, rdev-n þ rdev-s, rdev-n þ rdev-s1, or the control were tested for the presence of antibodies against ibv at 1 week (n ¼ 25), 2 weeks (n ¼ 25), 3 weeks (n ¼ 25), 4 weeks (n ¼ 15), and 5 weeks (n ¼ 15) post-vaccination. there were very significant differences in the antibody titers of the vaccinated groups as compared to that of the control group (p < 0.01). all single vaccinated groups were compared to the corresponding covalent vaccinated group. significant differences in antibody titers are indicated with dashes. of the s1 and s2 subunits. the s1 subunit induces efficient cellular immunity , while the n protein mainly induces cross-reactive antibodies (ignjatovic and galli, 1993) , which may be one of the reasons for the difference in antibody levels among the rdev-n, rdev-s and rdev-s1 groups. to evaluate the cellular immune responses induced by the rdevs, we detected the percentages of cd4 þ cd3 þ and cd8 þ cd3 þ t-lymphocytes in peripheral blood samples collected from vaccinated chickens on postvaccination days 3, 7, 14, and 21. the results showed that cd8 þ tlymphocytes increased and the causative ratio of cd4 þ /cd8 þ tlymphocytes decreased at day 7 post-vaccination in each vaccinated group, suggesting the induction of cytotoxic t lymphocytes (ctls). reportedly, cd4 þ t-lymphocytes induce and enhance the immune response by secreting cytokines, while most ctls were shown to be cd8 þ cells, which play a major role in the control of viral infection. collisson et al. (2000) reported that ibv-specific ctl activity was dependent on the s and n proteins of ibv, which was consistent with the results of the present study. moreover, chickens infected with ibv gray strain showed ctl responses as early as 3 days and peaked at 10 days postinfection (collisson et al., 2000) , consistent with our results on the detection of cd8 þ t-lymphocytes. in this study, the antibody responses and increases in cd8 þ tlymphocytes levels were detected as early as 7 days postvaccination, suggesting efficacious delivery of the foreign protein as a vaccine vector to the host, which elicited both humoral and cellular immune responses. chen et al. (2010) reported partial protection of chickens against a recombinant fowlpox virus expressing the ibv s1 protein with only 25% of birds showing clinical signs. in this study, clinical signs were detected in a significantly lower percentage of birds that were vaccinated with rdev-s, rdev-s1, or rdev-n after challenge with virulent strain ck/ch/ldl/091022 when compared with the control group, suggesting vaccination with these three viruses conferred better protection. in addition, a lower percentage of birds vaccinated with rdev-s showed clinical signs after challenge when compared with birds vaccinated with the other two viruses, demonstrating that better protection was conferred by vaccination with rdev-s. furthermore, although covalent vaccination with rdev-n & rdev-s or rdev-n & rdev-s1 provided the same clinical protection (20% clinical signs) as chickens in the rdev-s group, vaccination with the covalent vaccines offered better protection in regard to viral shedding of virulent ibv. we did not test a live ibv vaccine in the present study because no commercial lx4-type (qxlike) vaccine is currently available. in our previous study, we found that the h120 commercial vaccine did not provide efficacious protection against the ck/ch/ldl/091022 strain of ibv, resulting in a morbidity rate of 40% (sun et al., 2011) , which was higher than that observed in chickens vaccinated with rdev-s (20%), rdev-n (30%), dev-n & rdev-s (20%), or rdev-n & rdev-s1 (20%). the mortality induced by ibv ck/ch/ldl/091022 infection in the control group in this study was higher than that reported by sun et al. (2011) , which may be due to the higher dosages of the challenged viruses used in that experiment as compared to the present study (10 6 vs. 10 4.8 eid 50 per chicken, respectively). in a previous study, immunization with a recombinant fowlpox that coexpressed the s1 protein of ibv and chicken interleukin 18 provided complete protection against ibv infection in chickens (chen et al., 2010) . therefore, the coexpression of immunomodulatory factors, such as cytokines, may improve the efficacy of rdevs to induce protection against ibv infection. hence, future studies are warranted to determine whether the use of combinations of our rdev vaccines or boosting with a live or inactivated vaccine may induce better efficacious protection against ibv infection in poultry. in addition, the protection provided by our rdev vaccine against other ibv serotypes, such as the massachusetts serotype, should also be evaluated. development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex infectious bronchitis virus s2 gene sequence variability may affect s1 subunit specific antibody binding proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo coronavirus avian infectious bronchitis virus infectious bronchitis construction and immunogenicity of a recombinant fowlpox vaccine coexpressing s1 glycoprotein of infectious bronchitis virus and chicken il-18 cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry the long view: 40 years of infectious bronchitis research transmission of table 3 ratio of 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attenuation, safety, and efficacy of an infectious bronchitis virus ga98 serotype vaccine development and validation of rt-pcr tests for the detection and s1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples antigenic domains on thepeplomer protein of avian infectious bronchitis virus: correlation with biologicalfunctions phylogeny of antigenic variants of avian coronavirus ibv antigenicity of thepeplomer protein of infectious bronchitis virus characterization of the genes encoding ul24, tk and gh proteins from duck enteritis virus (dev): a proof for the classification of dev molecular characterization of the genome of duck enteritis virus recombinant duck enteritis virus works as a single-dose vaccine in broilers providing rapid protection against h5n1 influenza infection a duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against h5n1 avian influenza virus infection in ducks 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chickens. vaccine 32, 664e670. glossary ibv: infectious bronchitis virus dev: duck enteritis virus rdev: recombinant duck enteritis virus cef: chicken embryo fibroblast rt-qpcr: rt and real-time pcr elisa: enzyme ratio of cd4 þ and cd8 þ t-lymphocytes 3 dpv a 7 dpv 14 dpv 21 dpv 5 dpc rdev-n 0.80 ± 0.06 b 0.64 ± 0.06a 0.72 ± 0.08 0.72 ± 0.09 0.72 ± 0.12 rdev-s 0.92 ± 0.13 0.74 ± 0.10 0.85 ± 0.18 0.85 ± 0.14 0.89 ± 0.20 rdev-s1 0.82 ± 0.12 0.71 ± 0.08 0.82 ± 0.09 0.80 ± 0.14 0.80 ± 0.14 rdev-n & rdev-s 0.93 ± 0.16 0.73 ± 0.8.4 0.96 ± 0.24 0.81 ± 0.25 0.90 ± 0.33 rdev-n & rdev-s1 0.82 ± 0.14 0.76 ± 0.08 0.87 ± 0.20 0.83 ± 0.15 0.89 ± 0.22 control 0.87 ± 0.20 0.86 ± 0.17 0.92 ± 0.24 0.80 ± 0.19 0.86 ± 0.44 key: cord-259738-yuqc6dk0 authors: tang, mengjun; wang, hongning; zhou, sheng; tian, guobao title: enhancement of the immunogenicity of an infectious bronchitis virus dna vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2 date: 2008-03-07 journal: j virol methods doi: 10.1016/j.jviromet.2008.01.017 sha: doc_id: 259738 cord_uid: yuqc6dk0 a dna vaccine against infectious bronchitis virus (ibv) can induce specific humoral and cell-mediated immunity. however, compared to conventional vaccines, dna vaccines usually induce poor antibody responses. to develop a more potent ibv dna vaccine formulations, a monocistronic vector encoding the nucleocapsid protein of ibv and a bicistronic vector separately encoding the nucleocapsid protein and immune-stimulatory interleukin-2 were constructed. when the dna vaccines were administered to the quadriceps muscle of chickens, the induced humoral and cellular responses were evaluated. there was a significant difference in elisa antibody levels elicited by either monocistronic or bicistronic dna vaccines. the percentage of cd3(+), cd3(+)cd8(+) and cd3(+)cd4(+) subgroups of peripheral blood t-lymphocytes in chickens immunized with bicistronic dna vaccine were higher than those in chickens immunized with monocistronic dna vaccine. when chickens were challenged with a virulent strain of ibv, the protective efficacy could be enhanced significantly after immunization with bicistronic dna vaccine. these results demonstrated that bicistronic dna vaccine is an effective approach to increase ibv dna vaccine immunogenicity. infectious bronchitis (ib) is an acute and highly contagious respiratory disease of chickens. it is still a major health problem in the chicken industry in the world. vaccination to control ib has been practiced for over half a century (bijlenga et al., 2004; cavanagh and naqi, 2003; cavanagh, 2003) . such conventional vaccines, although generally effective, do have some disadvantages. attenuated vaccines, which generally induce long-lasting immunity, have a risk of insufficient attenuation and/or genetic instability (cook et al., 1986) . the limitations of inactivated vaccines include high cost for manufacturing and lack of long-term immunity. therefore a new generation of ib vaccines is called for. infectious bronchitis virus (ibv) is an enveloped coronavirus that contains an unsegmented, single-stranded, positive-sense rna genome. in addition to the internally localized nucleocapsid (n) protein, the virion is composed of spike protein, membrane protein and a small envelope protein. n protein carries epitopes inducing cross-reactive antibodies and is the most abundant virus-derived protein produced throughout infection (seah et al., 2000; boots et al., 1992; yu et al., 2001) . the carboxyl end of n protein has ctl epitopes for mhc compatible chickens . chickens inoculated with dna plasmids expressing the carboxyl end of n were protected from ibv infection . in recent years, the development of dna vaccines as a potentially safe alternative has been explored. dna immunization is an important vaccination strategy that has many characters desirable for an ideal vaccine, including induction of broad immune responses, long-lasting immunity and simple and cheap production. experimental dna vaccines against viral, bacterial, and parasitic disease have been described (tacket et al., 1999; strugnell et al., 1997; kalinna, 1997) and dna vaccines have been licensed for two nonhuman applications: one for west nile virus for horses (powell, 2004) and the other for infectious hematopoietic necrosis virus for salmon (lorenzen and lapatra, 2005) . hence dna vaccines appear to be a useful technology. it is believed that delivery of only a single dna plasmid of antigen is not optimal to protect against infection. adjuvants are used widely in various vaccine formulations for the enhancement of immune responses. among the adjuvants, cytokines have been explored extensively to augment the potency of dna vaccines (gurunathan et al., 2000; reyes-sandoval and ertl, 2001; scheerlinck, 2001; barouch et al., 2004; calarota and weiner, 2004; stevenson, 2004; barouch, 2006) . interleukin-2 (il-2), initially known as t-cell growth factor, is a powerful immunoregulatory lymphokine which produced by lectin-or antigen-activated t-cells (morgan et al., 1976) . it is secreted by mature t-lymphocytes upon stimulation and certain t-cell lymphoma cell lines constitutively. plasmid il-2 has been investigated as a potential vaccine adjuvant in several studies and has been shown to increase dna vaccine protective immunity against pathogens (diane and carlos, 2004; rompato et al., 2006; bu et al., 2003) . in the present study, a bicistronic plasmid encoding the nucleocapsid protein and immune-stimulatory interleukin-2 has been constructed and its immunogenicity and protective effect in chickens has been evaluated. it has been shown that the delivery of a bicistronic plasmid containing n gene and il-2 can accelerate specific antibody induction with an increase t-cell responses. the use of bicistronic vector may enable more efficient delivery of both antigen and cytokine in dna vaccination and promote synergistic responses. the specific-pathogen-free (spf) chicken embryos were purchased from shangdong institute of poultry science, shandong, pr china. chickens were hatched and housed in a specificpathogen-free environment at the laboratory animal and resources facility, sichuan university. the nephropathogenic strain of ibv, saibk strain was propagated in the allantoic cavities of 10-day-old spf embryonated chicken eggs, and then harvested allantoic fluid 36 h post-inoculation. the 50% chicken infection dose (eid 50 ) was determined by inoculating serial 10fold dilutions of virus into 10-day-old spf embryonated chicken eggs. vero cells were cultured in dulbecco's modified eagle's medium (gibco) supplemented with 10% fbs, 100 u/ml penicillin and 100 g/ml streptomycin at ph 7.2 and were kept at 37 • c with 5% carbon dioxide. the pires-egfp/dsred bicistronic plasmid which was constructed and identified previously, was used to construct monocistronic and bicistronic dna vaccines. this plasmid contained two fluorescent protein genes which were inserted into the multiple cloning sites (mcs) located on either side of the internal ribosome entry site from the encephalomyocarditis virus (ecmv). the ibv n gene was subcloned from recombinant plasmid pibvn into egfp of pires-egfp/dsred between nhei and xhoi restriction sites, and generated the plasmid pires-n/dsred. the il-2 gene was amplified by pcr from the plasmid pdnail-2 as a template. the forward primer (5 -ccaggatccaccatgatgtg aag-3 ) and reverse primer (5 -gaagcggccgcagattagttagc-3 ) specific for the il-2 gene were employed. the pcr product of il-2 was digested with bamhi and noti and ligated into similarly digested pires-n/dsred, then the bicistronic plasmid pires-n/il2 was created. the plasmid pires-n/dsred was digested with bamhi and noti, gel purified and blunt ended to create monocistronic pires-n. identification of the recombinant plasmids was performed by double enzymes digestion and dna sequencing. six-well tissue culture plates were seeded with vero cells (10 6 /well), and the cells were grown until they were about 70% confluent. the purified plasmids, pires-n, pires-n/il-2 and pires were transfected respectively into the vero cells with lipofectamine according to the manufacturer's instructions (invitrogen, ca, usa). the expression products were identified after 36-48 h. the transfected cells were harvested after 36 h and total cellular rna was prepared from the transfected cells by trizol reagent (gibco brl, usa). the transcription products were detected by reverse-transcription polymerase chain reaction (rt-pcr) with specific primer sets for the n gene and il-2 as listed. n forward primer: 5 -catctcgagtctt-ttatcatggcaagc-3 , reverse primer: 5 -ggcgaattca-ttagagttcattttcac-3 ; il-2 forward primer: 5 -ccaggatccaccatgatgtgcaaag-3 , reverse primer: 5 -gaagcggccgcagattagttagc-3 . the medium was aspirated 48 h after transfection, and the cells were washed once with phosphate-buffered-saline (pbs), fixed with 100% acetone for 10 min at −20 • c, then washed three times for 5 min each with pbs. thereafter, transfected cells were incubated at 37 • c for 1 h with the antibody, which was antiserum of rabbit to ibv. then the cells were washed twice for 5 min each with pbs and incubated for a further 1 h at 37 • c with the secondary fitc-conjugated goat-anti-rabbit igg antibody that included 0.05% azovan blue (purpose of using azovan blue was to identify the positive vero cells and negative vero cells). the positive vero cells expressing n protein were stained green; the negative vero cells were stained red. the cells were washed twice with pbs and analyzed the expression of the recombinant plasmid by fluorescence microscopy. the plasmids pires-n/il2, pires-n, pires were amplified in escherichia coli jm 109 and extracted using the alkaline lysis method as described previously (sambrook et al., 1989) . after purification by peg8000 precipitation, the plasmids were resuspended in phosphate-buffered saline (pbs, ph 7.2) and kept at −20 • c until used for immunization. for vaccination, the chickens were randomly divided into four groups (n = 20 each). the 7-day-old chickens were injected intramuscularly into the quadriceps muscle with 150 g of plasmid pires-n/il2 (group 1). a second group was given 150 g of plasmid pires-n (group 2). other groups included chickens administered with 150 g of empty vector pires (group 3), and chickens injected with 0.5 ml pbs only (group 4). all groups were boosted with an equivalent dose at 21 days after the initial inoculation. pre-vaccination sera were collected from all vaccinated chickens. blood was also collected before booster vaccination as well as before challenge. sera were stored at −20 • c for serologic analysis. total serum immunoglobulin g (igg) specific for ibv was measured by indirect enzyme-linked immunosorbent assay (elisa) as described previously, with brief modifications: elisa plates were coated with ibv lysate at 5 g/ml in carbonate buffer, ph 9.6, for overnight at 4 • c and blocked with 10% non-fat dried milk in pbs at 37 • c for 3 h. serum samples were tested in 1:20 dilution in 10% dried milk in pbst. igg against ibv was revealed with horseradish peroxidaselabeled goat-anti-chicken conjugate diluted 1:2000 in pbst. the substrate solution used was tmb microwell peroxidase. after 20 min of incubation in the dark, the reaction was stopped by the addition of 100 l of 2 m h 2 so 4 , and the optical density at 450 nm was measured in an elisa microplate reader. sera were run in duplicate. negative and positive control sera were included in each assay. total serum immunoglobulin g (igg) specific for ibv are represented as the optical density. peripheral blood samples from immunized chicken were collected from the jugular vein in 2.5 ml syringes preloaded with 0.2 ml of sodium heparin to prevent clotting on day 7 after the boosting vaccination. peripheral blood mononuclear cells were isolated from each blood sample by ficoll-hypaque density gradient centrifugation. pbmc were adjusted to 1 × 10 7 cells/ml. the 100 l of samples (1 × 10 6 cells) were incubated for 1 h at room temperature with antibody as follows: mouse anti-chicken cd4-pe, mouse anti-chicken cd8-fitc, mouse anti-chicken cd3-sprd (bd biosciences pharmingen). leukocyte samples were triply labeled with cd3, cd4 and cd8 antibodies. the samples were processed on fluorescence activated cell sorter. all of the chickens were challenged with 100eid 50 of the ibv saibk strain in 0.1 ml by the nasal-ocular route at 15 days after the boosting immunization. the challenged chickens were examined daily for signs of clinical illness such as coughing, sneezing, ataxia, dyspnea or death for 2 weeks. dead chickens were necropsied to confirm death by ibv infection. the challenged chickens generally began to show clinical signs from 4 to 10 days after challenge. chickens in each group were euthanized at 14 days post-infection. necropsies were performed immediately postmortem and kidney tissues were collected for further detection of virus. the kidney tissues were incised individually from either the dead or euthanized chickens at 14 days post-challenged. the virus in the kidney tissues of the challenged chicken was detected by rt-pcr. total rna was extracted using trizol ls reagent and subjected to rt-pcr using primers directed to the 3 untranslated region (forward primer: 5 -gatgaggagaggaacaatgc-3 ; reverse primer: 5 -tgggcgtcctagtgctgt-3 ). total protection was defined as negative for the presence of virus in the kidney. data were analyzed using the one-sided student's t test. differences were considered statistically significant with p < 0.05. the monocistronic and bicistronic dna vaccine plasmids encoding ibv n protein and il-2 were constructed using bicistronic plasmid pires-egfp/dsred as shown in fig. 1 . to confirm the transcription of constructs pires-n/il2, pires-n in a eukaryotic system, the plasmids were transfected respectively into vero cells. total rna was extracted from transfected cells at 38 h and analyzed by rt-pcr for the presence of each corresponding mrna. the predicted rt-pcr products were of 1.3 kb in size for n and 0.6 kb for il2, all of which were confirmed by gel electrophoresis. no specific band of a similar size was seen in any of the mrna samples in the absence of reverse transcription (fig. 2) . this result showed that constructs encoding n and il-2 gene can be transcribed successfully in the eukaryotic system. the expression of pires-n/il2, pires-n was demonstrated by indirect immunofluorescence assay. after transfection with lipofectamine, the transfected cells displayed positive signals for the protein and located cytoplasm, where there was green fluorescence. expression of n protein was not detected in transfected empty plasmid control cells (fig. 3) . this result shows that constructs encoding n protein can be successfully expressed in the eukaryotic system. the plasmids pires-n/il2, pires-n induced detectable antibodies to ibv ag in chickens one week after injection and the levels increased with subsequent vaccination. there was no specific antibody response in the group of chickens receiving pbs and pires plasmid. there was a significant difference in elisa antibody levels (p < 0.05) elicited by either monocistronic or bicistronic dna vaccines since the 14th-day after first inoculation. the result suggests that the bicistronic pires-n/il2 can enhance humoral responses (fig. 4) . peripheral blood lymphocytes were analyzed by flow cytometry on day 7 after the boosting immunization. the percentage of cd3 + and cd4 + cd3 + t-lymphocytes significantly higher (p < 0.05) was observed from chickens immunized with pires-n/il2 than the pires-n group. the percentage numbers of cd8 + cd3 + t-lymphocytes subgroups of the pires-n/il2vaccinated group were higher than the pires-n-vaccinated group but no significant difference (p > 0.05) (fig. 5) . mortality, kidney infection and percent protection after challenge of chickens were summarized in table 1 . chickens that started to show clinical signs or died from viral infection did so beginning on day 5 after challenge. the chickens immunized with either control vector pires or pbs were not protected and developed coughing, nasal discharge, dyspnea. the death rate of the pires and pbs immunized chickens was 55 and 65% at 14 days after challenge, respectively. the death rate of the pires-n/il2 dna vaccine immunized chickens was only 5%, lower than that of the chickens injected with the pires-n dna vaccine. to evaluate the level of protective response after challenge, the collected kidney samples were analyzed by rt-pcr. pcr results indicated that 20 and 50% of birds vaccinated with the pires-n/il2 and pires-n plasmids were positive for the presence of virus in the kidney, respectively. all chickens immunized with either control vector pires or pbs were positive in rt-pcr test. the protection percent of group that vaccinated with the pires-n alone was higher than that of the empty vector or pbs. the group vaccinated with the pires-n/il2 dna vaccine (80%) had the highest protection rate in all vaccinated groups. this suggests that the plasmid expressing both n protein and il-2 offers enhanced resistance against a virulent ibv challenge. in order to increase the efficiency of this immunization procedure, cytokine was co-expressed with the viral protein. this strategy has already been used successfully to enhance the effect of dna vaccination procedures (li et al., 2006; chow et al., 1997; henke et al., 2004) . the pires-egfp/dsred bicistronic plasmid which was constructed and identified previously was used to construct monocistronic and bicistronic dna vaccines. the plasmid pires-egfp/dsred enabled the simultaneous translation of two genes of interest from the same rna transcript. each gene is cloned into one of the multiple cloning sites on either side of the internal ribosomal entry site of the encephalomyocarditis virus (emcv). the entire construct is under control of the cytomegalovirus (cmv) immediate-early promoter allowing the expression of two individual proteins from one plasmid. in this study, an experimental immunization strategy was developed and tested against ibv. two recombinant plasmids pires-n/il2, pires-n were constructed. these recombinant plasmids were inoculated in chickens and tested in a protection-challenge experiment, demonstrating that vaccination with the co-expression plasmid pires-n/il2 can induce stronger immune response than vaccination with pires-n. thus, it seems that vaccination with a bicistronic dna vaccine expressing both ibv n protein and il-2 may elicit potent immune response. in this study, the level of specific antibodies developed in pires-n/il2 group was higher than that of pires-n group. however, the precise role of antibodies for the control of ibv infection remains controversial. some reports have shown that circulating antibody titer did not correlate with protection from ibv infection (gelb et al., 1998; gough and alexander, 1979; raggi and lee, 1965) . other studies demonstrated that humoral immunity plays an important role in disease recovery and virus clearance (cook et al., 1991; thompson et al., 1997; toro and fernandez, 1994) . the antibody-dependence of the mechanism of protection against the disease remains unclear, suggesting an important role of the t-cell response in efficacy protection. in order to evaluate recombinant plasmids induced t-cell response, peripheral blood lymphocytes were analyzed by flow cytometry. results of t-lymphocytes subgroup detection indicated, that the percentage numbers of cd3 + , cd4 + cd3 + and cd8 + cd3 + t-lymphocytes subgroups in pires-n/il2 vaccinated chickens group were higher than those in the pires-n vaccinated chickens. this demonstrated that il-2 has the ability to stimulate t-cell growth. it has been shown that cellmediated immunity to ibv is also induced and is believed to be a protective mechanism in ibv infection. cd8+ ctl are critical in the control of infectious bronchitis in poultry dhinakar and jones, 1997; seo et al., 2000) . cd4+ t-cell responses may increase the proliferation, maturation and functional activity of cd8+ ctl, providing increased help for b-cells and directly producing antiviral cytokines. by increasing the number of t-cells at the same site being able to respond to n antigen, there might have been a limiting effect on viral replication leading to better protection. to investigate the level of protection elicited by pires-n/ il2, vaccinated chickens were challenged with a nephropathogenic strain of ibv. chickens that received the pires-n/il2 plasmid dna were better protected than that administered with the plasmid pires-n. mortality of pires-n/il2 group was lower than that of pires-n group. the protection rate of pires-n/il2 group was the highest in all the vaccination groups, possibly indicating protective immunity overcome by virus aggressiveness. these results suggested that vaccination with the co-expression plasmid of n protein gene and il-2 may have increased the protection rate against challenge. results of immune response and viral challenge showed that the group inoculated with pires-n/il2 provided stronger immune response and better protection rate than pires-n vaccinated group. this indicated that il-2 might effectively enhanced humoral-mediated immune and cell-mediated immune responses to some extent. this was consistent with the result of previous reports (li et al., 2006; chow et al., 1997; henke et al., 2004) . these results demonstrated that bicistronic dna vaccine is an effective approach to increase ibv dna vaccine immunogenicity. our results showing the induction of both antibody and t-cell responses against the ibv challenge in chickens demonstrate that the delivery of antigens and cytokines via bicistronic vectors is feasible in the 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chicks from acute infection dna vaccines and adjuvants dna vaccine for bacterial infections phase i safety and immune response studies of a dna vaccine encoding hepatitis b surface antigen delivered by a gene delivery device systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens avian infectious bronchitis: specific lachrymal iga level and resistance against challenge study of protection by recombinant fowl poxvirus expressing c-terminal nucleocapsid protein of infectious bronchitis virus against challenge this work was supported by the foundation of chinese national programs for high technology research and development (project number 2006aa10a205). key: cord-001083-vy1nxax2 authors: malagnac, fabienne; fabret, céline; prigent, magali; rousset, jean-pierre; namy, olivier; silar, philippe title: rab-gdi complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes date: 2013-09-19 journal: plos one doi: 10.1371/journal.pone.0073772 sha: doc_id: 1083 cord_uid: vy1nxax2 in the model fungus podospora anserina, the payip3 gene encoding the orthologue of the saccharomyces cerevisiae yip3 rab-gdi complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. inactivation of payip3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. long and short forms of payip3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). the frameshift has been conserved over the evolution of the pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild. all living cells have to accurately convert gene information into proteins in a complex multistep process. translation is suspected to be the key step where errors may occur. one of the main issues is to keep translating the correct reading frame from the start codon to the stop codon. the reading frame is defined by the start codon, as the genetic code is unpunctuated; any shift leads to an aberrant protein. spontaneous frameshift errors occur at low levels in comparison to missense errors. the ribosome has developed several mechanisms to allow proper translocation of the trnas in the different sites. signals are present on some mrnas and induce an alternative reading of the genetic code by diverting the standard rules. these elements are very efficient at disrupting ribosome accuracy, increasing error rate from background (,5610 25 ) to 50% [1] . such programmed events are known as recoding. among the recoding sites identified, programmed frameshifting (prf) are the most frequently found signals. +1 frameshifting motifs are widespread and regulate several important cellular functions in both eukaryotes and prokaryotes [2, 3] whereas 21 frameshifting events are currently mainly limited to viral genomes [4] , with very few exceptions such as dnax in prokaryotes or the edr/peg10 and ma3 genes in eukaryotes [5, 6, 7, 8] . 21 frameshifting motifs are minimally formed by a slippery sequence of the general structure x xxy yyz (the initial reading frame is indicated) where trnas shift reading frame, and a stimulatory element (mainly a secondary structure) [9,10] located 5-9 nucleotides (nt) downstream of the slippery sequence. in the filamentous fungus podospora anserina, it was hypothesized about 30 years ago that translation accuracy changes during cell differentiation allowing the production of key regulatory proteins required for the development of this model fungus [11] . indeed, tampering with accuracy of the p. anserina cytosolic translation apparatus results in impaired development [12, 13] . especially, decreasing the error rate triggers abnormal ascospore formation [14, 15] and the development of an epigenetic cell degeneration mechanism known as crippled growth [16] . the regulatory proteins, whose diminished expression could be responsible for both alterations as hypothesized by picard-bennoun, are still unknown. moreover, altering translation accuracy results in altered lifespan [17, 18] , through presently unknown mechanism(s) [15, 17, 19, 20] . while sequencing the p. anserina genome, we identified potential candidates regulated by recoding that could explain the importance of maintaining a certain level of translational error [21] . among these candidate genes, a 21 frameshifting site located in the p. anserina orthologue of the saccharomyces cerevisiae yip3/pra1 protein appears conserved in pezizomycotina. yip3 is a rab-gdi displacement factor involved in endoplasmic reticulum to golgi transport. it is conserved in animals [22] and plants [23] . we present here a functional analysis of this factor in p. anserina showing that its production is indeed regulated by a programmed translational 21 frameshift, which appears widely conserved in filamentous ascomycetes. all the p. anserina strains used in this study derived from the ''s'' (uppercase s) wild-type strain that was used for sequencing [21, 24] . the latest genome sequence and est derived from the s strain are available at http://podospora.igmors.u-psud.fr. construction of the dmus51::su8-1 strain lacking the mus-51 subunit of the complex involved in end-joining of broken dna fragments was described previously [25] . dna integration in this strain proceeds almost exclusively by homologous recombination. standard culture conditions, media and genetic methods for p. anserina have been described [26] and the most recent protocols can be accessed at http://podospora.igmors.u-psud.fr/more.php; the m2 minimal medium is a medium in which carbon is supplied as dextrin, and nitrogen as urea. the methods used for nucleic acid extraction and manipulation have been described [27, 28] . transformation of p. anserina protoplasts was carried out as described previously [29] . for the frameshifting measurement, the fy1619 s. cerevisiae strain was used (mata ura3-52 trp1d63 his3d200 leu2d1). the reporter plasmid for frameshifting measurement was the pac vector [30] . the podospora target sequences, corresponding to either the 21 frameshift sequence (podo-1) (59-gcttttt ccggggaggtggtctaggtggtcggccacgagctcc or the in-frame control sequence (podo0) (obtained by addition of a g at the 59 end) were inserted at the msci restriction site located at the junction between bgalactosidase and the firefly luciferase coding sequences. hybrid sequences consisting of the ibv (infectious bronchitis virus) slippery site with either the ibv spacer sequence (ibv-podo-1 sp6) (59-tatttaaacgggtacgggaggtggtcaaggtggtc ggccacgagctcccaaattttcgcccc-39) or the podospora spacer sequence (ibv-podo-1 sp2) (59-tatttaaaccgg ggaggtggtctaggtggtcggccacgagctcccaaat tttcgcccca-39) followed by the podospora pseudoknot were also cloned into the pac msci site, as well as a sequence consisting of the ibv slippery site out-of frame with the ibv spacer sequence and the p. anserina pseudoknot (ibv-podo0) (59-ttatttaa acgggtacgggaggtggtcaaggtggtcggccacgag ctcccaaattttcgccccat-39). the b-galactosidase and firefly luciferase activities were quantified in the same crude extract as described previously [31] for standard growth conditions. quantifications were the mean of six independent measurements. the efficiency, defined as the ratio of firefly luciferase activity to b-galactosidase activity, is expressed as percentage, and calculated by dividing the firefly luciferase/b-galactosidase ratio obtained from the 21 frameshift construct by the same ratio obtained with the in-frame control construct [30] . statistical significance was determined using the mann-whitney test (xl stat 2007 software). payip3 was inactivated by replacing the payip3 cds with a hygromycin b-resistance marker. the flanking regions of the payip3 gene were amplified by pcr, using the primers 8470gf (59-aagcttccggatccgagaataacc-39) and 8470gr (59-tctagacggtttggagaaggaaaagg-39) for the upstream sequence, and 8470df (59-ctcgagaccattaacgcccgttgttt-39) and 8470dr (59-aagcttcttcgcgaccttctcaa-39) for the downstream sequence. two primers contain sites for restriction enzymes to help in cloning. the pcr products were digested with xbai/hindiii for the upstream region and with hindiii/xhoi for the downstream region and cloned into the pbchygro vector [13] digested with xbai and xhoi. the plasmid recovered was then linearized with hindiii and introduced into the p. anserina dmus51::su8-1 strain by transformation. numerous transformants were obtained. selected transformants were crossed with the wild-type strain to remove the dmus51::su8-1 marker and obtain mat+ and mat2 strains carrying the deletion. southern blot analysis confirmed that for two transformants the payip3 gene was correctly deleted ( figure s1 ). one such mutant was selected for further phenotypic analyses. to complement the payip3 d mutant, the wild-type gene was amplified by pcr using the phusionh high-fidelity dna polymerase (fermentas), p. anserina wild-type genomic dna and primers 8470df and 8470gr. the product was cloned into the pbc-phleo vector [13] , to yield ppayip3 + . sequencing of the insert proved that no mutation occurred in payip3 during amplification and cloning. the ppayip3 + plasmid was then introduced by transformation into the payip3 d mutant. twentysix transformants, all with a wild-type phenotype, were recovered. two were selected for further studies and crossed with wild-type. the phleomycin-resistant payip3 d f1 progeny carrying the payip3 + transgene had a wild-type phenotype, demonstrating that restoration of the phenotype was due to the introduction of a wild-type copy of the payip3 gene. the corrected version of the payip3 cds was pcr amplified using the phusionh high-fidelity dna polymerase, p. anserina payip3 c -gfp genomic dna (see below) and primers 8470df and 8470rl (59-agcggccgcttaactgacacaaacgacaatcg-39). because the payip3 c allele was amplified from a gfp-tagged version it has no stop codon. therefore the pcr product was cloned into the pbcphleo vector [13] along with the rib2 terminator harboring a taa codon in its 5 prime end, to yield ppayip3 c . sequencing the insert proved that no mutation occurred in payip3 c during amplification and cloning. the ppayip3 c plasmid was then introduced by transformation into the payip3 d mutant. 19 transformants, all with a payip3 d mutant phenotype, were recovered. two were selected for further studies and crossed with wild-type. analysis was performed on f1 progeny with the payip3 c payip3 d genotype. the truncated version of payip3 cds was pcr amplified using the phusionh high-fidelity dna polymerase, p. anserina wild-type genomic dna and primers 8470df and 8470rc (59-atctagactagaccacctccccggaaaaagcc-39). the pcr product was cloned into the pbc-phleo vector [13] , along with the rib2 terminator to generate the payip3 t plasmid. sequencing the insert proved that no mutation occurred in payip3 t during amplification and cloning. the ppayip3 t plasmid was then introduced by transformation into the payip3 d mutant. thirteen transformants, showing a range of phenotype intermediates between the wild-type and the payip3 d mutant, were recovered. three of them were selected for further studies and crossed with wild-type. analysis was performed on the f1 progeny with the payip3 t payip3 d genotype. to construct a c-terminal gfp fusion of the frameshifted protein, the 535 bp 39-end fragment of the 39 orf of the payip3 gene was pcr-amplified using the orf39w-bgl2 (59-gaa-gatctggctctagtgcatccaggacac-39) and orf39c-sma1 (59-tttcccgggtcgctttccttctagcagtacc-39) oligonucleotides, containing, respectively, the bglii and smai sites (underlined in the sequence). after enzymatic digestion, the fragment was cloned into the bglii and smai sites of the pegfphygro vector, resulting in the pyip3 + -gfp vector. pegfp-hygro contains the hygromycin b resistance gene from pbc-hygro inserted into the noti site of the pegpf-1 vector from clontech. a c-terminal gfp fusion with the frameshifted protein was artificially obtained using the pyip3 c -gfp vector, which was constructed by cloning a 2 kb fragment of the payip3 gene, in which the frameshift was eliminated. to this end, two pcrfragments corresponding to the 59 orf (860 bp) and the 59-end of the 39 orf (1208 bp) were amplified, respectively, with the orf59w-sal1 (59-acgtgtcgaccggttcagccgttctttc ggagc-39) (sali site underlined) and phase-c (59-cctcc ccggaaaaagccctcatcgataggcttg-39) oligonucleotides, and with the phase-w (59-caagcctatcgatgaggg ctttttccggggagg-39) and orf39c-sma1 oligonucleotides. phase-w and phase-c oligonucleotides are complementary and contain the slippery sequence (indicated in italic) of the 21 frameshifting site. they also carry a supplemental nucleotide (in bold) compared to the genomic sequence in order to place the 39 orf in frame with the 59 orf. both fragments were pcr-joined, saliand smai-digested and cloned into the correspondingly digested pegfp-hygro vector to yield the pyip3 c -gfp vector. a last c-terminal gfp fusion was constructed with only the 59 orf. a 869 bp fragment of the the 59 orf was pcr-amplified using the orf59w-sal1 and the orf59c-sma1 (59-agacccgggc-cacctccccggaaaaagcctc-39) (smai site underlined) oligonucleotides. after digestion with both the sali and smai enzymes, and cloning into the sali-smai-digested pegfp-hygro, the pyip3 t -gfp vector was obtained. all constructs were verified by sequencing and then transformed into the dmus51::su8-1 strain. dna was extracted from selected transformants, pcr-amplified so as to sequence the inserted chimaeric genes. for each of the three plasmids (pyip3 + -gfp, pyip3 c -gfp and pyip3 t -gfp), two transformants, in which a correct integration of the chimaeric gfp transgenes had occurred, were selected and crossed with wild-type. in the progeny, mat+ and mat2 strains carrying the gfp chimaeric genes and devoid of the dmus51::su8-1 were selected for observation. gfp localization was identical in the two transformants of all three constructs. the anti-gfp monoclonal antibodies from roche applied science (catalog number 11814460001) were used for the detection of gfp-fusion proteins by western blot analysis. proteins were extracted as described [32] . pictures were taken with a leica dmire 2 microscope coupled with a 10-mhz cool snap hq charge-coupled device camera (roper instruments). they were analyzed with imagej. the gfp filter was the gfp-3035b from semrock (excitation: 472 nm/30, dichroïc: 495 nm, emission: 520 nm/35). the trees were constructed using phyml [33] with the default parameters [34] and 100 bootstrapped data sets. trees were visualized with the itol server [35] . the structure of the -1 translational frameshifting motif of payip3 as seen in figure 1a , the mrna of the p. anserina payip3 gene (cds number pa_1_8470) overlaps two open reading frames (orfs). the small one located at the 59 end of the messenger is similar to the s. cerevisiae yip3 gene (ynl044w), while the larger one at the 39 end is not present in this yeast (fig. 1b) . this second orf has no known functional domain and is only found in the genomes of pezizomycotina, a large group of ascomycota fungi (see phylogenetic analysis below). translation from the first 59 orf should produce a 19.5 kda protein similar to yip3. it is possible to generate a larger protein of 61.6 kda in p. anserina that would encompass both the small 59 and large 39 orfs by hypothesizing a -1 frameshift. analysis of 12 independent cdnas obtained during the p. anserina genome sequencing project showed that the mrna sequence was identical to that of the gene except for a 173 bp intron located in the 59 untranslated region [21] . this indicates that the putative frameshift is not corrected by either rna editing or splicing and thus it had to occur during translation. we could not find a canonical frameshift signal; however, examination of the payip3 sequence suggested that frameshifting could happen at codon nu 170, since there is at this position the sequence ''u uuu ucc'', a potential slippery sequence [10] . despite the fact that this slippery sequence does not match the consensus x xxy yyz motif, this sequence would still allow trna tandem slippage by pairing of the trna ser (iga) to the -1 codon (uuc) since g-u base pairing between codon and anticodon is possible. moreover, there is a sequence capable of forming a stem-loop and possibly a pseudoknot two nt downstream of the potential slippery sequence (fig. 1c ), a feature that should increase frameshifting frequency [9, 10] . such close vicinity between the slippery sequence and the stimulatory element is rather unusual for a 21 frameshifting stimulatory element. however the g-c stretch at the base of stem 1 is reminiscent of the very well-studied ibv pseudoknot [36] . despite these unusual features we were able to detect the long frameshifted protein fused with gfp, demonstrating that this 21 frameshifting motif is indeed functional (see below). in our previous study [21] , we observed that the yip3 -1 frameshifting motif is conserved during evolution, at least in pezizomycotina. the availability of additional fungal genome sequences now makes it possible to refine the phylogenetic analysis. to this end, the yip3 orthologues from representative species, whose genome sequences were available in public databases and covering the entire diversity of the eumycota, were manually annotated. the sequences of the 59 short and 39 large orfs of the pezizomycotina proteins, along with those of other fungi, were aligned and phylogenetic trees were constructed ( figures s2 and s3 ). both trees were compatible with the known evolution of the eumycota and pezizomycotina (fig. 2) . in most fungi, yip3 is encoded by a single gene (fig. 2) . the presence of a c-terminal extension was detected in all species of pezizomycotina, except for ascosphaera apis and arthrobotrys oligospora (fig. 2) . examination of the structure downstream the frameshift sites showed that it was highly conserved in most pezizomycotina and compensatory changes could be detected in the pseudoknot structure ( fig. 3a and 3b) . otherwise, changes were restricted to the predicted loops (fig. 3b) . however, in the case of tuber melanosporum, the adopted structure may not be a pseudoknot, but rather a simple stem-loop. interestingly, in two clades, the capnodiales (eight species investigated) and the ophiostomatales (one species investigated), the c-terminal extension was not in the 21 frame but in the +1 frame ( fig. 2 and fig. 3c ). the uuuuu slippery sequence and the pseudoknot were conserved in grosmania clavigera, the ophiostomatales, while the uuuuu sequence was changed to uuucu and only a small stem loop could be predicted in the case of mycosphaerella graminicola, a capnodiales (fig. 3c ). this could indicate that a single trna slippage would occur in this sequence instead of the classical tandem trna slippage. deletion of payip3 uncovers a growth phenotype but evidences no role in ascospore formation and crippled growth development to define the role of payip3 in the physiology of p. anserina, the gene was inactivated by replacing its entire coding sequence by a hygromycin b resistance marker, i.e., the deleted gene is unable to produce either the short or the long forms of payip3 (see materials and methods). the payip3 d mutant strain was investigated during the entire life cycle of the fungus and no difference was detected during the reproductive phase, i.e., kinetics and modalities of fruiting body development, ascospore genesis and germination. on the contrary, mycelium defects appeared. first, the mycelium of payip3 d grew slightly more slowly than wild-type (6+0.2 mm/d instead of 7+0.2 mm/d). however, growth resumed at the same speed as wild-type after 3 to 4 days of incubation. this was accompanied by an altered pattern in the repartition of the fruiting bodies (perithecia; fig. 4 ) when grown on m2 minimal medium. indeed, on this medium the wild-type produced perithecia mainly as a 1 cm-thick ring with an internal diameter of 1 cm, whereas in the mutant the ring was 0.7 cm thick the presence (species in black) and absence of the 39 orfs (species in grey) of yip3 was mapped on the most probable phylogenetic tree of the eumycota [43, 46] . the most parsimonious scenario of evolution is a single appearance of the 39 orf after the split of the orbiliomycetes from the other pezizomycotina (slanted arrow) and a secondary loss in a. apis (cross). * denotes the two groups for which a+1 frameshift is hypothesized and 62 the species in which a duplication of yip3 has occurred. doi:10.1371/journal.pone.0073772.g002 and its internal diameter was 0.7 cm. moreover, although the kinetics of perithecium development was identical after fertilization, perithecia from mat+/mat2 heterokaryotic mutant cultures yielded ascospores half a day before wild-type cultures, suggesting that fertilization occurred earlier in the mutant, in line with the smaller diameter of the ring of fruiting bodies. finally, when grown on a medium with wood shavings as sole carbon source, fertility of the mutant was diminished and fewer ascospores were produced, while growth with whatman paper (i.e. pure cellulose) as carbon source was only marginally modified (fig. 4) . significantly, crippled growth and life span were not modified in the mutant (data not shown). to assess whether the ring and wood shaving phenotypes were due to deletion of payip3, a wild-type copy of the payip3 gene was introduced by transformation into the payip3 d mutant. as seen in figure 5 , complete restoration of the wild-type phenotype was observed in transformants carrying an ectopic copy of payip3 + , whereas the complemented strains formed a wild-type ring of perithecia and produced as much ascospores as wild-type on wood shaving medium, demonstrating that inactivation of payip3 was responsible for all the phenotypes observed (compare payip3 d with payip3 + ). the mutant was also transformed with truncated and corrected alleles of the payip3 gene. the payip3 t truncated allele was obtained by removing the 39 orf and produced only the 19.5 kda polypeptide. the payip3 c corrected allele was created by inserting a c in codon nu168, which is just upstream of the frameshift site, and produced the 61.6 kda polypeptide resulting from the fusion of the proteins produced by the 39 and 59 orfs. introduction of payip3 c did not result in the restoration of a wildtype phenotype: ring and ascospore production on wood shaving medium was identical to that observed with the payip3 d mutant (fig. 5) . a heterogeneous restoration depending on the transformants was observed with the payip3 t allele. recovery ranged from complete (e.g., the t1 transformant had a wild-type phenotype) to inexistent (e.g., the t2 transformant has a payip3 d mutant phenotype, fig. 5) , while others presented a modified repartition of perithecia and diminished fertility on wood shaving medium (e.g., the t3 transformant had a more diffuse ring and produced reduced amount of ascospores on wood shaving medium). integrative transformation in p. anserina results mostly in non-homologous insertion. variability in the complementation observed with the truncated allele could be explained by influence of the insertion point on the expression of the transgene. to check whether expression of both the truncated and corrected form in the same strain could rescue a complete wild-type phenotype, we constructed by genetic crosses strains carrying both the corrected and truncated alleles of payip3. as seen in figure 5 , these strains exhibited the same phenotypes as those expressing the truncated allele alone, indicating that co-expression of both forms is not sufficient to recover a wild-type phenotype. to determine the in vivo expression level and localization of the short and long forms of yip3, we constructed plasmids carrying chimaeric versions of the wild-type, truncated and corrected alleles by adding in frame the egfp cds at the 39 end of payip3 (see materials and methods). transformation of the three plasmids into p. anserina resulted in their integrations at the payip3 chromosomal locus by a single crossing-over, generating the modified loci depicted in fig. 6a . in the case of the wild-type payip3 + -gfp transgene only a small ,500 pb truncated payip3 cds was present downstream of the chimaeric gene, while for the two other constructs a complete cds was retained. however, these copies had a 59-untranslated region truncated just before the intron (fig. 1) , and thus lacked the promoter region as well as 350 pb of the 59-utr of the payip3 mrna. to ensure that the recovered gfp-transgenes were expressed, we monitored by western blotting with an anti-gfp antibody the production of the proteins from payip3 + -gfp, payip3 t -gfp and payip3 c -gfp (fig. 6b) . a doublet of bands, likely due to different levels of posttranslational modification observed for proteins with reticulum/golgi localization, was detected at ,45 kda for payip3 t -gfp, as well as a band of about 88 kda for payip3 + -gfp and payip3 c -gfp. these sizes are expected for the fusion proteins, showing that they were actually produced in p. anserina. although present in low amounts, the detection of a band of high molecular weight with the payip3 + -gfp construct demonstrated that the 61.6 kda-long form of the protein is naturally produced by the wild-type allele. when analyzed on m2, the strains carrying the payip3 + -gfp and payip3 t -gfp transgenes grew and presented a ring of perithecia as did the wild-type strain, while those carrying payip3 c -gfp were similar to the payip3 d mutant (fig. 7) . to test whether the phenotype of payip3 c -gfp resulted from lack of activity of the remaining downstream wild-type cds or to a dominant negative effect of the payip3 c -gfp copy, we constructed by genetic crossings balanced heterokaryon with the following genotype: payip3 c -gfp lys2-1/payip3 + leu1-1. these had a wildtype phenotype (fig. 7) , showing that the payip3 c -gfp phenotype was recessive. the same expected result was observed with a payip3 d -gfp lys2-1/payip3 + leu1-1 heterokaryon used as control. therefore, the payip3 c -gfp protein displayed no dominant negative effect. hence as observed for the ectopic payip3 c protein, payip3 c -gfp is not functional and the downstream copy microscopic examination revealed a clear expression of the wild-type fusion payip3 + -gfp protein in the hyphae confirming that the recoded form is efficiently expressed in vivo in the host. this longer protein seems to display the same localization as the smaller form (payip t -gfp; fig. 8 ). the corrected in-frame form (payip3 c -gfp) is more difficult to observe in the hyphae, as expected if it is present in lower amounts. in all three strains, the fluorescence was concentrated in foci compatible with a reticulum/golgi localization as observed for the orthologous yip3/ pra1/pra2 protein [37, 38] . intriguingly, while we did not detect fluorescence in the centrum of fruiting bodies from the payip3 + -gfp and payip3 c -gfp strains, a clear staining was observed with the payip3 t -gfp protein, suggesting that only this short form of the protein is present in this tissue. unfortunately it is not possible to evaluate frameshifting efficiency by western-blot as the small peptide is not tagged with gfp in the payip3 + -gfp construct. moreover western-blot results from payip3 c -gfp suggest that the longer form could be unstable, due to the weak intensity of the band (fig. 6) , which corresponds to 100% of corrected protein. to determine if the identified payip3 recoding sequence was able to promote 21 frameshifting, it was inserted between the b-galactosidase and firefly luciferase open reading frames of a yeast s. cerevisiae reporter plasmid [30] . the 21 frameshifting efficiency was quantified as described in materials and methods. the level was estimated to be 1%, which in s. cerevisiae indicated a low but true -1 frameshift event. we also replaced the slippery sequence by the very well characterized slippery sequence from the ibv 21 frameshift (t tta aac) in frame or out-of-frame. positioning the ibv slippery sequence out-of-frame results in a significant drop in frameshifting efficiency (0.3%), indicating that the 1% obtained with the payip3 sequence is significant. however, despite having tested several spacer distances between the ibv slippery sequence and the payip3 pseudoknot, it is clear that this pseudoknot is unable to stimulate frameshifting in the ibv sequence to the same extent as does the ibv pseudoknot does ( figure s4 ). this confirms the unusual nature of the frameshifting event that occurs on this sequence. we present evidence that the p. anserina gene orthologous to s. cerevisiae yip3, payip3, is expressed in a complex fashion, whereby two proteins are produced with a single mrna through a translational -1 frameshifting event. the mrna produced by this gene encompasses two orfs, the 59 orf is highly conserved and is similar to the cds of yip3, while the 39 orf is less conserved and present only in pezizomycotina. the two orfs can be expressed in p. anserina as a single polypeptide if a 21 frameshift occurs during translation. we were able to reveal in vivo a fusion protein corresponding to the translation of the two orfs. we know from the analysis of est that the mrna does not undergo any modification (splicing, editing); therefore, only a translational event can explain the fusion protein. several translational recoding events such as ribosome hopping or frameshifting could explain how the fusion protein is synthesized. however, two characteristic features reminiscent of frameshifting motifs are found in the overlapping sequence between the two orfs: i) a pseudoknot that is an mrna structure commonly stimulating 21 frameshifting [9, 10] ; ii) a slippery sequence. this sequence does not match the 21 frameshifting slippery sequence consensus; nevertheless, the slippage of the trna ser (iga), which base pairs to the ucc codon in the initial frame, can occur on the -1 codon (uuc) since g-u base pairing between codon and anticodon is possible. another unconventional feature of this new frameshifting site is the proximity of the slippery sequence to the stimulatory pseudoknot. indeed the slippery sequence is located only 2 nt upstream of the pseudoknot whereas the spacer region is usually around 5 to 9 nt. to gain further details about this new frameshifting site we tested this motif in the yeast s. cerevisiae with a dual reporter system. frameshifting efficiency was estimated as 1%, which is low but significant since frameshifting efficiency dropped to as low as 0.3% when the reading frame in which the ribosome encounters this slippery sequence was changed. moreover our western-blot results suggest that frameshifting efficiency could be higher in the natural host. to better characterize the frameshifting mechanism the slippery site was replaced by a canonical slippery sequence from the ibv pseudoknot. despite efforts to position the ibv slippery sequence either at 6 nt from the pseudoknot (the size of the ibv spacer region), or at 2 nt (the size of the payip3 pseudoknot) the ibv frameshifting efficiency in yeast (10-15%) was not reached and only a moderate increase with a 2 nt spacer was observed. this indicates that the payip3 pseudoknot is unable to stimulate frameshifting as does the ibv pseudoknot with a 6 nt spacer. however we cannot rule out that the slight increase observed with figure 7 ). (b) cellular extracts from transformants with the indicated genotypes were separated on a 10% acrylamide 1 mm-thick gel and probed with an anti-gfp antibody. the left part results from a short exposition time that reveals the control and the payip3 t peptides. the right part is a longer exposition time to reveal the proteins produced from payip3 + and payip3 c transgenes. brackets and arrow, respectively, indicate the short and the longer proteins specifically revealed by the anri-gfp antibody. controls: 59 kda fusion protein of su12 ribosomal protein in frame with gfp (su12-gfp, [48] ). doi:10.1371/journal.pone.0073772.g006 the 2 nt spacer is significant. this tentatively suggests that the payip3 pseudoknot may slightly improve basal frameshifting of the ibv slippery sequence when positioned 2 nt downstream. it is always difficult to extrapolate the functional role from a predicted structure, as the pseudoknot can fold in an inactive form [39] . however one plausible explanation comes from the lack of conservation of the potential slippery sequence between the two organisms, suggesting that the frameshifting event does not occur by a dual trna slippage as for ibv but more probably by a single p-trna slippage, similarly to frameshifting found in bacterial transposable elements [40] . this would also explain why the payip3 pseudoknot is unable to fully stimulate dual trna slippage on the ibv sequence. this is also in accordance with the fact that this pseudoknot displays several unusual features. it is positioned very close to the slippery sequence (only 2 nt), and there is a highly conserved bulge of 3 nt in stem 1 of the pseudoknot. such a 3 nt bulge has already been described for the edr frameshifter pseudoknot [6] but remains very unusual. mutagenesis of edr frameshifting supports a tandem trna slippage model and the bulge found in edr pseudoknot does not play any role in frameshifting. the situation could be very different for payip3 frameshifting as a trans-acting factor could bind this pseudoknot to stimulate frameshifting in the host. this frameshifting is not conserved in s. cerevisiae so this unknown factor would be absent in this species explaining the inability of the pseudoknot to stimulate frameshifting at the ibv slippery site. whatever the mechanism, this frameshifting site is functional in p. anserina. it is worth mentioning that up to now the only two eukaryotic genes (ma3 and edr/peg10), that use a -1 frameshift to extend the size of a protein, are derived from domesticated retroviruses. no such signature is found in payip3 suggesting that this will be the first cellular gene using a -1 frameshifting event to extend the protein length and not related to a retrovirus. in s. cerevisiae, yip3 facilitates the dissociation of endosomal rab-gdi complexes [41] . however, yip3 may have multiple functions in the cell, which are as yet not well characterized [37] . in p. anserina, deletion of payip3 triggers three phenotypes. indeed, on m2 standard medium, the mutants initiate their growth slightly more slowly than wild-type and differentiate a smaller ring of fruiting bodies that produce ascospores half a day earlier. they are also impaired in their ability to produce abundant ascospores on medium with wood shavings as sole carbon source. at the present time, the molecular defects underlying these phenotypes are unknown, but defects in protein routing may account for them. in particular, degradation of wood shavings relies on the secretion of many enzymes that act synergistically, and impairment in secretion can alter the ability to degrade lignocellulose. remarkably, the mutants are not affected in their ability to complete maturation of ascospores (although their yield is reduced) and to undergo crippled growth, the two main developmental phenotypes triggered by increasing translation accuracy. likewise, their lifespan is not modified, as typically seen in accuracy mutants. therefore, payip3 is not the factor produced by a recoding event and hypothesized to regulate either one of these phenomena [11, 15, 16, 17] . some of the other p. anserina genes found to be potentially regulated by recoding [21] could be involved. the possible role of payip3 frameshifting in p. anserina is not clear at the present time. indeed, expression of the 19.5 kda alone can restore a wild-type phenotype, when present at the payip3 chromosomal location (fig. 5) . however, when present at an ectopic location, complementation may not be complete, likely due to abnormal expression of the transgenes integrated at an ectopic location (fig. 5) , unlike what is observed for the wild-type allele with frameshift. moreover, expression of the 61.6 kda polypeptide alone is unable to restore a wild-type phenotype, indicating that it is inactive. it does not appear to act as a dominant negative form (fig. 5) . finally, expressing both the 19.5 kda and 61.6 kda forms within the same cells from different transgenes does not improve phenotypic rescue. several explanations can be proposed. possibly, the laboratory conditions tested are not appropriate to detect an effect of the lack of frameshift. redundancy from another factor along with payip3 may be masking an effect of the frameshift product. the 19.5 kd and 61.6 kd forms may need to be present in defined ratio to observe an effect. another possibility would be that doing an error during translation rather than producing two different polypeptides is important. this could allow for spatial or temporal regulation of payip3. interestingly, while expression of the 19.5 kda form is detected in the perithecium centrum, expression of the 61.6 kda form was not detected either from the wild-type allele or the corrected allele. possibly, the c-terminus of the 61.6 kda polypeptide acts as a degradation signal in the centrum. this situation is reminiscent of the s. cerevisiae pde2 gene. indeed this gene undergoes translational stop codon readthrough producing a short and a long protein. the longer protein is destabilized and quickly degraded by the proteasome [42] . in the case of payip3 this degradation signal is tissue specific adding a supplementary layer of regulation. the reason for the lack of the 61.6 kda in the centrum of fruiting bodies is not clear, as we detected no change in the ascospore maturation and ejection processes in the mutants. possibly, a protein degradation mechanism exists in the centrum so as to ensure that only certain proteins are transmitted to the progeny. the 61.6 kda protein may be recognized and specifically degraded, without causing any harmful effect. whatever the role of the recoding, it seems important from an evolutionary point of view as it is conserved in all the pezizomycotina that we have investigated except for a. oligospora and a. apis. a. apis is a fungus that infects beehives and may present features associated with parasitism, such as simplified metabolism. more interesting is the fact that a. oligospora belongs to the orbiliomycetes, a class of pezizomycotina, which is believed to be the first to have diverged during evolution [43] . if correct, the most parsimonious explanation of the phylogenetic pattern of the c-terminal extension would be its origin after the split of the orbiliomycetes from the other pezizomycotina and its removal from the ancestors of a. apis due to their parasitic lifestyle (fig. 7) . this split likely occurred more than 400 million years ago [44] . during this time, the 21 frameshift was conserved except in two orders for which a+1 frameshift may occur. in the ophiostomatales, the frameshift site is well conserved, i.e., a slippery sequence and a pseudoknot are present, yet only a+1 frameshit can join the 59 and 39 orfs. in the capnodiales, the slippery sequence and the pseudoknot are not conserved, but the two orfs are conserved in a+1 frameshift configuration. this confirms that recoding during translation is important for the regulation of yip3 in filamentous ascomycetes. a recent report [45] shows that recodings are involved in metabolic control in a wide range of fungi through the production of distinct polypeptides in the targeting of enzymes to different compartments. here, we show that a gene involved in routing proteins is also subjected to translation recoding control, possibly adding a level of complexity to the metabolic network. reprogrammed genetic decoding in cellular gene expression autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme expression of peptide chain release factor 2 requires high-efficiency frameshift non-canonical translation in rna viruses a functional -1 ribosomal frameshift signal in the human paraneoplastic ma3 gene characterization of the frameshift signal of edr, a mammalian example of programmed -1 ribosomal frameshifting programmed ribosomal frameshifting generates the escherichia coli dna polymerase iii gamma subunit from within the tau subunit reading frame the gamma subunit of dna polymerase iii holoenzyme of escherichia coli is produced by ribosomal frameshifting frameshifting in alphaviruses: a diversity of 39 stimulatory structures rna pseudoknots and the regulation of protein synthesis does translational ambiguity increase during cell differentiation? genetics of ribosomes and translational accuracy in podospora anserina two new easy-to-use vectors for transformations ribosomal suppressors and antisuppressors in podospora anserina: altered susceptibility to paromomycin and relationships between genetic and phenotypic suppression eef1a controls ascospore differentiation through elevated accuracy, but controls longevity and fruiting body formation through another mechanism in podospora anserina propagation of a novel cytoplasmic, infectious and deleterious determinant is controlled by translational accuracy in podospora anserina increased longevity of ef-1 alpha high-fidelity mutants in podospora anserina a site-specific deletion in mitochondrial dna of podospora is under the control of nuclear genes deletion and dosage modulation of the eef1a gene in podospora anserina: effect on the life cycle informational suppressor alleles of the eef1a gene, fertility and cell degeneration in podospora anserina the genome sequence of the model ascomycete fungus podospora anserina targeting rab gtpases to distinct membrane compartments the pra1 gene family in arabidopsis les phénomènes de barrage chez podospora anserina. i. analyse génétique des barrages entre souches s and s the crucial role during ascospore germination of the pls1 tetraspanin in podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes contribution à l'étude génétique d'un ascomycète tétrasporé: podospora anserina (ces.) rapid methods for nucleic acids extraction from petri dish grown mycelia transformation by integration in podospora anserina.i. methodology and phenomenology nonsense-mediated decay mutants do not affect programmed -1 frameshifting versatile vectors to study recoding: conservation of rules between yeast and mammalian cells a mitotically inheritable unit containing a map kinase module a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood phylogeny.fr: robust phylogenetic analysis for the non-specialist interactive tree of life (itol): an online tool for phylogenetic tree display and annotation characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot saccharomyces cerevisiae rab-gdi displacement factor ortholog yip3p forms distinct complexes with the ypt1 rab gtpase and the reticulon rtn1p pra isoforms are targeted to distinct membrane compartments an equilibrium-dependent retroviral mrna switch regulates translational recoding programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of trnas yip3 catalyses the dissociation of endosomal rab-gdi complexes translational readthrough of the pde2 stop codon modulates camp levels in saccharomyces cerevisiae orbilia ultrastructure, character evolution and phylogeny of pezizomycotina dating divergences in the fungal tree of life: review and new analyses cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi a multigene phylogeny of olpidium and its implications for early fungal evolution freiburg rna tools: a web server integrating intarna, exparna and locarna in vivo labelling of functional ribosomes reveals spatial regulation during starvation in podospora anserina we thank sylvie françois for her expert technical assistance and anne-lise haenni for correcting the manuscript. key: cord-282314-9cua2jzg authors: albanese, grace a.; lee, dong-hun; cheng, i-hsin n.; hilt, deborah a.; jackwood, mark w.; jordan, brian j. title: biological and molecular characterization of arkga: a novel arkansas serotype vaccine that is highly attenuated, efficacious, and protective against homologous challenge date: 2018-10-01 journal: vaccine doi: 10.1016/j.vaccine.2018.08.078 sha: doc_id: 282314 cord_uid: 9cua2jzg almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (ibv) using live attenuated vaccines mass administered by spray at day of hatch. although many different types of ibv vaccines are used successfully, the arkdpi serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. in this study, we examined a different ark vaccine strain (ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. further attenuation of the ark99 vaccine was achieved by passage in embryonated eggs but arkga p1, p20, and p40 (designated arkga after p1) were still too reactive to be suitable vaccine candidates. however, arkga p60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. in addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. the full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the illumina platform and the data showed single nucleotide polymorphisms (snps) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. arkga p60 accumulated the most snps in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. these results indicate that the arkga p60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic ark-type virus. avian infectious bronchitis virus (ibv) is a gammacoronavirus that causes an economically significant upper respiratory tract disease in chickens [1] . because of its prevalence and infectivity, nearly all commercial poultry in the u.s. are vaccinated for ibv in a serotype-specific manner [2, 3] . ibv vaccines are developed by passaging a pathogenic field virus in embryonated eggs until the virus has lost its pathogenicity in chickens. during these repeated rounds of embryo passage, the pathogenic field virus will accumulate mutations that result in an adaptation for replication in embryos. conversely, the outcome of this adaptation is a decreased affinity for chicken tissues and therefore a reduced virulence in https://doi.org/10.1016/j.vaccine.2018.08.078 0264-410x/ó 2018 elsevier ltd. all rights reserved. abbreviations: ark99, arkansas 99; arkdpi, arkansas delmarva poultry industry; arkga, arkansas georgia; cas, chorioallantoic sac; c t , cycle threshold; eid 50 , 50% embryo infective dose; ibv, infectious bronchitis virus; mhv, murine hepatitis virus; nsp2, nonstructural protein 2; nsp3, nonstructural protein 3; p, passage; pbs, phosphate-buffered saline; qrt-pcr, quantitative real-time reverse-transcriptase polymerase chain reaction; rt-pcr, reverse-transcriptase polymerase chain reaction; sars-cov, severe acute respiratory syndrome coronavirus; sd, standard deviation; sem, standard error of mean; snp, single nucleotide polymorphism; spf, specific-pathogen free; us, united states; usda, united states department of agriculture. chickens [4] [5] [6] . live attenuated vaccines stimulate both humoral and cellular immunity, resulting in high levels of protection, and can be mass applied by spray [3, 7] . immunity resulting from vaccination with live attenuated ibv vaccines prevents replication of homologous virulent challenge virus within only a short time following vaccination [7] . of the vaccines used in the u.s., the arkansas delmarva poultry industry (arkdpi) serotype vaccine has been shown to be highly variable in its protective ability and is frequently isolated from vaccinated chicks [8] [9] [10] . ideally, when mass applying an ibv vaccine, a high percentage of chicks should be infected with actively replicating virus (vaccine coverage) by days 7-10 post-vaccination, followed by a gradual decline in viral load. however, it has been shown that the arkdpi vaccine has an atypical vaccine coverage and replication pattern when mass applied by spray, and previous data from our laboratory suggests that the percentage of chicks infected with vaccine virus by 10 days post-vaccination only reaches 15-25% [11, 12] . multiple replication cycles also occur in the bird (indicated by viral load and clinical signs in chicks), resulting in ''rolling" reactions at different time points post-vaccination [13] . our previous research has shown that to achieve an adequate proportion of infected chicks with arkdpi vaccine and eliminate rolling replication cycles, a 100x dose is required [12] . the atypical vaccine coverage and cycling observed following arkdpi vaccination is a product of the multiple minor genetic subpopulations in the vaccine bottle [14] . it has been previously shown that several serotypes of ibv vaccines contain genetic subpopulations and the subpopulations are often recovered in chickens following vaccination, even though these vaccines show a typical infection and replication cycle and protect from challenge [15] . with arkdpi, the major population in the vaccine contains multiple, distinct amino acid changes in the spike protein that increase binding affinity in the embryonated egg but decrease binding affinity to mature chicken cells [9, 16] . conversely, the minor populations, which have the opposite spike protein binding profile, are more suited to infect and replicate in chickens [16, 17] . however, these minor subpopulations are only a fraction of the total genetic population contained in the vaccine bottle. thus, the proportion of infected chicks is very low and the time to reach peak infection and replication is delayed [12] . for these reasons, chickens do not develop adequate immunity following arkdpi vaccination. although using one of the viral subpopulations with binding affinity for chicken cells directly as a vaccine will induce a protective immune response, these subpopulations cannot be maintained through multiple passages in embryonated chicken eggs, which is required to propagate ibv vaccine. research has been performed to homogenize the arkdpi viral population by adapting it for growth in chicken embryo kidney cells, however this vaccine model has not yet been shown to be commercially feasible [18] . while arkdpi is the only commercially available ark-type ibv vaccine today, it is not the only ark-type ibv vaccine ever produced. the arkansas 99 (ark99) strain was the first ark-type virus to be attenuated for use as a vaccine. when originally mass applied in the field, it caused a severe vaccine reaction in young broilers, and was therefore discontinued when arkdpi was developed [19, 20] . the purpose of this study was to reevaluate the original and additionally attenuated ark99 vaccine by multiple serial passaged in embryonated eggs as a potential ark-type vaccine candidate. in addition, we investigated the mechanisms of attenuation of this vaccine by sequencing the genome and performing snp analysis during the subsequent embryo passages. this study led to development of a new, more attenuated yet still efficacious vaccine strain designated arkansas georgia (arkga). ark99 vaccine is no longer produced nor is usda license maintained by any vaccine manufacturer. an archived reference sample of live ark99 vaccine was obtained from a commercial source and passaged once in 9-to-11 days of incubation specific-pathogen free (spf) chicken embryos as described below. the university of georgia egg-passaged virus, now designated arkga, was then used for further experimentation. different egg passages, beginning at egg passage 1 (p1) and going to p60, were used in this study for consecutive experiments. a pathogenic arkansas serotype challenge virus from our laboratory was also used in this study. spf embryonated chicken eggs were purchased from charles river laboratories (north franklin, ct) and incubated to 9-to-11 days of development for virus passage, titration, and isolation experiments. commercial non-vaccinated broiler chickens were used in the vaccination experiments as described below. arkga was serially passaged 60 times by inoculating 9-to-11day-old spf embryonated chicken eggs in a 0.1 ml volume via the chorioallantoic sac (cas) route [21] . inoculated eggs were incubated at 37°c for 48 h, at which point the embryos were humanely euthanized, and chorioallantoic fluid was collected for subsequent passage into additional 9-to-11-day-old embryos. embryos were candled daily and mortality determined to be from non-viral origin was discarded. viruses were titrated at different egg passage levels using the following protocol: 10-fold serial dilutions of the virus were made in sterile deionized water and each dilution was inoculated into five 10-day-old embryonated spf chicken eggs (0.1 ml/egg). inoculated eggs were incubated at 37°c for 7-days and embryos were examined for ibv-specific lesions. embryo mortality within 24-h post-inoculation was considered nonspecific and not included in virus titer calculations. virus titers were calculated by the method of reed and muench [22] and expressed as the 50% embryo infectious dose (eid 50 ). in addition to titration of embryo passages, vaccine and challenge viruses for the arkga p1 and p60 experiments were also titrated following dilution for inoculation into chickens to confirm the inoculation dose. one hundred one-day-old broiler chicks were vaccinated with the arkga p1 vaccine candidate in a 7 ml spray volume using a commercial vaccine spray cabinet and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in horsfal-bauer isolation units as controls. at 7, 10, 14, 17, 21, 24, and 28 days post-vaccination, all vaccinated chicks were swabbed in the choanal cleft for qrt-pcr analysis of viral load. clinical signs corresponding to ibv vaccine reactions were also recorded on those days [3] . on day 30 post-vaccination, 20 vaccinated and 5 non-vaccinated chickens were challenged with pathogenic arkansas serotype virus in a 0.1 ml eyedrop application, while an additional 5 vaccinated and 5 non-vaccinated chickens were held as non-challenged controls. five days post-challenge, clinical signs were scored and all chickens were swabbed and euthanized for necropsy. tracheas were collected at necropsy for ciliostasis scoring. 2.6. experiment 2. evaluation of infection and replication of arkga p20, p40, and p60 vaccine candidates and protection from challenge 2.6.1. trial 1. arkga p20 the arkga p1 vaccine candidate was further attenuated by 19 additional embryonated egg passages, yielding the arkga p20 vaccine candidate. one hundred one-day-old broiler chicks were vaccinated using a spray cabinet with the arkga p20 vaccine candidate in an 18 ml spray volume and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in isolators as controls. swabs were taken at 3, 5, 7, 10, and 14 days post-vaccination to assess viral load and vaccine coverage in chicks, and clinical signs were recorded. the arkga p20 vaccine candidate was passaged an additional 20 times in embryonated eggs to produce the arkga p40 vaccine candidate and another vaccination trial was conducted as described in trial 1. the arkga p40 vaccine candidate was passaged an additional 20 times in embryonated eggs to further attenuate the virus, producing arkga p60. one hundred one-day-old broiler chicks were spray vaccinated with the arkga p60 vaccine candidate in an 18 ml spray volume and placed in an isolation house on fresh litter. ten additional non-vaccinated chicks were placed in isolation units as controls. at 3, 5, 7, 10, 14, 17, 21, 24, and 28 days postvaccination, all vaccinated chicks were swabbed in the choanal cleft for qrt-pcr analysis of viral load as previously described. clinical signs corresponding to vaccine reactions were also recorded on those days. on day 30 post-vaccination, 20 vaccinated and 5 non-vaccinated chickens were challenged with pathogenic ark-type ibv in a 0.1 ml eyedrop application, while an additional 5 vaccinated and 5 non-vaccinated chickens were held nonchallenged as controls. five days post-challenge, clinical signs were recorded, and all chickens were swabbed in the choanal cleft palate and euthanized for necropsy. tracheas were collected at necropsy for ciliostasis scoring. viral rna was extracted from 50 ll of choanal swab fluid using the magmax-96 rna isolation kit (ambion inc., austin tx) on a kingfisher flex magnetic particle processor (thermo scientific, waltham, ma) per the manufacturer's protocol. quantitative real-time rt-pcr (qrt-pcr) was conducted using an applied biosystems 7500 fast real-time pcr system (life technologies, carlsbad, ca) and the agpath-id tm one-step rt-pcr kit (ambion inc.) per the manufacturer's recommendations. primers and probe for the qrt-pcr were previously published [23] and consist of a forward primer ibv5 0 gu391 (5 0 -gct ttt gag cct agc gtt-3 0 ), a reverse primer ibv5 0 gl533 (5 0 -gcc atg ttg tca ctg tct att g-3 0 ) and a taqman ò dual-labeled probe ibv5 0 g probe (5 0 -fam-cac cac cag aac ctg tca cct c-bhq1-3 0 ). cycle-threshold (c t ) values above the limit of detection for each run (determined by a standard curve) were considered negative [24] . all positive samples were used to determine the total percent positive for each group. post-challenge viral load data was presented as relative viral load values made between the groups within an experiment and were not absolute virus genome copy numbers. clinical signs were scored based on a method described by jackwood et al. [25] . scoring was conducted on a scale from 0 to 3, where 0 = negative, 1 = mild signs, 2 = watery eyes and some mucus in the nares, and 3 = watery eyes, mucus in the nares, and tracheal rales. ciliostasis scoring was conducted by examining five rings approximately 1 mm thick cut from each chicken trachea representing the proximal, middle and distal portion. cilia activity was observed with an inverted microscope (olympus, center valley, pa). scoring was conducted based on the method by cook et al. wherein: 0, all cilia beating; 1, 75% of cilia beating; 2, 50% of cilia beating; 3, 25% of cilia beating; 4, no cilia beating. each ring was scored by 3 individuals independently. mean scores per group were calculated and relationships between groups were analyzed statistically [26] . routine virus isolation techniques were used for detection of ibv challenge virus in 9-to-11 days of incubation embryonated spf chicken eggs. briefly, 2 ml of ice-cold pbs were added to the choanal swab fluid to match the stipulations of the u.s. code of federal regulations, title ix (9-cfr) [27] . pbs from the swabs was filter sterilized and 0.2 ml of each sample were inoculated into the cas of 6 embryonated chicken eggs. eggs were candled daily (24-72 h deaths were discarded) for 7 days and the number of deaths and embryo lesions consistent with ibv infection was recorded. complete genome sequencing was performed on arkga p1, p20, p40, and p60 to detect changes occurring within the viral genome during attenuation. virus stock was filtered with a 0.2 lm syringe filter. viral rna was extracted from samples using the direct-zol rna miniprep kit (zymo research) and treated with dnase i (new england biolabs). the sispa method was used for random amplification of rna as previously described [28] . complementary dna (cdna) was synthesized using superscript iv (invitrogen/ thermo scientific). double stranded cdna (dsdna) was generated from cdna templates using second strand cdna synthesis kit (applied biological materials inc.). complete genome sequencing at a 50â depth of coverage was conducted using the nextera xt dna sample preparation kit (illumina) and miseq sequencer (illumina) according to manufacturer's instructions. de novo and directed assembly of genome sequences was carried out using the mira3 sequence assembler and geneious r8 program (www.geneious.com). non-synonymous substitutions in the assembled sequence reads were compared to consensus sequence at 5% of minimum variant frequency using geneious r8 program. whole genome consensus sequences were entered into the gen-bank database, with accession numbers as follows: ark99 pathogenic field virus mh779860, arkga p1 mh779856, arkga p20 mh779857, arkga p40 mh779858, arkga p60 mh779859. for arkga p1, p20, p40, and p60 vaccination trials, viral rna from 5 choanal cleft palate swabs each from days 7, 10, and 14 post-vaccination was purified and amplified for sequencing of the s1 region of the genome. briefly, viral rna was purified using the zymo direct-zol rna miniprep kit (zymo research, irvine ca). s1 gene sequences were amplified by rt-pcr using the titan one-step rt-pcr system (roche diagnostics, indianapolis, in) and previously published primers: news1oligo5 0 [29] and degenerate3 0 [30] . rt-pcr reactions were analyzed on a 1% w/v agarose gel and bands of the correct size were excised and dna was purified from the gel fragment using the genejet gel extraction kit (thermo scientific, waltham, ma). sanger sequencing was performed by the georgia genomics facility, university of georgia, athens, ga. the s1 sequences were assembled and compared using the dnastar suite of programs (dnastar, madison wi). the s1 amino acid sequences of viral rna isolated from vaccinated chickens was compared to the consensus s1 sequences of the vaccine virus for p1, p20, p40, and p60 obtained from whole genome sequencing to detect changes in the viral population after replication in chicken tissues. statistical analysis was performed using prism v.6.0. (graphpad software, inc., la jolla, ca). for experiments 1 and 2, postvaccination viral load determined by qrt-pcr was compared between all vaccinated chickens within each collection time point via analysis of the mean and standard error of the mean (sem). post-challenge clinical signs, ciliostasis, and viral load between challenge groups were analyzed using an alpha of 0.05 with ordinary one-way anova and tukey's multiple comparisons test. 3.1.1. arkga p1 whole genome sequence analysis and comparison with ark99 pathogenic field virus the reference sequence used for comparison of the arkga passages was the full genome of the ark99 pathogenic field virus (unpublished sequence). in the arkga p1 vaccine consensus sequence, mutations occurred in polyprotein 1a, the spike gene, and in a non-coding region (fig. 1) . only the mutation occurring in the spike gene resulted in an amino acid change (table 1) . arkga p1 was titrated in embryos prior to vaccination and was determined to have an eid 50 of 1x10 6 /ml. appropriate dilutions were performed to achieve the desired vaccination dose of 1x10 3.5 eid 50 per bird, and this was confirmed by back-titration in embryonating eggs. fig. 2 shows viral load and vaccine infection rate (coverage) post-vaccination with arkga p1. viral load in chickens was high by day 7 post-vaccination, and remained constant until 14-days post-vaccination, when it began to decrease ( fig. 2a) . arkga p1 vaccine candidate coverage was 100% by day 7 post-vaccination and remained constant throughout the course of the experiment (fig. 2b) . clinical signs were also recorded at these time points and tracheal rales were observed in 60% of the chicks vaccinated with arkga p1 vaccine candidate at 10 days post-vaccination, which is consistent with previous reports for ark99. titration of pathogenic ark type challenge virus showed an eid 50 of 1x10 7 /ml. challenge virus was diluted to a dose of 1x10 3.5 per bird prior to inoculation and confirmed by backtitration in embryos. data collected at five days post-challenge is shown in fig. 3 . all groups showed significantly less clinical signs than the non-vaccinated and challenged group (fig. 3a) , and all groups had significantly reduced ciliostasis scores compared to the non-vaccinated and challenged group (fig. 3b) , as expected. relative viral load was also significantly reduced in all groups when compared to the non-vaccinated and challenged group (fig. 3c ). plotting the ct values from individual samples taken from each group shows that 4/20 chickens in the arkga p1 vaccinated and challenged group were positive by qrt-pcr (fig. 3d ). virus isolation was not performed for this trial. 3.1.3. analysis of the arkga p1 s1 sequence isolated from vaccinated birds table 2 shows important amino acid positions that were noted to change in the s1 gene region during the arkga passages. in the arkga p1 s1 sequence, there was no difference in viral sequence between the vaccine and the virus isolated from choanal cleft palate swabs of vaccinated chickens. 3.2.1. arkga p20 whole genome sequence analysis and comparison with previous arkga passages between p1 and p20, all 3 mutations that occurred between the ark99 pathogenic field virus and arkga p1 were lost, and a new mutation was gained in a non-coding region of the p20 consensus sequence (table 1 , fig. 1 ). arkga p20 was titrated and shown to have an eid 50 of 1 â 10 6.5 / ml. as seen in arkga p1, for chicks vaccinated with arkga p20, viral load and vaccine coverage were typically high early postvaccination (fig. 4) . however, thirty percent of chickens vaccinated with arkga p20 showed severe clinical signs (rales) on day 10 post-vaccination, which is reduced from the previous trial but still much higher than what would be accepted by the commercial poultry industry. because of the excessive clinical signs seen post-vaccination, the arkga p20 vaccination trial was ended prior to challenge, and back-titration of the diluted vaccine was not performed. non-coding c ? t none analysis of the arkga p20 s1 sequence isolated from vaccinated birds sanger sequencing of the s1 subunit of the spike gene in postvaccination swabs showed two amino acid positions that changed between the vaccine and the swab viral material (table 2) . at amino acid position 198 where there was a lysine present in the vaccine, 20% of the swabs showed a threonine. at position 200, the vaccine had a glycine, but 7% of the swabs sequenced showed a change to aspartic acid. 3.3.1. arkga p40 whole genome sequence analysis and comparison with previous arkga passages in arkga p40, the consensus whole genome sequence showed 4 mutations compared to the reference ark99 field virus sequence. a change in the nonstructural protein 2 (nsp2) region of polyprotein 1a resulted in an amino acid substitution. a substitution also occurred in the s2 region of the spike gene. two other snps were noted in non-coding regions; one of which was at nucleotide position 24,512 that was maintained from p20 (table 1, fig. 1 ). prior to vaccination, arkga p40 was titrated and determined to have an eid 50 of 1 â 10 8 /ml. in chicks vaccinated with arkga p40, viral load and vaccine coverage were lower at days 3 and 5 post-vaccination than in the p20 trial but peaked by day 7 postvaccination (fig. 5) . clinical signs were reduced to 10% of chicks with tracheal rales at 10 days post-vaccination, which was less than with p20, but was still considered too pathogenic for a commercial poultry vaccine. for this reason, the arkga p40 vaccination trial was terminated at this point, and back-titration of the diluted vaccine was not performed. in arkga p40, the s1 sequence comparison between the vaccine virus and viral rna from swabs of vaccinated birds showed changes in the same two amino acid positions, 198 and 200, that were seen in p20. in p40 the frequency of change was increased to 67% in position 198 and 60% in position 200 (table 2) . 3.4.1. arkga p60 whole genome sequence analysis and comparison with previous arkga passages numerous snps were seen in the arkga p60 consensus sequence compared to the ark99 pathogenic field virus. five snps occurred in polyprotein 1ab, 4 of which resulted in amino acid substitutions. the nsp2 snp from p40 was maintained, and two polyprotein 1ab snps occurred in the nonstructural protein 3 (nsp3) region. in p60, snps were seen in s1 as well as s2 of the table 2 s1 amino acid sequence comparison of arkga vaccine virus and viral rna isolated from 5 choanal cleft palate swabs on days 7, 10, and 14 post-vaccination. included are mutations recorded for amino acid positions in the s1 sequence that were maintained to arkga p60. arkga passage s1 amino acid position vaccine amino acid reisolated vaccine amino acid and frequency of change spike gene, although the s2 snp seen in p40 was not detected. of note, a snp was detected at nucleotide position 23,809 in s2 that generated a stop codon, resulting in truncation of s2 by 8 amino acids. additional snps were detected towards the end of the genome, including in the membrane, 5b, and nucleocapsid proteins ( table 1 , fig. 1 ). arkga p60 was diluted from a determined eid 50 of 1 â 10 7 /ml to a vaccination dose of 1 â 10 3.1 eid 50 per bird, as shown by back-titration in embryos. arkga p60 viral load and vaccine coverage are shown in fig. 6 . viral load in chicks was high soon after vaccination, though coverage was lower than expected on days 3 and 5 post-vaccination. by 7 days post-vaccination, coverage had reach 93% and peaked at 100% on day 14 post-vaccination. by 21 days post-vaccination, chickens began to clear the vaccine virus, indicated by reduced viral load and coverage (fig. 6) . only 3% of chicks vaccinated with arkga p60 showed clinical signs (snicks), which was deemed acceptable for an ibv vaccine. back-titration of diluted ark type challenge virus indicated a dose of 1 â 10 3.4 eid 50 per bird. data from five-days postchallenge with pathogenic ark virus are presented in fig. 7 . all groups showed significantly reduced clinical signs (fig. 7a) , ciliostasis scores (fig. 7b ) and viral loads (fig. 7c ) compared to the non-vaccinated/challenged group. when analyzing the individual viral load values, 5/20 of the vaccinated and challenged birds were positive by qrt-pcr. it should also be noted that the arkga p60 vaccinated/nonchallenged group had 2/5 chickens positive for virus (fig. 7d) . virus isolation post challenge was consistent with the results found by qrt-pcr ( table 3 ). all of the non-vaccinated and nonchallenged group swabs were negative for virus isolation. in the vaccinated/non-challenged group, one of the swabs was found to be positive with an embryo death at 120-h post-inoculation. all 5 of the other embryos in this set died by 72-h post-inoculation however, indicating a possible bacterial contamination in that sample. in the vaccinated/challenged group, 3/19 of the swabs were found to be positive for ark-type challenge virus. all embryos in the 20th swab sample died at 48 h post-inoculation, so that sample could not be analyzed. all 5 of the non-vaccinated/challenged bird swabs were positive for ibv. to ensure that virus isolation positives in challenged groups were indeed challenge virus and not residual vaccine, the spike gene of samples from both challenged groups was sequenced. in all instances, sequence matched the arkansas challenge virus, indicating it was not residual vaccine. in the vaccinated/nonchallenged group, no sequence could be obtained from qrt-pcr positive samples. when comparing the s1 amino acid sequence between the arkga p60 vaccine and the virus re-isolated from vaccinated birds, multiple changes were seen. the two amino acid changes at positions 198 and 200 seen in p20 and p40 were 100% predominant in re-isolated virus in p60. in addition, at amino acid positions 117 and 385, the p60 vaccine virus population had two substitutions occur compared to prior vaccine passages. the amino acids in virus isolated from swab material showed a reversion to previous vaccine sequences at these positions ( table 2) . in this study, serial passage of the arkga vaccine virus in embryonated eggs resulted in numerous changes to the viral genome. over 60 passages, the arkga virus accumulated 15 snps, 10 of which were located in the replicase and spike genes, which have been attributed to ibv attenuation [31] . most notably, arkga p60 had snps in the nsp2, nsp3, and both s1 and s2 gene regions. changes in nsp2 and nsp3, which are part of the viral replicase complex, have been shown to impact viral replication and pathogenesis [32] [33] [34] [35] [36] [37] [38] . in the spike gene, which is the major determinant of cell tropism and plays a role in viral attenuation [39, 40] , 5 amino acid changes occurred in the s1 and s2 subunits. one mutation in s2 resulted in truncation of spike by 8 amino acids, likely shortening the cytoplasmic domain. changes in the cytoplasmic tail of ibv and murine hepatitis virus (mhv) have been shown to affect endocytosis signaling and regulation of the levels of spike at the surface of infected cells and may reduce infectivity [41, 42] . in severe acute respiratory syndrome coronavirus (sars-cov), a truncation of 8 amino acids from the carboxyl terminus of s resulted in reductions in cell fusion and cell surface expression and faster endocytosis compared to wild type [43] . as this truncation was only seen in arkga p60 and not in previous passages, further investigation is needed to fully understand the ramifications of this truncation. additionally, a mutation was detected in the nucleocapsid gene, which may be related to reduced replication efficiency [36] . the genomic changes seen in arkga over serial embryo passage can be correlated to the changes in performance dynamics seen in vaccinated chickens. during passage of arkga, the virus became more attenuated, showing reduced reactivity in vaccinated broiler chickens. different passages of the arkga vaccine were evaluated for infection, replication, vaccine reaction, and efficacy in broiler chicks. experimental vaccine and challenge trials showed that the arkga p1 vaccine had suitable infection and replication, and induced adequate protection from challenge, but was too pathogenic, causing a severe vaccine reaction in the majority of chicks. further passages in embryonated eggs reduced the severity of the vaccine reaction to 30% for p20, 10% for p40, and 3% for p60. this further attenuation did not adversely affect infection or replication characteristics of the vaccine, as the relative viral load in chicks did not change throughout the trials. arkga p60 vaccine coverage was slightly less than expected shortly after vaccination but reached 100% by day 14. this may be attributed to the s1 amino acid changes in position 117 and 385 seen between the vaccine and swab sequences in p60, as the s1 sequences re-isolated from chickens had reverted to the more fig. 7 . experiment 2. trial 3. arkga p60 vaccinated and non-vaccinated clinical signs, ciliostasis scores, and viral loads in chickens post-challenge. clinical sign scores were calculated based on severity where 0 = negative, 1 = mild signs, 2 = watery eyes and some mucus in the nares, and 3 = watery eyes, mucus in the nares and trachea (tracheal rales). ct = cycle threshold. clinical sign scores, ciliostasis scores, and relative viral load were compared between challenge groups using ordinary one-way anova (a = 0.05) with tukey's multiple comparisons test. the -/-, arkga/-, and -/ark groups each contained 5 chickens. the arkga/ark group contained 20 chickens. pathogenic p1 sequence. in the arkga p1 vaccine trial a higher vaccine coverage was seen, indicating that these amino acid positions may have an impact on rate of infection. it is also possible that the changes in the replicase gene and the s2 mutations impacted the performance of arkga p60. infection and replication patterns in all trials were predictable and ''typical" of what would be expected of an ibv vaccine. this stands in contrast to the infection and replication cycles of the arkdpi vaccine, which shows very low vaccine coverage and multiple replication cycles during the life of the bird following spray vaccination [13] . one explanation for this is in the s1 portion of the spike gene. the arkga p1 spike sequence contains a histidine at position 43, which has been previously shown to significantly increase spike protein binding to chicken tracheal tissues, and an asparagine deletion at position 344, which has been shown to influence the ability of antibodies to recognize the protein. conversely, the arkdpi s1 contains a tyrosine at position 43 and an asparagine at position 344, which may be attributed to its reduced ability to bind and replicate in chicken tissues [16] . the presence of a minor virus subpopulation capable of inducing a protective immune response in arkdpi results in a very low dose of protective vaccine virus when mass vaccinating chickens and consequently poor protection [12] . although arkga was found to have subpopulations, as evidenced by the snps observed within each passage, which is quite typical for ibv vaccines [15] , those subpopulations were able to produce a protective immune response when mass applied. the arkga vaccine at p1 and p60 was effective at protecting chickens from a pathogenic ark ibv challenge. clinical signs and viral loads post-challenge were significantly lower than nonvaccinated and challenged groups, and all vaccinated birds passed the ciliostasis test. again, this stands in contrast to previous arkdpi vaccine and challenge experiments that showed that chickens were clearly not protected from challenge after arkdpi vaccination by spray [12] . the attenuated arkga vaccine described herein is a significant improvement over the current commercially available arkdpi vaccine when comparing infection and replication following spray application and induction of protective immunity following homologous challenge. the arkga (p60) is also genetically distinct, making it possible to distinguish the arkga vaccine from the arkdpi vaccine or pathogenic viruses. further molecular investigation is needed to fully evaluate the amino acid changes seen in pass 60, but these changes do not seem to impact the effectiveness of the vaccine. in conclusion, the arkga vaccine developed herein is safe when given to 1-day old broilers by spray, and it induces an efficacious immune response against homologous challenge. the authors declare that there were no conflicts of interest. all experiments in this research were conducted in accordance with animal care and use protocols approved by the university of georgia iacuc committee. grace a. albanese participated in the research and article preparation. dong-hun lee performed the complete genome sequencing and de novo and directed assembly of genome sequences and coronavirus avian infectious bronchitis virus vaccination against infectious bronchitis virus: a continuous challenge. vet micro modification of the avian coronavirus infectious bronchitis virus for vaccine development the early history of infectious bronchitis development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review diseases of poultry data from 11 years of molecular typing infectious bronchitis virus field isolates comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different arkansas delmarva poultry industry-derived infectious bronchitis virus vaccines epidemiological and experimental evidence for immunodeficiency affecting avian infectious bronchitis infectious bronchitis virus field vaccination coverage and persistence of arkansas-type viruses in commercial broilers minimum infectious dose determination of the arkdpi infectious bronchitis virus vaccine delivered by hatchery spray cabinet evaluation of infectious bronchitis virus arkansas-type vaccine failure in commercial broilers different evolutionary trajectories of vaccinecontrolled and non-controlled avian infectious bronchitis viruses in commercial poultry avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination polymorphisms in the s1 spike glycoprotein of arkansas-type infectious bronchitis virus (ibv) show differential binding to host tissues and altered antigenicity rapid selection in chickens of subpopulations within arkdpi-derived infectious bronchitis virus vaccines kidney celladapted infectious bronchitis arkdpi vaccine is stable and protective report: arkansas 99, a new infectious bronchitis serotype a new serotype of infectious bronchitis virus responsible for respiratory disease in arkansas broiler flocks a laboratory manual for the isolation, identification and characterization of avian pathogens a simple method of estimating fifty per cent endpoints development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-pcr and correlation with virus detection in embryonated eggs avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus title 9, code of federal regulations, standard requirements for ibv vaccines use of sequence-independent, single-primer-amplification (sispa) for rapid detection, identification, and characterization of avian rna viruses further development and use of a molecular serotype identification test for infectious bronchitis virus redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de072 strain of infectious bronchitis virus identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi purification, crystallization and preliminary x-ray analysis of nonstructural protein 2 (nsp2) from avian infectious bronchitis virus the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3 genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication genomic characteristics and changes of avian infectious bronchitis virus strain ck/ch/ldl/97i after serial passages in chicken embryos changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism sequence changes of infectious bronchitis virus isolates in the 3 0 7.3 kb of the genome after attenuating passage in embryonated eggs contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion revised the article. i-hsin n. cheng and deborah a. hilt participated in the research. mark w. jackwood and brian j. jordan participated in the study design and in the research and article revision. all authors have approved the final article. this research was supported by the university of georgia research funding and by sponsorship from merck animal health. key: cord-290638-7ro72sv3 authors: lenstra, johannes a.; kusters, johannes g.; koch, guus; van der zeijst, bernard a.m. title: antigenicity of the peplomer protein of infectious bronchitis virus date: 1989-01-31 journal: molecular immunology doi: 10.1016/0161-5890(89)90014-x sha: doc_id: 290638 cord_uid: 7ro72sv3 abstract to study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited dnase digestion and inserted in the escherichia coli expression plasrnid pex (stanley and luzio, 1984). the antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. the polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. the n-terminal region of one of the two subunits, s2, was recognized by all polyclonal sera and by two monoclonal antibodies. this clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. the epitopes found as antigenic pex expression products do not coincide with the regions in the s1 subunit that have been found to contain hypervariable sequences. we suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pex system. the relevance of our finclings for vaccine development is discussed. coronaviruses act as causative agents of a number of infectious diseases in domesticated animals (for a review about these positive stranded rna viruses, see siddell et al., 1983; sturman and holmes, 1983) . conventional vaccines do not exist or are rendered ineffective by antigenic variation. this latter mechanism is thought to be relevant for the epidemiology of infectious bronchitis virus (ibv), which causes a highly contagious respiratory disease in chickens. after recent outbreaks of the disease, new serotypes have been isolated that may have originated from the live attenuated strains used for vaccination (kusters et al., 1987) . obviously, recombinant-dna expression products or peptide vaccines would offer a valuable alternative for conventional vaccines. the antigenic properties of ibv or other coronaviruses most likely reside in the peplomer (cavanagh et al., 1984) , a club-shaped spike projecting from the viral membrane and consisting of a dimer or trimer of the e2 peplomer protein (cavanagh, 1983) . analysis of the amino acid sequences of the peplomer proteins from representatives of the three main coronavirus serotypes (de groot et a[., 1987) has led to a model, in which two §author to whom correspondence should be addressed. abbreviations: elisa, enzyme-linked immunosorbent assay; ibv, infectious bronchitis virus; mab, monoclonal antibody; pbs, phosphate-buffered saline. long cc-helices are located in the c-terminal half of the monomer subunit. in the oligomer, those helices intercalate and form a coiled-coil structure, which constitutes the stalk of the peplomer. the transmembrane segment at the c-terminal end serves as membrane anchor. the n-terminal half is thought to form the spherical outer part of the peplomer. in ibv, proteolytic cleavage results in an n-and a c-terminal subunit, designated sl and s2, respectively. sl is thought to be the most relevant part for the antigenic properties of ibv (cavanagh et al., 1986) . it is recognized by strongly neutralizing monoclonal antibodies (mockett et al., 1984; koch et al., unpublished) . furthermore, isolated sl is capable of eliciting neutralizing antisera (cavanagh et al., 1986) . by comparing the amino acid sequences of different strains, it was found that most amino acid substitutions, likely to reflect antigenic variation, have occurred in the sl subunit (niesters et ul., 1986; binns et al., 1986; kusters et al., unpubljshed) . in this study, we have localized epitopes by expressing parts of the peplomer sequence in a prokaryotic expression system, and measuring the antigenicity of the expression products (stanley and luzio, 1984) . most probably, this method detects epitopes that are essentially conformation independent. antigenic regions were almost exclusively found in the s2 subunit, suggesting that the antigenic variation in sl reflects nonformation dependent epitopes. one immunodominant region has been localized near the n-terminus of s2. this region was recognized by all polyclonal antisera tested as well as by two neutralizing monoclonal antibodies, and contains at least two different epitopes. the sequence of this region may be the starting point for the development of peptide vaccines. schuell). the hinfl ends of hl were made blunt by treatment with the klenow fragment of dna polymerase. depending on the desired reading frame, the fragments were ligated in compatible sites of pex i, pex2 or pex3 (stanley and luzio, 1984) . which had been treated with calf intestine phosphatase if linearized by a single digestion. the cdna clones p39 and p42 of the peplomer gene of ibv strain m41 in the vector puc9 have been described previously (niesters et al., 1986) . rabbit antisera against m41 were raised by intramuscular injection of purified virus (1 mg protein), lysed by triton x-100, in freund's complete adjuvant, followed by the same dose in freund's incomplete adjuvant after two and three weeks, respectively. mouse antiserum was raised by intraperitoneal injection of 100 pg virus protein, followed by a second dose after 1 month. chicken antisera were collected from convalescent (chickens 1 and 3) or hyperimmune (chickens 2 and 4) animals, infected with ibv strain m41 (chickens 1 and 2) or strain d207 (chickens 3 and 4). recombinant plasmids were introduced into e. coli strain pop 2136 via the caclz transformation procedure. recombinants expressing the ibv gene fragments were selected by immunoscreening with a rabbit antiserum or, when the expression product was not recognized antigenically, by hybridization followed by gel electrophoresis of the protein expression products. as hybridization probe, a mixture of hindiiiihinfi fragments of the insertion from plasmid p39 was used, which could be isolated free from the 2.7-kb puc fragment by gel electrophoresis. the preparation of the monoclonal antibodies 26.1 and 31.5 against ibv has been described previously (koch et al., 1986) . alkaline phosphatase-conjugated goat anti-(mouse igg, h + l) and goat anti-(rabbit igg, fc) were from promega (madison); anti-(chicken igg, h + l) conjugates were from nordic, tilburg, the netherlands (horseradish peroxidase) or from miles (alkaline phosphatase). as pex host strain, e. coli pop 2136 was used, which was constructed by dr raibaud (institut pasteur, paris). unless mentioned otherwise, all dna manipulations used were done essentially as described by maniatis et al. (1982) or davis et al. (1986) . genomic cdna clones p39 and p42 were cut with restriction enzymes as indicated in fig. 1 tris-hc1 (ph 7.5) 1.5 mm mncl, and 0.1 mg/ml bsa at 22 c for 6 min (first 5 /tl of the reaction mixture) or 10 min (remaining 5 ~1). as judged by gel electrophoresis and autoradiography after end-labelling with the klenow fragment of dna polymerase, fragments of 2&500 bp were generated. these fragments were made blunt-ended by treatment with t4 dna polymerase, followed by treatment with the klenow fragment. after sequential extractions with phenol/chloroform and ether, the fragments were ligated directly in pex2 dna, which had been cut with smai and dephosphorylated with calf intestine phosphatase. the reaction was carried out overnight this rapid cloning procedure obviates the use of linkers (haymerle ez al., 1986 ) but generates nevertheless a sufficient number of clones from a low amount of insert dna. immunoscreening of bacterial colonies transferred to nitrocellulose membranes (ba85 from schleicher and schuell) was performed as described (stanley, 1983) , using a gelatin/triton x-1oojpbs buffer and an e. coli lysate to quench aspecific reactions. the dilutions of the primary antisera were 1: 1000 for polyclonal sera and 1: 100 or 1:500 for ascites fluids containing monoclonal antibodies. binding of antisera to colonies was visualized using alkaline phosphatase-conjugated secondary antisera (diluted 1:5000) and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates (blake et al., 1984) . to localize dnase fragments from pex clones that were positive after immunoscreening or to check the correct reading frames for the expression of restriction fragments, dna from pex/ibv recombinants was isolated by the alkaline-lysis method, cut with two restriction enzymes flanking the insertion and with hind111 to cleave the pex vector sequence. the resulting mixture of fragments was ligated in m13mp9 dna cut with the appropriate enzymes. subsequently, the nucleotide sequence of the insertion was determined by the dideoxynucleotide termination method and compared to the sequence of the ibv m41 peplomer gene. expression of the pex fusion gene, leading to the synthesis of a hybrid b-galactosidase protein, was induced in a 5 ml exponential culture (odmm ca. 0.25) by incubation at 42°c for 90 min. hybrid proteins were purified as triton x-100 pellet (stanley and luzio, 1984) . cells were spun down (10 min at 5ooog), resuspended in 100 ~1 15% (w/v) sucrose, 50mm tris-hci (ph 8.0), 50mm edta, and treated with lysozyme (1 mg/ml) for 10 min. after addition of 140 ~10.2% (v/v) triton x-100 in 10 mm tris-hcl (ph 8.0) 1 mm edta, the suspension was sonicated in a bath sonicator for 15 min. the lysozyme digestion as well as use of the sucrose buffer appeared essential for the purity of the final preparation. after spinning down the insoluble hybrid protein (10 min at 10,ooog) and removing the viscous supernatant as completely as possible, the pellet was resuspended in triton x-100 buffer. sonification, centrifugation and removal of the supernatant were repeated (l-2 times) until no viscosity remained. finally, the pellet was resuspended in 250~1 pbs. after adding concentrated sds-containing lysis buffer (laemmli and favre, 1973) , hybrid proteins were spotted via a manifold on nitrocellulose membranes or fractionated by sdspolyacrylamide gel electrophoresis (7.5%) followed by western blotting. hybrid proteins thus immobilized were analysed as described for the filters of the immunoscreening (see above). binding of chicken antisera was visualized using peroxidase-conjugated secondary antisera (diluted 1: 1000) and 3'-3' diaminobenzidine and h,oz as substrates. in the pex expression system, foreign gene fragments are inserted at the 3' end of a cro-iacz expression vector (stanley and luzio, 1984) . expression of this gene is under control of the lambda p, promotor and induced by inactivation of the temperature sensitive cl repressor at 42°c. the resulting expression products precipitate inside the cell and are thus protected against proteolysis. after solubilization, the product of the foreign gene fragment, linked to the c-terminal residue of the /?-galactosidase part, may be recognized by appropriate antisera. figure 1 shows the hinfl, pst i, sau3ai and sau3ai/psti fragments that have been excised from the genomic cdna plasmids p39 or p42 (niesters et al., 1984) and expressed in pex plasmids. the hinfl site in the region encoding the n-terminal signal sequence of the peplomer protein allowed the cloning of the 5' part of the coding sequence without the stop codons just upstream of (and in frame with) the atg start codon. as judged from an sds-polyacrylamide gel [ fig. 2(a) ], the hybrid proteins have sizes predicted by the lengths of the insertions. exceptions were ps4, which contains the stop codon of the peplomer gene, and the 144 bp fragment p4, which was inserted twice, both fragments expressed in tandem and in the same reading frame. analysis of the sequence showed that only in the case of hl and p4, hybrid proteins of (almost) the same size could be obtained after translation in a wrong reading frame; the proper reading frames of these clones have been checked by sequencing. fragment pl starts with a stretch of 12 dg residues (originating from ~42); apparently, this did not disturb the reading frame by ribosome slippage along the homopolymer track (stanley, 1983) . the ibv peplomer sequences contained in the hybrid proteins are listed in table 1 . only residue 1064 has not been overlapped by an expression product. further, a reactive region exactly on the junction of two fragments or in the gap between p4 and p5 can only be detected in an overlapping fragment if this fragment does not contain other reactive regions. in the theoretically worst case, the reactivity of 8% of the total peplomer sequence would not be detected, assuming an epitope size of 10 residues. in fig. 2(b) , a western blot of the ibv/pex hybrid proteins, incubated with a rabbit antiserum, is shown. only three regions were recognized strongly, one contained in p2, ~4, psl and ps2, the second in p3 and ~4, and the third in ps4. weaker signals were observed for the regions contained in p4, in p5 while not in ps3, and in the overlap of s5 and p6, respectively. have been carried out with another rabbit antiserum, with a mouse antiserum and with four antisera from chickens, infected with ibv strain m41 (chickens 1 and 2) or with strain d207 (chickens 3 and 4) . the binding patterns are summarized in fig. 3 . because of high background signals of the chicken antisera, only strong signals cquld be scored. apparently, each antiserum recognized a different set of epitopes. the number of the positions of the restriction-enzyme fragments of the peplomer gene are shown in fig. i . the fragments were ligated to linearized pex 1, -2 or -3 as indicated. the last column indicates which parts of the amino acid sequence of the peplomer protein are synthesized by the recombinants after induction of the expression. antigenic regions as defined by the restriction fragments varies from 1 (mouse) to 6 (rabbit 1). one region, corresponding to fragment ps2, was recognized by all homologous and heterologous antisera. (koch et al., unpublished results) . none of these sera bound any of the pex products, most likely because the epitopes of these antisera are conformation dependent. however, the lack of binding of other, non-neutralizing monoclonal antibodies recognizing conformation independent epitopes on the peplomer protein suggests that other factors than the conformation dependence may affect the reactivity of monoclonal antibodies with pex hybrid proteins. koch et al. (1986) described a number of monoclonal antisera with weak neutralizing activity that bound to the s2 subunit on western blots. one of these, mab 26.1, recognized all ibv strains tested and has a neutralization titre of about 10'. figure 2 (c) shows a western blot of hybrid proteins screened with this mab. the strong binding to fragments p2, s4, psl and ps2 localizes the epitope of mab 26.1 between residues 390 and 612, the same region that is recognized by all polyclonal sera. since subunit s2 starts at residue 538, the epitope must lie in the n-terminal region (residues 538 to 612) of s2. the same localization has been found for the epitopes of another s2-specific mab 31.5. to further localize the epitope of mab 26.1, a library of small random dnase fragments in pex has been constructed. after immunoscreening, 0.2% of the colonies were found to react positively with mab 26.1. six clones were selected for sequence analysis after subcloning in ml3 replicative-form dna. the results are summarized in fig. 4 . all clones contain nucleotides 1620-1673, restricting the localization of the epitope of mab 26.1 to residues 541-558. figure 5 shows the antigenicity of four expression products of the dnase fragments to mab 26. antisera and fragments are listed in table 2 . the antiserum from chicken 1 binds to the expression products of the same fragments as mab 26.1, indicating the recognition of the same or an adjoining epitope. the results with antiserum of chicken 2 were clouded by a particularly high background reaction, but the clear positive reactions with two of the fragments indicate a binding to the same region. interestingly, the negative reactions of fragments (15841682) and (1617-1673) demonstrate that the epitope(s) of mab 31.5 and of the scra of rabbit i, rabbit 2 and mouse are located partially or completely in the region 562-574. the positions of the dnase defined regions reactive with the polyclonal sera are graphically represented in fig. 3 by short horizontal lines. the delineations of the individual epitopes are indicated in fig. 4 by heavy lines, the dashed line indicating the possibility that the region 541-561 contributes to the epitopes recognized by the mab 31.5 and by the rabbit and mouse antisera. in indirect elisas on western blots with intact viral proteins, mabs 26.1 and 31.5 have different specificities (koch et al., 1986. unpublished results). mab 26.1 recognizes the $2 subunit of all strains tested, while mab 31.5 only binds to s2 of the h120/b222/m41 serotype. the same specificity was found with pex expression products: mab 26.1 binds to the hybrid proteins of strains m41, d207 and d1466; mab 31.5 only to m41 products. an accurate definition of the epitope of mab 26.1 and other epitopes in the same region, based on the sequences of several ibv strains, on pepscan peptide synthesis (geysen et al., 1984) and on competition experiments will be published elsewhere (kusters et al.; koch et al., in preparation) . in the pex system, the expression product of an inserted dna fragment constitutes the c-terminus of a hybrid /l-galactosidase protein, which precipitates in the cell. prior to the assay of its antigenicity, the hybrid protein is solubilized by sds and transferred to a nitrocellulose filter. presumably, epitopes found by cross-reaction with antisera are essentially conformation independent. the localization of epitopes within stretches of 10-20 residues in several coronaviruses (fig. 4 , unpublished results) demonstrates that the binding of antibody does not depend on particular flanking sequences, and that any nativelike folding of the epitope in the hybrid protein is confined to the same small region. classically, continuous epitopes are localized by testing the antigenicity of peptides (atassi, 1984) . prokaryotic expression of subgenomic fragments in pex or in i.gtll (nunberg et ul., 1984; mehra et al., 1986 ) offers a convenient alternative and is not limited to small proteins. we have combined the expression of large (13&1400 bp) restriction fragments to localize antigenic regions, and of small dnase fragments to delineate epitopes more accurately. another strategy is the subcloning of small kusters et al., luytjes et al., unpublished results) . this obviates the sequence analysis of dnase fragments, but depends on the presence of suitable restriction sites or on the availability of homologous sequences. another new and complementary approach to the localization of continuous epitopes is the synthesis of a large number of peptides coupled to a solid phase, the so-called pepscan (geysen et al., 1984) . this method has been used in conjunction with the pex expression system for the localization of a neutralization epitope of the peplomer protein of mouse hepatitis virus (luytjes et al., unpublished results). however, not all monoclonal antibodies that cross-react with pex expression products bind to pepscan peptides spanning the antigenic region (posthumus and meloen, unpublished results). for instance, mab 26.1 did not recognize any of the overlapping nonapeptides spanning the n-terminal region of the s2 subunit. whether this should be explained by the lengths of the peptides or by local conformations in the hybrid proteins will be the subject of further study. the reactivity patterns of the different polycionaf antisera (fig. 3 ) outline the variability of the humoral immune response against the ibv peplomer protein at the molecular level. there are also a number of other noteworthy features. firstly, all antisera recognize in the 1162-residue sequence a relatively small (l-6) number of regions, predominantly in the s2 subunit; the actual number of epitopes per antiserum depends on the degree of clustering of epitopes within the antigenic regions. so, if the whole surface of the proteins is potentially antigenic (berzofsky, 1985; geysen et al., 1987) , this is clearly not the case for the determinants detected as antigenic pex hybrid proteins. secondly, the n-terminal part of the s2 subunit is recognized by all polyclonal antisera tested and by a number of monocional antibodies. within this region, the monoclonal and polyclonal antibodies recognized different epitopes ( table 2 ). the mabs have neutraiization titres ranging from 10 to 1000 (koch et al., 1986 ) suggesting a role of this region in the neutralization of the infection. thirdly, the regions found to be antigenic do not coincide with the hypervariable regions in the sl subunit (niesters et al., 1986; binns et al., 1986) . these regions are supposed to be part of neutralization epitopes that are the targets of antigenie variation. in fact, although the sl subunit is thought to induce the protective immunity against ibv (cavanagh et al., 1986) we found it to be relatively, if not completely, devoid of antigenicity (fig. 3) . as the most obvious explanation, we propose that the neutralization epitopes in st are conformation dependent and cannot be mimicked by pex hybrid proteins. this would also explain the lack of reaction of the neutralizing mabs that were directed against epitopes in si. apparently, these epitopes are of the same category as the conformation dependent neutralization epitopes of the haemagglutinin of influenza virus (wiley et al., 1981) . in contrast, the epitopes localized in the n-terminal region of s2 may be comparable to the epitopes of foot-and-mouth disease virus, detectable by cross-reaction with peptides (geysen et al.. 1984; rossman et al., 1985) . respectively. the rabbit antisera were generously provided by ms n.m.c. bleumink-pluvm and dr h. g. m. niesters (veterinary faculty). the experimental contributions of antigenic structures of proteins intrinsic and extrinsic factors in protein antigenic structures comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6/82 with that of ibv beaudette a rapid, sensitive method for detection of alkaline phosphatase-conjugated antiantibodies on western blots coronavirus ibv: structural characterization of the spike protein by virtue of their continuous epitopes often correspond to relatively inherent cross-reactivity with short peptides, these coronaviruses use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid chemistry of antibody binding to a protein efficient construction of cdna libraries in plasmid expression vectors using an adaptor strategy antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies molecular epidemiology of infectious branchitis virus in the netherlands mokectttar cloning, a laboratory manual. cold spring harbor laboratories efficient mapping of protein antigenic determinants monoclonal antibodies to the sl spike and membrane proteins of avian infectious bronchitis virus strain massachusetts m41 the pcplomer protein sequence of the m41 strain of coronavirus ibv and its comparison with beaudette strains epitopes on the peplomer protein of infectious bronchitis virus strain m4i as defined by monoclonal antibodies method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein ~~70 the biology of coronaviruses solubilization and immune-detection of p-galactosidase hybrid proteins carrying foreign antigenic determinants ~onst~ction of a new family of high efficiency bacterial expression vectors: identification of cdna clones coding for human liver proteins antigenic crossreactivity between proteins and peptides: new insights and applications correlation between segmental mobility and the location of antigenic determinants in proteins frankenberger identification of the antibodv-combining site of hone e mobile regions in the protein structure (westhof et regions are the prime candidates for the development of peptide vaccines (van regenmortel, 1987) . thus, for the n-terminal region of the ibv s2 subunit. structural considerations may outweigh the relatively weak neutralization, if compared with the epitopes in sl (mockett et al., 1984; koch et al., further, m41 sequences cross-react with chicken antisera against d207, belonging to a different serotype, (fig. 3, table 2 ) and vice versa (not shown). apparently, this region is not subject to antigenic variation. this would be another advantage of the epitopes found in 52.thirdly, the recognition of the n-terminal s2 sequence by several polyclonal antisera indicates that this region is strongly immunodominant.this would favour a response in all vaccinated animals.these considerations may become relevant as soon as the technology for designing effective peptide vaccines becomes available. key: cord-293651-96cmduez authors: callison, scott a.; hilt, deborah a.; boynton, tye o.; sample, brenda f.; robison, robert; swayne, david e.; jackwood, mark w. title: development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens date: 2006-08-28 journal: j virol methods doi: 10.1016/j.jviromet.2006.07.018 sha: doc_id: 293651 cord_uid: 96cmduez it is important to rapidly differentiate infectious bronchitis virus (ibv) from disease agents like highly pathogenic avian influenza virus and exotic newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. in this study, we report the development and testing of a real-time rt-pcr assay using a taqman(®)-labeled probe for early and rapid detection of ibv. the assay amplifies a 143-bp product in the 5′-utr of the ibv genome and has a limit of detection and quantification of 100 template copies per reaction. all 15 strains of ibv tested as well as two turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. evaluation of the assay was completed with 1329 tracheal swab samples. a total of 680 samples collected from ibv antibody negative birds were negative for ibv by the real-time rt-pcr assay. we tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for ibv by real-time rt-pcr, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. we also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of ibv and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. in addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for ibv in the field. the percentage of birds infected with the ibv vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. this observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. the real-time rt-pcr test described herein can be used to rapidly distinguish ibv from other respiratory pathogens, which is important for control of this highly infectious virus. the test was extremely sensitive and specific, and can be used to quantitate viral genomic rna in clinical samples. infectious bronchitis (ib) is a highly infectious disease of the upper-respiratory tract in chickens (cavanagh and naqi, 2003) . it can also affect the kidneys and reproductive tract. it is of economic importance to the poultry industry due to the high morbidity and production losses associated with the disease. the etiologic agent of ib is infectious bronchitis virus (ibv), an enveloped, positive-sense, single-stranded rna virus that belongs to the coronaviridae family. the virus contains four structural proteins; nucleocapsid surrounding the viral rna, an integral membrane glycoprotein, a small envelope protein, and a spike glycoprotein located on the surface of the viral envelope, which contains epitopes that induce virus-neutralizing and serotype-specific antibodies. although control of ibv is primarily through the use of live attenuated vaccines, the disease is difficult to control because different serotypes of the virus do not cross-protect. therefore, it is imperative to quickly and accurately detect the presence of the virus within an infected poultry flock so that subsequent flocks can be properly vaccinated. it is also important to rapidly differentiate ibv infections from other upper-respiratory diseases like avian influenza, newcastle disease, infectious laryngotracheitis, and avian mycoplasmosis so that appropriate measures against those diseases can be taken in a timely manner. current diagnostic assays for ibv include virus isolation in embryonating eggs, tracheal organ culture, or cell culture immunoassays, and molecular assays that detect the viral rna (gelb and jackwood, 1998) . virus isolation is considered the reference standard; however, it is expensive and time consuming because several passages may be required to detect the virus. immunoassays use ibv-specific monoclonal antibodies to detect the virus in direct or indirect fluorescent antibody and enzymelinked immunosorbent assay (elisa) formats. although faster and simpler than virus isolation, immunoassays tend to lack specificity and sensitivity and none detect all strains or types of ibv karaca et al., 1992; naqi et al., 1993) . molecular assays for the detection of ibv are commonly used because they provide highly specific and sensitive results in a timely manner. molecular assays use the reverse transcriptase-polymerase chain reaction (rt-pcr) to detect viral rna directly from a clinical sample or from virus isolated in a laboratory host system. when rt-pcr is used to amplify the spike glycoprotein of ibv, it can be coupled with restriction fragment length polymorphism (rflp) or nucleic acid sequencing to identify the type of the virus (cavanagh et al., 1990; jackwood et al., 1997; keeler et al., 1998; kingham et al., 2000; kwon et al., 1993) . the constant threat of globally important diseases like avian influenza and newcastle disease, which must be differentiated from ib, in diagnostic investigations, makes it extremely important to rapidly identify the causative agent of any upperrespiratory disease or changes in egg shell quality and egg production in chickens. in this study, we developed a taqman ®based real-time rt-pcr assay for rapid detection of ibv viral rna directly from clinical samples. we examined the sensitivity and specificity of the test and evaluated it using known negative, known positive, and clinical samples from commercial chickens. virus isolation was performed in specific pathogen-free (spf) 9-11-day-old embryonating chicken eggs as previously described (gelb and jackwood, 1998) . other viral and mycoplasma strains used in this study are listed in table 1 . extraction of rna from clinical samples was performed with the high pure rna isolation kit (roche, indianapolis, in) or the rneasy kit (qiagen, valencia, ca). extractions from allantoic fluid and tracheal swabs taken from experimentally infected birds as well as rna extracted for sensitivity and specificity analysis and for test evaluation was conducted with the magmax 96 total rna isolation kit (ambion, austin, tx) following the manufacturer's suggestions. a sequence alignment ( fig. 1 ) of the 5 -untranslated region (utr) of six strains of ibv (genbank accession nos. ay392051, m95169, ay392049, ay392054, ay392050, ay851295) was used to identify conserved sequences within this region to design primers and a probe for the real-time rt-pcr assay. a forward primer ibv5 gu391 (5 -gct ttt gag cct agc gtt-3 ) located at nucleotide positions 391-408 of the ibv m41 strain genome sequence (genbank accession no. ay851295); a reverse primer ibv5 gl533 (5 -gcc atg ttg tca ctg tct att g-3 ) located at nucleotide positions 533-512 of the ibv m41 strain genome sequence, and a taqman ® dual-labeled probe ibv5 g probe (5 -fam-cac cac cag aac ctg tca cct c-bhq1-3 ) located at nucleotide positions 494-473 of the ibv m41 strain genome sequence were designed to amplify and detect a 143-bp fragment of the 5 -utr gene. the primers were synthesized by integrated dna technologies (coralville, ia), and the probe was synthesized by biosearch technologies (novato, ca). primers and probe were utilized in a 25 l reaction containing 12.5 l of quantitect probe rt-pcr 2× mix (qiagen, valencia, ca), 0.25 l of rt enzyme (qiagen), primers to a final concentration of 0.5 mol, probe to a final concentration of 0.1 mol, 2.75 l of water, and 5 l of rna template. the reaction was conducted in a smartcycler (cepheid, sunnyvale, ca) at 50 • c for 30 min; 95 • c for 15 min with optics off; 40 cycles of 94 • c for 1 s followed by 60 • c for 60 s with optics on. for each reaction, the cycle threshold (c t ) number was determined corresponding to the pcr cycle number at which the fluorescence of the reaction exceeded 30 u of fluorescence, which is the default value for the smartcycler. runoff rna transcripts corresponding to the first 735 nucleotides of the mass 41 ibv genome were generated to use as standards in the assay. transcripts were generated from a plasmid containing the first ∼5500 bp of the mass 41 genome of ibv downstream of a t7 promoter, which was created using the 5 -primer a-beau u30 (5 -tacactagccttgcgc-taga-3 ) and 3 -primer a-beau l5447 (5 -gcacgcca-aagtcccatag-3 ). briefly, rna was extracted from the mass 41 strain of ibv using the high pure rna isolation kit (roche diagnostics corporation). the purified rna was resuspended in diethylpyrocarbonate (depc) treated water and used in the rt-pcr reaction as previously described (jackwood et al., 1997; lee and jackwood, 2001) . the amplified product was cloned into the pcr-xl-topo vector (invitrogen inc., carlsbad, ca) per the manufacturer's directions and sequenced to verify its accuracy. the plasmid was linearized at base pair 735 relative to the start of the genome with xmn i (new england biolabs, beverly, ma). the linearized dna was gel purified and used as template with a ribomax t7 in vitro transcription system (promega, madison, wi) per the manufacturer's recommendations. the length of the runoff rna transcripts was verified by agarose gel analysis, and the concentration was determined using a biophotometer (eppendorf, hamburg, germany). ten-fold serial dilutions of the runoff rna transcripts containing 2 × 10 6 to 2 × 10 1 copies of template per microliter were made and 5 l of each dilution was used as template in the assay. the limit of detection/quantification and reproducibility of the assay were determined by six independent runs. the limit of detection was defined as the lowest rna concentration yielding a c t value where the fluorescence of the reaction exceeded 30 u of fluorescence. the limit of quantification was defined as the lowest rna concentration on the standard curve that maintained linearity. the average number of ibv genome copies for each group of experimental samples was estimated using the equation derived from the standard curve. to test the specificity of the assay, rna or dna from 11 different pathogens known to infect the avian upper-respiratory tract (table 1) was used in the test. we further tested the specificity of the assay using negative samples obtained from 340 commercial layer chickens housed from 1 day of age in positive pressure horsfal isolation units at poultry diagnostic and research center (pdrc, athens, ga). the birds were monitored for serum antibody titers to ibv using the proflock ® ibv elisa kit (synbiotics). a total of 123 serum samples were collected at 3 weeks (62 samples) and 6 weeks (61 samples) of age. sterile polyester tipped applicators (mdci ltd., west sussex, u.k.) were used to take tracheal swabs from each bird at 3 and 6 weeks of age (680 total samples). each swab was placed into a 1.5 ml microcentrifuge tube containing 1 ml of sterile 1× pbs (ph 7.4). the tubes were mixed using a bench-top vortex, and stored at −70 • c until needed for rna extraction. the sensitivity of the assay was determined using clinical tracheal swab samples submitted to delaware (agriculture research laboratory, university of delaware, georgetown de 19947, usa) and maryland (maryland diagnostic laboratory, salisbury, md 21801, usa) laboratories. clinical samples consisting of 229 tracheal swabs from commercial chickens experiencing upper-respiratory disease, submitted as routine diagnostic cases to the delaware (georgetown, de) and maryland (salisbury, md) state diagnostic laboratories were tested by the real-time rt-pcr assay, as well as, by virus isolation at those laboratories (gelb and jackwood, 1998) . the sensitivity was calculated with the formula: se = tp/(tp + fn), where; se = sensitivity, tp = true positives, which were samples positive by virus isolation (gelb and jackwood, 1998 ) and by the real-time rt-pcr assay, and fn = false negatives, which were samples positive by virus isolation and negative by the real-time rt-pcr assay. the real-time ibv rt-pcr test was evaluated with known positive samples obtained from ibv inoculated specific pathogen-free (spf) chickens divided into four groups of 32 chickens each and housed in four positive pressure horsfal isolation units located in four separate filtered air positive pressure rooms at pdrc (athens, ga). at 1 day of age, blood and tracheal swab samples were taken from two birds in each group. the next day, each bird was inoculated intranasally with 50 l of the arkansas dpi strain of ibv with the following titers; group 1 no virus, group 2 was given 1.6 × 10 1 embryo infectious dose 50 (eid 50 )/ml, group 3 was given 1.6 × 10 3 eid 50 /ml, and group 4 was given 1.6 × 10 5 eid 50 /ml. the birds were examined twice daily for clinical signs and tracheal swabs were taken from five birds in each group on 1, 5, 10, 14, 21, and 28 days post-inoculation (d.p.i.). tracheal swabs were placed in 200 l of 1× pbs (ph 7.4) and stored at −70 • c until used for rna extraction. in addition, a total of 300 tracheal swab samples (100 birds swabbed at 3, 7, and 14 days post-vaccination) from a commercial broiler flock that was spray vaccinated with a combination mass/ark commercial vaccine at 14 days of age were also tested. after collection, each tracheal swab was placed into 1.5 ml microcentrifuge tubes containing 1 ml of sterile 1× pbs (ph 7.5). the tubes were mixed using a bench-top vortex and stored at −70 • c until used for rna extraction. the primers and taqman ® probe targeted a highly conserved region of the 5 -utr (fig. 1) and amplified a 143-bp product (data not shown). the probe was designed to anneal to the same strand as the ibv5 gl533 primer. this strategy allowed for the fewest guanine residues within the probe sequence and placed the 5 -end of the probe only 17 bp away from the 3 -end of the ibv5 gl533 primer. the detection and quantification limits were determined using c t values obtained for each reaction containing from 10 7 to 10 2 copies of the standard rna. the values were plotted against the log of the number of template copies and a linear equation (y = −0.282x + 11.861) with a r 2 value = 0.997 was generated (fig. 2) . the assay maintained linearity for at least six orders of magnitude. using the slope from the linear equation, the overall efficiency of the assay was estimated to be 91.43%. fig. 2 . the assay standard curve was generated by plotting the c t values vs. log 10 of 10-fold serial dilutions (10 7 -10 2 ) of standard rna corresponding to the 5 -utr of the ibv genome. an overall reaction efficiency of 91.43% was estimated using the standard curve slope as indicated by the formula. the assay was negative below 100 template copies. therefore, the limit of detection and quantification were both determined to be 100 template copies. the standard deviation of the mean c t values obtained for each reaction containing from 10 7 to 10 2 copies of the standard rna calculated from six independent runs, ranged from 0.123 to 0.517 cycles. the assay amplified rna from all 15 ibv strains and the two tcov strains listed in table 1 , whereas no amplification signal was observed from any of the other pathogens tested. a search of the genbank database using the blastn analysis program (http://www.ncbi.nlm.nih.gov/blast/) indicated that 16 additional strains of ibv would likely also be detected by the assay (saibbk, dq288927; h52, ay392048; partridge/gd/s14/2003, ay646283; peafowl/gd/kq6/2003, ay641576; cal99, ay514485; k1699, ay561728; cu994, ay561727; cu570, ay561726; cu805, ay561725; cu-t2, ay561724; cu510, ay561723; cu705, ay561722; ko751, ay561721; ma5, ay561720; lx4, ay338732; bj, ay319651). three hundred and forty commercial layers not exposed to ibv, were negative for ibv antibodies (61 serum samples) by commercial elisa (synbiotics) at 6 weeks of age when the experiment was terminated. maternal antibodies were detected in 1 of 62 serum samples taken from those birds at 3 weeks of age. a total of 680 tracheal swabs (340 swabs at 3 weeks of age and 340 swabs at 6 weeks of age) taken from those birds were negative for ibv in the real-time rt-pcr assay. a total of 181 out of 229 (79.04%) clinical samples submitted to the delaware and maryland diagnostic laboratories were positive by the ibv real-time rt-pcr assay, whereas only 63 out of 229 (27.51%) samples were positive by vi. the two tests were the same for 109 out of 229 (47.60%) samples. only one sample was ibv real-time rt-pcr negative and vi positive, while 119 samples were ibv real-time rt-pcr positive and vi negative. using virus isolation as the reference standard, the calculated sensitivity for the assay was 0.98. known positive tracheal swabs taken from three groups of chickens given different doses of the arkansas dpi strain of ibv and one group of negative control birds are presented in fig. 3 . the mean copy number for each group was calculated using a standard curve (fig. 2) . at 1 d.p.i., viral load in the trachea correlated with the dose given to each group with a 28fold difference of viral rna measured between groups 2 and 3, and a 64-fold difference between groups 3 and 4. at 5 d.p.i., the virus load in the trachea plateaued at approximately 1 × 10 7 rna copies regardless of dose. at 14 d.p.i., birds that received the lowest dose of virus (group 2), had the highest copy number of viral genomes (approximately 10,000 copies), whereas birds in group 4, which received the highest dose of virus, had the lowest number of viral rna copies (approximately 600 copies). three of the samples from the negative control birds in group 1, fig. 3 . the viral load in each sample was quantified using the standard curve (fig. 2 ) and the average viral genome copy number per group was calculated. group 1 (1a) received no virus, group 2 (2a) received a 50 l dose of virus with a titer of 1.6 × 10 1 eid 50 /ml, group 3 (3a) received a 50 l dose of virus with a titer of 1.6 × 10 3 eid 50 /ml, and group 4 (4a) received a 50 l dose of virus with a titer of 1.6 × 10 5 eid 50 /ml. error bars indicate ± 1 s.d., and d.p.i. = days post-inoculation. had recorded c t values of 35.4, 28.25, and 36.84, at 1, 5, and 21 d.p.i., respectively. two of those samples (35.4 and 36.84) were below our calculated limit of detection and thus considered negative. the other sample was retested and also found to be negative (data not shown). tracheal swabs taken from an ibv spray vaccinated commercial broiler chicken flock at 3 days post-vaccination, had 58 out of 100 samples test positive, whereas at 7 days postvaccination, 65 out of 100 samples were positive and at 14 days post-vaccination, 83 out of 100 samples were positive. in this report we present the development and evaluation of a real-time taqman ® -based rt-pcr assay for the detection and quantification of ibv genomic rna directly from tracheal swabs. the target region for the assay was the highly conserved 5 -utr of the ibv genome, which resulted in amplification of all ibv strains tested. it also amplified the 5 -utr of two strains of tcov. this result was not unexpected because tcov is closely related to ibv and likely arose from a recombination event within the spike glycoprotein gene (guy, 2000; jackwood et al., 2004) . all other tcov genes including the 5 -utr appear to be similar to ibv (guy, 2000) . with the exception of the closely related tcov, the assay was specific to ibv and did not detect any of the other pathogens tested. like all coronaviruses, ibv generates a 3 -co-terminal nested set of viral mrnas when it replicates in the cell. generally, only the full-length viral genome is packaged in the virus particle. by targeting the highly conserved extreme 5 -end of the genome, but not the leader sequence (nucleotides 1 to 64), which is found on all viral subgenomic mrnas, the assay only utilizes full-length viral rna as template, which should make it more sensitive than other rt-pcr methods that target the spike gene, and more accurate as a quantification tool for viral load in biological samples. the limit of detection/quantification and reproducibility of the assay was evaluated by generating a standard curve with rna run-off transcripts. the standard curve was generated from six independent runs on samples containing from 10 7 to 10 2 copies of rna and the limit of detection and quantification were both determined to be 100 template copies. using the standard curve equation, the efficiency of the assay was calculated to be 91.43%. in addition, the assay appears to be highly reproducible based on standard deviations of the c t values, which ranged from 0.123 to 0.517 cycles. we found that the real-time rt-pcr assay was extremely sensitive in a diagnostic laboratory setting. of the clinical samples submitted to the delaware and maryland diagnostic laboratories, 79% were positive for ibv by the real-time assay whereas only 27% of those samples were positive by virus isolation. the window of time that ibv can be detected following infection with the real-time rt-pcr test described herein is 21 days post-infection, which likely represents a significant improvement over virus isolation and explains the higher rate of detection in the tracheal swab samples. for virus isolation, it is generally recommended that tissues such as cecal tonsil and kidney or cloacal swabs be taken in addition to tracheal swabs because the virus is known to persist there (gelb and jackwood, 1998) . not only does that practice significantly increases the work load it also can yield misleading results because vaccine viruses can also persist in those tissues and be shed in the feces (cavanagh and naqi, 2003) . it is important to evaluate any new diagnostic test with a number of known negative and positive samples. known negative samples, obtained from commercial layer chicks maintained in isolation units and monitored for antibodies to ibv by elisa, all tested negative for ibv in the real-time rt-pcr assay. since we wanted to insure the birds remained free of ibv, we choose commercial layer chicks with maternal antibodies to the virus as an added insurance against infection. for known positive samples, we used spf chicks with no maternal antibodies and infected them with three different dosages of the arkansas dpi strain of ibv. an additional negative control group was also maintained during that experiment. three samples from the negative control group had c t values of 35.4, 28.25, and 36.84 . after retesting, all of the samples were below our calculated limit of detection and considered negative. cross-contamination of samples especially during the rna extraction process is a real concern with any rt-pcr reaction, but it is recognized that precautions should be taken to limit contamination at every step of the procedure. the viral load in the trachea of birds given different dosages of ibv showed that regardless of the initial dose of virus, a similar maximal amount of viral rna was detected at 5 d.p.i. more studies will be necessary to determine the course of virus replication in vivo, but it appears that the tracheal epithelium can support a finite amount of virus and that virus replication continues until that limit is reached. for the dosages used in our study, the maximum level of viral load was reached within 5 days post-infection. it is interesting to note and quite appropriate that the requirements for efficacy of ibv vaccines outlined in section 113.327 title 9 of the code of federal regulations (http://www.access.gpo.gov/nara/cfr/waisidx 99/9cfr113 99. html) requires that detection of challenge virus be conducted 5 d.p.i. we also observed that the level of viral rna in the trachea of birds, which received a high dose of virus, declined more quickly than levels in birds receiving a low dose of the virus. loss of ciliated columnar epithelium and presumably the associated virus replicating in those cells, is a common lesion with ibv infection. birds receiving a high dose of ibv can develop clinical signs and lesions within 24 h, whereas natural spreading virus generally requires 36 h or more before clinical signs and lesions occur (cavanagh and naqi, 2003) . presumably, high dosages of the virus would accelerate the loss of the epithelial cells in the upper-respiratory tract leading to a more rapid decline in viral load. when spray vaccinated commercial broilers were examined for ibv by the real-time rt-pcr assay we found that only 58 of 100 birds were positive at day 3 post-vaccination. by day 7, 65 of 100 birds were positive and by 14 days post-vaccination 83 of 100 birds were positive for ibv. the dynamics of vaccine spread in commercial chicken flocks following spray vaccination in the field is not known. our data indicates that approximately half of the birds in the flock were initially exposed to the vaccine (day 3 post-vaccination data), which then spread to other birds in the flock so that by 14 days post-vaccination, more than 80% were exposed. more data will be required to verify this result, but it appears that exposure to ibv vaccine by in-house spray vaccination could be improved. initially it could be considered advantageous for ibv vaccines to spread in a marginally exposed flock providing a mechanism for all the birds in the flock to become immunized, however; vaccine spread in a flock also presents opportunities for back passage of these rapidly mutating coronaviruses potentially leading to a reversion to pathogenicity. it is important to rapidly differentiate infectious agents that cause respiratory diseases similar to low pathogenicity avian influenza and lentogenic and mesogenic newcastle disease so that veterinarians can quickly respond to outbreaks. in addi-tion, ibv remains an extremely important disease in commercial chickens and quickly diagnosing it is critical for control of this highly infectious virus. the real-time rt-pcr assay for ibv described herein can be conducted directly on tracheal swabs without the need for virus isolation. and although it does not differentiate between different types of ibv it is extremely sensitive and specific, and can be used to quantitate viral rna in clinical samples. molecular basis of the variation exhibited by avian infectious bronchitis coronavirus (ibv) infectious bronchitis, diseases of poultry infectious bronchitis, a laboratory manual for the isolation and identification of avian pathogens turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review molecular analysis of tcov, sars-cov, and ibv: how are they related further development and use of a molecular serotype identification test for infectious bronchitis virus a monoclonal antibody blocking elisa to detect serotype-specific infectious bronchitis virus antibodies production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s-1) gene identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s-1 gene differentiation of infectiousbronchitis virus serotypes using polymerase chain-reaction and restrictionfragment-length-polymorphism analysis origin and evolution of georgia 98 (ga98), a new serotype of avian infectious bronchitis virus a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes the authors would like to acknowledge the generous gifts from the following people: dr. erica spackman (usda/ars, athens, ga)-rna from aiv strains, dr. darrell kapczynski (usda/ars, athens, ga)-rna from apv and ndv strain b1, dr. maricarmen garcia and sylva riblet (pdrc, university of georgia, athens, ga)-dna from iltv, adenovirus st-1, and mg strains. this work was supported through specific cooperative agreement #58-6612-2-0219 with usda/ars from department of defense supplemental homeland defense funds of cris project #6612-32000-041-00x. key: cord-281526-7t9e4lgn authors: yin, lijuan; zeng, yuyao; wang, wei; wei, ying; xue, chunyi; cao, yongchang title: immunogenicity and protective efficacy of recombinant fusion proteins containing spike protein of infectious bronchitis virus and hemagglutinin of h3n2 influenza virus in chickens date: 2016-09-02 journal: virus res doi: 10.1016/j.virusres.2016.07.010 sha: doc_id: 281526 cord_uid: 7t9e4lgn infectious bronchitis (ib) is an acute and highly contagious viral respiratory disease of chickens and vaccination is the main method for disease control. the s1 protein, which contains several virus neutralization epitopes, is considered to be a target site of vaccine development. however, although protective immune responses could be induced by recombinant s1 protein, the protection rate in chickens was still low (<50%). here, we generated fused s1 proteins with ha2 protein (rs1-ha2) or transmembrane domain and cytoplasmic tail (rs1-h3(tm)) from hemagglutinin of h3n2 influenza virus. after immunization, animals vaccinated with fusion proteins rs1-ha2 and rs1-h3(tm) demonstrated stronger robust humoral and cellular immune responses than that of rs1 and inactivated m41 vaccine. the protection rates of groups immunized with rs1-ha2 (87%) were significantly higher than the groups inoculated with rs1 (47%) and inactivated m41 vaccine (53%). and chickens injected with rs1-h3(tm) had similar level of protection (73%) comparing to chickens vaccinated with rs1 (47%) (p = 0.07). our data suggest that s1 protein fused to the ha2 or tm proteins from hemagglutinin of h3n2 influenza virus may provide a new strategy for high efficacy recombinant vaccine development against ibv. avain infectious bronchitis (ib) was first described in 1931 as a highly contagious disease and thereafter was found to be caused by an infectious bronchitis virus (ibv), which belongs to coronavirus genus within the coronaviridae (cavanagh, 2007; sjaak et al., 2011) . though both live-attenuated and inactivated vaccines are used worldwide to control the disease, ibv occasionally outbreaks in endemic areas (bijlenga et al., 2004; cavanagh, 2003) . the use of attenuated live-vaccines elicits local, systemic and cell-mediated immunity to the virus (cavanagh, 2003; raj and jones, 1997) , but poses a risk of residual pathogenicity associated with vaccine backpassage in flocks (abro et al., 2012; mckinley et al., 2008; mckinley et al., 2011) . though the inactivated ib vaccines are relatively safe, they are efficacious only when used as boosters after priming vaccination with live vaccines (ladman et al., 2002) . thus to develop a safer, more efficacious and economic vaccine candidate is of great interest for scientific research. infectious bronchitis virus (ibv) is an enveloped virus containing a single-stranded positive-sense rna genome of 27.6 kb (wickramasinghe et al., 2014) . the genome of ibv encodes four structural proteins, including spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins (cavanagh, 2007) . people have found that the spike (s) protein, the largest structural protein, constitutes the characteristic club-shaped 16-21 nm projections that emerge from the virion surface and presents a corona-like appearance under electron microscopy (cook et al., 2012) . in addition, the s protein was suggested to have haemagglutinating activity that the entry of the viruses is mediated by sialic acid binding activity of the s protein (schultze et al., 1992) . the s protein is a class i fusion peptide, in which the variable s1 domain is involved in host cell attachment and the conserved s2 domain mediates fusion of the virion and cellular membranes (bosch et al., 2003) . the s1 protein contains the primary neutralizing epitopes that can induce neutralization, hemagglutination inhibition (hi) and serotype-specific antibodies (cavanagh et al., 1986a,b; ignjatovic and galli, 1994; ignjatovic and galli, 1995 koch et al., 1990) , thus making it a main target when designing new ibv vaccines. the s protein alone is considered to be sufficient to induce good immunity (cavanagh, 2007) . however, when the s1 subunit of ibv was recombined using baculovirus, although protective immune responses were induced with multiple inoculations, the percentage of protected chickens was still less than 50% (song et al., 1998) . thus, to induce better immunogenicity against ibv infections, manipulation of the s protein is considered to be a good strategy when designing new ibv vaccines. our previous research demonstrated that various influenza h1, h5 and h9 hemagglutinins (has) proteins containing replaced h3-wt tm showed increased thermal stability and immunogenicity (liu et al., 2014) . it is intriguing for us to know whether recombinant hemagglutinin and s protein can also induce better immunogenicity and protective efficacy against ibv infections. in this study, we first investigated whether the recombinant s protein could induce better immune responses. therefore, we generated a recombinant s1 (rs1) protein and two recombinant s1 fusion proteins, rs1-h3(tm) (fused with the transmembrane (tm) and cytoplasmic tail (ct) of influenza h3n2 ha protein) and rs1-ha2 (fused with the ha2 domain of influenza h3n2 ha protein). our results suggest that the immunogenicity and protection efficacy of these recombinant fusion proteins have been enhanced in chickens. sf9 insect cells were cultured in serum-free sf900ii medium (gibco, grand island, ny, usa) at 27 • c. the virulent m41 strain (china institute of veterinary drug control, ivdc) was propagated in 10-day-old specific pathogen free (spf) chicken embryos. eid 50 for the ibv m41 was calculated according to the reed-muench method as described previously (reed and muench, 1938) . the a/swine/guangdong/01/1998(h3n2) was isolated and maintained by our laboratory. all primers used in this study were synthesized by invitrogen and summarized in table 1 . a bac-to-bac baculovirus expression system was used for the production of recombinant expression bacmids and baculoviruses. briefly, ibv-m41 s1 gene, h3(tm) and ha2 fragments (genbank accession number fj830855.1) from h3n2 strain (a/swine/guangdong/01/1998) were first amplified and cloned into pmd-18t vector (takara) and then amplified sequentially. s1-h3(tm) and s1-ha2 fusion genes were then generated by overlapped pcr and cloned between the sali and hindш sites of pfastbac1 vector (invitrogen) to generate recombinant shuttle plasmids (fig. 1a) . the shuttle vectors were then chemically transformed into competent dh10bac tm escherichia coli cells (invitrogen). all clones were verified by sequencing (invitrogen). recombinant baculovirus generation and infection were performed as previously described (liu et al., 2013) . briefly, the obtained recombinant bacmids were transfected into sf9 cells and incubated for 3 days. the target recombinant baculoviruses (rbvs) were then harvested from the supernatant. the structure of the recombinant fusion proteins are shown in fig. 1b. cell lysates were separated on 10% sds polyacrylamide gels, and then electrophoretically transferred to polyvinylidene difluoride membranes (millipore, billerica, ma). membranes were blocked and subsequently detected with chicken polyclonal sera (china institute of veterinary drugs control) against ibv virus at a 1:2000 dilution, horseradish peroxidase (hrp)-conjugated anti-chicken secondary antibody at a 1:5000 dilution (ptglab, usa) and commercial ecl kit (pierce). for recombinant proteins expression, sf9 cells were infected with recombinant or wild-type baculoviruses and cultured for 3 days, and the cells were collected, ultrasonicated, and then centrifuged at 12,000 × g for 30 min at 4 • c. the supernatants were further centrifuged at 120,000 × g for 3 h at 4 • c. the resulting precipitates were resuspended in pbs and loaded onto a discontinuous sucrose gradient of 30%, 40%, 50%, and 60% sucrose for recombinant proteins enrichment, and ultracentrifuged at 65,000 × g for 16 h at 4 • c. fractions were collected from the gradient interphases as described previously (liu et al., 2014) , and their recombinant proteins contents were analyzed using coomassie blue stained sds-page electrophoretogram. formalin-inactivated m41 virus was purified and concentrated by ultracentrifugation as previously described (kong et al., 2010) . the overall protein concentrations of recombinant proteins and inactivated ibv were determined using bca protein assay kit (pierce, company); and the protein concentrations of the three recombinant proteins (rs1 protein, rs1-h3(tm) protein and rs1-ha2 protein) and s1 protein concentration of inactivated ibv were determined using sds-page gel electrophoresis by genesnap and genetools from syngene software with bsa as standard. ninety 10-day-old spf chickens were collected from the spf experimental animal center (dahuanong animal health products co., ltd., guangdong, china), and housed in individual isolators under positive pressure, then randomly divided into six groups (n = 15 chickens/group). recombinant proteins, inactivated m41 virus, sf9 cell lysate (infected by wild-type baculoviruses), and pbs were emulsified with montanide tm isa 71 vg at a 3:7 ratio respectively (w:w; antigens:adjuvant) as recommended by the manufacturer (seppic, paris, france) and then were used in intramuscular injection. group 1 chickens were immunized with 5 g rs1 protein per chicken. group 2 chickens were injected with 5 g rs1-h3(tm) protein per chicken. group 3 chickens were vaccinated with 5 g rs1-ha2 protein per chicken. group 4 chickens received 5 g inactivated m41 (s1 protein) per chicken, as a positive control. for negative controls, animals in the remaining two groups were injected with either rwt (sf9 cell lysate infected by wild-type baculoviruses) or pbs. booster immunization, using the same antigen at inoculation, was conducted 2 weeks after the prime. two weeks after booster immunization, chickens from each group were challenged with 2 × 10 4 eid 50 of the ibv-m41 strain via the nasal-ocular route in 200 l pbs per chicken, and were observed daily for clinical symptoms over a week period and euthanised at 7 day after challenge. the trachea and kidney tissues of chickens in all groups were harvested for further detection of virus. all animal experiments were conducted in compliance with the institutional guidelines for animal protection rights in china. sera were collected from chickens at 0, 14 and 28 days after primary immunization for ibv-specific antibody detection. the purified m41 virosomes were made following the procedures as previously described (liu et al., 2013) , and used as antigen to detect the ibv-specific antibodies in an indirect elisa. briefly, the purified virosomes at a concentration of 3.5ug/ml were coated, and incubated with serial dilutions of each serum sample (37 • c for 1 h). the secondary donkey anti-chicken hrp-conjugated antibodies (ptglab, usa) were used at a 1:5000 dilution. the optical density value was 450 nm. the end-point titer was determined as the reciprocal of the final dilution giving a threefold optical absorbance of negative control as described previously (liu et al., 2014) . cellular immune response was assessed by counting the numbers of various types of functional t lymphocytes in vaccinated chickens. two weeks after the boosting immunization and prior to challenge, five chickens were selected randomly from each group and peripheral blood samples were collected from the brachial wing vein using heparinized syringes. peripheral blood mononuclear cells (pbmcs) were isolated and adjusted to 1 × 10 7 cells/ml. the samples (100 l; 1 × 10 6 cells) were incubated for an hour at room temperature with mouse anti-chicken cd4-fitc, cd8-rpe and cd3-sprd (southern biotech, birmingham, al, usa). flow cytometry was performed on a facs calibur instrument (becton dickinson, san jose, ca, usa) and data were analyzed with cel-lquest software (bd bio-sciences). tracheal and renal tissues of chickens were collected for detection of the challenge virus. total rnas were extracted from tissue suspensions using takara minibest viral rna/dna extraction kit (takara biotechnology, dalian, china) and subjected to rt-pcr (primescrip tm rt-pcr kit, takara bio, china) using primers directed at the nucleocapsid as previously described (zhang et al., 2014) . the absence of detectable virus in the tracheal and kidney was considered to have been protected by the vaccine against the challenge. fig. 1 . baculovirus construct for production of proteins. (a) construction of recombinant plasmids. pfbac1 contains polyhedrin promoter (pph), sv40 pa, sv40 polyadenylation signal; gentamicin; tn7r and tn7l. different target genes (s1, s1-h3 and s1-ha2) were inserted between pph and sv40 pa respectively. (b) construction of recombinant fusion proteins rs1, rs1-h3(tm) and rs1-ha2. for rs1-h3(tm) protein, rs1 protein was fused to the ct and tm domain of h3n2 ha protein; for rs1-ha2 protein, rs1 protein was fused to the ha2 domain of the ha protein of h3n2. the data were statistically analyzed by using a student's twotailed t-test when only two groups were compared or by one-way analysis of variance (anova) when more than two groups were compared. p values (p) less than 0.05 were considered statistically significant. different recombinant baculoviruses were constructed to express the target fused protein (fig. 1a) . to construct rs1-h3(tm) protein, recombinant s1 protein was fused to the cytoplasmic tail (ct) and transmembrane (tm) domain of hemagglutinin (ha) from influenza h3n2 subtype. for rs1-ha2 protein, rs1 protein was fused to the ha2 domain of the ha protein (fig. 1b) . to check whether the three recombinant proteins rs1, rs1-h3(tm), rs1-ha2 could be expressed in the sf9 cells, we analyzed protein expression in collected cell lysates from the recombinant baculovirus-infected sf9 cells. western blot under denaturing condition showed that these recombinant proteins were detectable as clearer distinct bands with molecular weight of 100 kda, 110 kda and 130 kda ( fig. 2a) . the molecular weight of the recombinant rs1 protein was comparable to the native protein of 100 kda (cavanagh et al., 1986a,b) . this result was further confirmed by sds-page using concentrated recombinant protein contents utilizing discontinuous sucrose gradient centrifugation (fig. 2b) , and these concentrated recombinant proteins were prepared for vaccination later in our study. to investigate whether the recombinant proteins could induce better immune response against ibv infection, we detected ibvspecific antibody in the sera of vaccinated chicken using indirect elisa. in general, two weeks after primary vaccination (day 14), ibv-specific antibody level increased in all groups besides rwt and pbs. the antibody level in chickens immunized with rs1-h3(tm) and rs1-ha2 increased significantly, comparing to the rs1 group and inactivated m41 group (fig. 3) . two weeks after booster vaccination (day 28), the levels of anti-ibv antibodies increased further in chickens immunized with inactivated m41 virus, rs1, rs1-h3(tm) and rs1-ha2. the inactivated m41 group developed a higher level of antibody than the rs1 group (p < 0.05). moreover, chickens vaccinated with rs1-h3(tm), rs1-ha2 had significantly higher antibody titers than animals in the rs1 group (p < 0.01), while fig. 3 . infectious bronchitis virus (ibv)-specific antibody titers in chickens sera. six groups of chickens (n = 15 chickens per group) were subcutaneously immunized with rs1, rs1-h3(tm), or rs1-ha2, respectively. inactivated m41 was used as a positive control, rwt (sf9 cell lysate infected by wild-type baculoviruses) and pbs were used as negative controls. sera from each chicken were collected and the ibv-specific antibody titers were determined by elisa. data is represented by the mean antibody titer +s.d. ratio of cd3 + cd8 + /cd3 + cd4 + t-lymphocytes 2 weeks after booster immunization a . ratio of cd3 + cd8 + /cd3 + cd4 + t-lymphocytes inactivated vaccine 0.46 ± 0.10a rs1 0.48 ± 0.05a rs1-h3(tm) 0.71 ± 0.03b rs1-ha2 0.73 ± 0.02b the difference between groups with the same letter (a or b) was not significant (p > 0.05). a the test was repeated five times in each group. data is presented by means ± sd. there was no difference between the groups of rs1-h3(tm) and rs1-ha2. these data indicate that the fused s1 proteins can boost better immune responses than inactivated m41 virus and s1 protein alone, resulting in a significantly increased antibody level. as cd3 + cd4 + helper t cells and cd3 + cd8 + cytotoxic t cells function as crucial components in cellular immunity, we counted the number of cd3 + , cd3 + cd4 + and cd3 + cd8 + t lymphocytes utilizing flow cytometry 14 days after the booster to measure immune response. levels of cd3 + and cd3 + cd8 + t lymphocyte increased significantly in group of rs1-ha2 and rs1-h3(tm) comparing to those groups inoculated with rs1, inactivated m41, rwt and pbs (p < 0.01). there was no significant difference between the rs1-h3(tm) and rs1-ha2 groups. and the percentages of the t lymphocytes were comparable between the rs1 and inactivated m41 vaccine groups (fig. 4) . further, the ratio of cd3 + cd8 + to cd3 + cd4 + t lymphcytes in rs1-h3(tm) (0.71 ± 0.03) and rs1-ha2 (0.73 ± 0.02) vaccinated groups were significantly higher than that in chickens immunized with rs1 (0.48 ± 0.05) and inactivated m41 (0.46 ± 0.10) (p < 0.05) ( table 2 ). these results suggest that though rs1 alone can elicit cellular immune responses in chickens, the recombinant rs1-h3(tm) and rs1-ha2 can significantly enhance the cell-mediated immune response. to further confirm our results, chickens were challenged with the virulent m41 strain. morbidity, tracheal infections, renal infections and protection rates after challenge are summarized in table 3 . in general, chickens immunized with inactivated m41 vaccine, rs1, rwt and pbs developed symptoms as dyspnea, coughing and rales on a different level. to further evaluate the level of protection after challenge, rt-pcr was performed to detect viral rna in the collected tracheas and kidneys. chickens vaccinated with rs1 (47%) presented a similar protection level comparing to chickens immunized with the inactivated m41 virus (53%). however, chickens immunized with rs1-ha2 were better protected (87%) comparing to chickens vaccinated with rs1 (47%) and inactivated m41 (53%) (p < 0.05), while chickens injected with rs1-h3(tm) had similar level of protection (73%) comparing to chickens vaccinated with rs1 (47%) (p = 0.07). there was a significant difference between the rs1-ha2-vaccinated group and the rs1-immunized group (p < 0.05) in the number of collected tracheas and kidneys that were positive for the virus. these data further confirm our previous data presenting better immunogenesis of the fused s1 proteins and suggest that these fused proteins, especially rs1-ha2 protein, can provide a new strategy for ibv vaccine development. the oligomeric spike (s) protein is a trans-membrane glycoprotein, usually forms dimers or trimers that extend from coronavirus membranes. this protruding virion protein is considered to be the main antigen and therefore mainly used to induce immunogenicity against coronaviruses (cavanagh, 1983; delmas and laude, 1990; lewicki and gallagher, 2002) . for instance, researches on severe acute respiratory syndrome (sars), which belongs to coronavirus, have suggested that recombinant trimeric full-length s proteins are highly immunogenic and able to induce an efficacious protective immune response (kam et al., 2007; zhou et al., 2006) . more recently, li et al. showed that the fused s protein with the foldon domain derived from t4 bacteriophage presented significantly greater potency in the induction of neutralizing antibodies than the s protein . these researches have suggested that the manipulated s protein could serve as major target for immune therapy and vaccine design against coronaviruses. once this integral protruding membrane protein is assembled, it undergoes endoproteolysis and cleaves to two noncovalently associated s1 and s2 fragments (cavanagh, 1983; cavanagh et al., 1986a,b) . the s1 domain contains the primary neutralizing epitopes for ibv, and thus is considered to be responsible determine its pathogenicity (wickramasinghe et al., 2014) . in 1998, song et al. showed that recombinant s1 (rs1) glycoprotein, expressed in a baculovirus expression system, could induce protective immunity in chickens against challenge with virulent nephropathogenic ibv, though multiple inoculations were required and the rs1 protein induced only 50% protection of the kidney (song et al., 1998) . other researchers displayed the s1 glycoprotein on the bacmam virus-based surface and presented that the bv-dual-s1 protein elicited stronger humoral and cell-mediated immune responses (zhang et al., 2014) . thus, enhancement of the antigenicity of the s1 fig. 4 . flow cytometry analysis of t lymphocyte subsets in chickens after immunization with rs1, rs1-h3(tm), rs1-ha2, inactivated m41, rwt (sf9 cell lysate infected by wild-type baculoviruses) and pbs. this test was performed two weeks after the boosting immunization. data are presented by means + sd. n = 5 chickens per group. the protection rate among the five groups are indicated with different letters. the difference among groups with the same letter (a-c)was not significant (p > 0.05). a morbidity was recorded for each day after challenge and is presented as total number of sick chickens in each group. b virus identified intrachea by rt-pcr detection. c virus identified in kidney by rt-pcr detection. d protection rate was determined by the number of unaffected chickens (without detection in tracheal and kidney)/total number of chickens in each group. glycoprotein is considered to be critical for the development of effective vaccines against ibv. our previous research presented that various influenza h1, h5 and h9 hemagglutinins (has) proteins containing replaced h3-wt tm showed increased immunogenicity (liu et al., 2014) . to improve the immunogenicity and protective efficacy of ibv s1 protein, here we generated two fusion proteins rs1-h3(tm) and rs1-ha2 from s1 protein of ibv and hemagglutinin of influenza virus h3n2. after challenged with virulent ibv m41 strain, our results demonstrated that fusion proteins performed better protection compared with inactivated m41 vaccine and recombinant rs1 protein alone, while the latter groups presented similar protection ratio. thus, an augment of the humoral and cellular immune responses was suggested to be induced by the fused rs1-h3(tm) and rs1-ha2 proteins. people suggested that humoral immunity is critical for disease recovery and virus elimination (cook et al., 1991) . consist with the immunity protective test, in this study, the level of specific antibodies induced by the recombinant rs1-h3(tm) and rs1-ha2 was higher than the rs1 and inactivated m41 group. one interesting thing is that, two weeks after booster vaccination (day 28), the antibody level induced by recombinant s1 protein was lower than that of inactivated virus in our study. it is possible that the inactivated m41 vaccine, which is derived from native viruses, comprised almost all the epitopes of ibv and thus presented better performance in inducing a ibv-specific antibody response after booster vaccination. it is generally known that cd3 + cd4 + helper t cells and cd3 + cd8 + ctls play a major role in inducing t cell-mediated cellular immunity (doherty et al., 1997) . some reports have demonstrated that the cd8 + ctl response are critical in the control of infectious bronchitis (ib) and more efficient than cd3 + cd4 + helper t cells in controlling respiratory virus infections (cook et al., 1991; thompson et al., 1997) . the cd4 + t-cell responses provide help for b-cells to mature and generate specific antibodies as well as directly antiviral cytokines, increase the proliferation, maturation, and functional activity of cd8 + ctl (doherty et al., 1997; kotani et al., 2000) . in this study, we found an significant increase of the cd3 + cd4 + and cd3 + cd8 + t lymphocyte subgroups in chickens immunized with rs1-h3(tm) and rs1-ha2, comparing to the rs1 and inactivated m41 vaccine groups. the ratio of cd3 + cd8 + to cd3 + cd4 + t lymphocytes in chickens immunized with rs1-h3(tm) or rs1-ha2 was greater than that in chickens inoculated with rs1 and inactivated m41 vaccine. these findings indicate the advantage of employing fused s1 protein to tms or ha2 of ha proteins in an effectively stimulating cellular immune responses, especially in promoting the ctl response, which is critical for virus clearance and recovery of chickens from ibv infections (collisson et al., 2000) . in summary, here we have successfully expressed two fused s1 proteins, rs1-ha2 and rs1-h3(tm) proteins, and demonstrated that these fusion proteins are superior to rs1 protein alone in terms of immunogenicity and protective efficacy. accordingly, our results suggest that the two recombinant fusion proteins rs1-ha2 and rs1-h3(tm) are suitable vaccine candidates for the development of vaccines against ibv. emergence of novel strains of avian infectious bronchitis virus in sweden development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection coronavirus ibv: structural characterization of the spike protein severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry effect of in ovo bursectomy on the course of an infectious bronchitis virus infection in line c white leghorn chickens the long view: 40 years of infectious bronchitis research assembly of coronavirus spike protein into trimers and its role in epitope expression effector cd4+ and cd8+ t-cell mechanisms in the control of respiratory virus infections the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens immune responses to structural proteins of avian infectious bronchitis virus antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions proteomic analysis of purified coronavirus infectious bronchitis virus particles kinetics of lymphocytic subsets in chicken tracheal lesions infected with infectious bronchitis virus protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/98 quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors immunogenicity and protection efficacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins recombinant influenza h1, h5 and h9 hemagglutinins containing replaced h3 hemagglutinin transmembrane domain showed enhanced heterosubtypic protection in mice avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus infectious bronchitis virus: immunopathogenesis of infection in the chicken a simple method of estimating fifty percent endpoints neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes infectious bronchitis virus variants: a review of the history, current situation and control measures induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens the avian coronavirus spike protein bacmam virus-based surface display of the infectious bronchitis virus (ibv) s1 glycoprotein confers strong protection against virulent ibv challenge in chickens a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord-296524-68gwusfe authors: barr, da; reece, rl; o'rourke, d; button, c; faragher, jt title: isolation of infectious bronchitis virus from a flock of racing pigeons date: 2008-03-10 journal: aust vet j doi: 10.1111/j.1751-0813.1988.tb14468.x sha: doc_id: 296524 cord_uid: 68gwusfe nan the chicken is the only confirmed natural host to infectious bronchitis virus (ibv) (hofstad 1984) . we report the isolation of ibv from a flock of racing pigeons and assess its significance. during the winter of 1985, a racing pigeon fancier in north eastern victoria observed an acute illness in his loft of 150 racing pigeons. affected birds had ruffled feathers, dyspnoea and excessive mucus at the commissures of the beak. eleven birds died during the first 24 h and 11 more over the next 2 days. seven birds were submitted for necropsy. the flock was treated with antibiotics. affected birds recovered over the next 2 to 3 weeks. at post-mortem examination, all birds were in average body condition. they had recently eaten but the linings of the oesophagus and crop were ulcerated. mucoid pharyngitis and tracheitis were noted. the lower intestines contained fluid. histological examination of oesophageal tissues confirmed that the birds were infested with trichomonads. four clarified suspensions from pooled samples of 5 tracheal mucosae and from 7 cloacal swabs were each inoculated into the allantoic cavity of sets of 5 nine-day-old fertile chicken eggs, allantoic fluids, from the third passage, were collected 72 h after inoculation of each set of eggs, clarified and pelleted in an ultra centrifuge. coronaviruses were seen when the pellets were examined by electron microscopy. ten days after inoculation, all eggs were opened and the embryos examined. embryos in each set were curled and stunted, changes which are characteristic of ibv. an aliquot of a tracheal isolate was passaged 3 times in eggs at 48 h intervals. fluorescence, shown to be specific for ibv, was seen in the aliantoic cells of each passage (endo and faragher, unpublished) . after concentration and treatment with phospholipase c type 1, allantoic fluid from the third passage haemagglutinated chicken red blood cells. the haemagglutination was inhibited by specific ibv antiserum, indicating ibv of sub-type b (faragher 1987) . the ibv was then sub-typed by plaque reduction serum neutralisation tests using serums specific for each of the 9 australian ibv subtypes (wadey and faragher 1981) , and shown to belong to sub-type b. twenty-six days after the onset of disease in the pigeons, samples of serum were obtained from 10 birds in the affected flock and also from 12 birds in an unaffected flock kept in a separate loft 4oom away. ibv haemagglutination inhibition (hi) antibody was detected at levels from 2 to 2'. the titres in serum from the unaffected and the affected pigeon flocks were similar. the pathogenicity of a cloacal ibv isolate was examined. four 4-week-old csiro spf chickens and four 8-week-old meat pigeons that were housed in the same cage were each inoculated by intranasal, intraocular and oral routes with allantoic fluid containing 10' eid,, of ibv. meat pigeons were chosen rather than racing pigeons to reduce the likelihood of previous exposure to ibv. a similar number of birds of both species was inoculated with phosphate buffered saline (pbs) and housed in a separate room. four days after inoculation, all the chickens inoculated with ibv had marked respiratory rales. a11 pigeons and those chickens inocuiated with pbs remained healthy for 18 d when all the birds were bled and killed. levels of ibv hi antibodies in the chickens increased from 2' before inoculation to 2' to 24 18 d after inoculation. this response is common following vaccination of chickens, whereas wild ibv stimulate higher antibody levels. no ibv hi antibody was detected in the pigeons. the ibv may have caused disease in the racing pigeons because their resistance was lowered by intercurrent disease. pigeons raced over long distances have been seen to shelter in open-sided caged-layer poultry sheds when they are attacked by raptors (j dark, personal communication). such direct contact may have been the route of transmission to the pigeons of an ibv shown to be of the same serotype as widely used australian ibv vaccine strains. a stray pigeon which the pigeon fancier had seen join his flock a week before the onset of disease may have been implicated in the transmission of the ibv. laboratory contamination as a source of the isolate was considered to be a remote possibility because no other ibv was isolated during the 5 days immediately preceding or following this case. we thank mr g beavis for submitting the pigeons and assisting with the investigation. diseuses of poultry key: cord-263476-ju7xqwa7 authors: xia, jing; he, xiao; yao, ke-chang; du, li-jing; liu, ping; yan, qi-gui; wen, yi-ping; cao, san-jie; han, xin-feng; huang, yong title: phylogenetic and antigenic analysis of avian infectious bronchitis virus in southwestern china, 2012–2016 date: 2016-08-13 journal: infect genet evol doi: 10.1016/j.meegid.2016.08.011 sha: doc_id: 263476 cord_uid: ju7xqwa7 the aim of this study was to decipher the molecular epidemiological and antigenic characteristics of infectious bronchitis virus strains (ibvs) isolated in recent years in southwestern china. a total of 24 field strains were isolated from diseased chickens between 2012 and 2016. phylogenetic analysis based on s1 nucleotide sequences showed that 16 of the 24 isolates were clustered into four distinct genotypes: qx (37.5%), tw (16.7%, twi and twii), mass (8.3%), and j2 (4.2%). the qx genotype was still the prevalent genotype in southwestern china. recombination analysis of the s1 subunit gene showed that eight of the 24 field strains were recombinant variants that originated from field strains and vaccine strains. a new potential recombination hotspot [atttt(t/a)] was identified, implying that recombination events may become more and more common. the antigenicity of ten ibvs, including seven field strains and commonly used vaccine strains, were assayed with a viral cross-neutralization assay in chicken embryonated kidney cells (cek). the results showed that the ten ibvs could be divided into four serotypes (massachusetts, 793b, sczy3, and scyb). sczy3 and 793b were the predominant serotypes. six of the seven field isolates (all except for ck/ch/scyb/140913) cross-reacted well with anti-sera against other field strains. in conclusion, the genetic and antigenic features of ibvs from southwestern china in recent years have changed when compared to the previous reports. the results could provide a reference for vaccine development and the prevention of infectious bronchitis in southwestern china. infectious bronchitis (ib) is a highly contagious disease in chickens that causes significant economic losses to the worldwide poultry industry (colvero et al., 2015) . the etiologic agent of ib is the infectious bronchitis virus (ibv), a member of the coronaviridae family, in the subfamily coronaviridae and genus gamma-coronavirus. the ibv genome is 27.6 kb and encodes at least four structural proteins, including the spike glycoprotein (s), membrane protein (m), small membrane protein (e), and nucleocapsid protein (n) (ujike and taguchi, 2015) . the major immunogen of ibv is the s1 subunit protein, which contains epitopes that can induce the production of specific neutralizing antibodies and the hemagglutination inhibition antibody. ibvs from different serotypes usually exhibit poor cross-protection (li et al., 2012) . due to the incomplete proofreading mechanism of the rna polymerase and the gene recombination during genome replication, ibv genomes are constantly evolving, and new ibv variant strains are always arising (baker and lai, 1990; lai, 1992) . since the early 1980s, ibv has been diagnosed in china by viral isolation. although the wide use of vaccine strains, such as h120, m41, 28/86, 4/91, and ma5, has successfully prevented ib epidemics on most farms, immune failure is still reported frequently as the result of infections with strains that differ serologically from the vaccine strains. therefore, continuing analysis of the genetic evolution and antigenic relatedness among field isolates and vaccine strains may provide critical insight for vaccine strain selection and vaccine development. our previous study revealed that isolates obtained between 2008 and 2009 from the sichuan province belonged mainly to a group of qx-like strains (79% qx-type; 5% twi-type) (zou et al., 2010) . in a later report from other researchers, qx-type and twi-type ibvs accounted for 37% and 37%, respectively, in sichuan area during 2011-2012 . while in southern china, picture was quite different, ck/ch/lsc/99itype was the predominant genotype and no qx-type strains were isolated during 2013 (mo et al., 2013) . so the genetic character of ibvs from china varied according to time and regions. for the antigenic features of ibvs isolated in recent years from china, less attention was given. a report showed that 28 ibvs from guangxi of china in 2009infection, genetics and evolution 45 (2016) infection, genetics and evolution j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m e e g i d 2011 could be divided into 6 serotypes (i-vi), but most of the isolates (24/28) was qx-genotype, ibvs from the qx-genotype may belong to different serotypes, and the serotype of ibvs varied according to time and regions (qin et al., 2014) . another report showed the serotype of twi-type strains were different from mass-type in taiwan in 2000 (wang and huang, 2000) . as the genetic and antigenic character of ibvs varied according to time and regions, and there is no official report on that of the ibvs from southwestern china in recent years, the molecular and antigenic characteristics of ibvs from southwestern china were not clear. the aim of this study was to decipher the genetic and antigenic characteristics of ibv strains circulating in commercial flocks in southwestern china in recent years. specific pathogen-free (spf) chicken embryos were obtained from beijing merial vital laboratory animal technology co., ltd. (beijing, china). m41 and h120 strains were obtained from the china institute of veterinary drug control (beijing, china). the 4/91 vaccine was supplied by internet international b.v. (boxmeer, nl). throughout 2012-2016, kidney, lung, and trachea samples were collected from broiler or layer chickens suspected of ib infection in southwestern china (table1). samples were homogenized in phosphatebuffered saline (pbs) containing 200 μg/ml penicillin and 100 μg/ml streptomycin in a ratio of 1:5-10. after filter sterilizing with a 0.22 μm filter membrane, 0.2 ml sample was inoculated into the allantoic cavity of 9-to 11-day-old spf embryos. the embryos were incubated at 37°c and examined twice daily for their viability. the allantoic fluids were harvested after 36 h incubation, and three blind passages were conducted. the presence of ibv was verified by reverse transcription-polymerase chain reaction (rt-pcr) of the n gene (zou et al., 2010) . the existence of other five pathogens, h9 subtype avian influenza virus (h9 aiv), newcastle disease virus (ndv), marek's disease virus (mdv), bacteria and coccidiosis in those samples were verified by following the methods of other reports (abu-akkada and awad, 2012; chen et al., 2012; li et al., 2010; rui et al., 2010; tian et al., 2011) . total rna was extracted from ibv-infected allantoic fluid with rnaiso plus (takara biotechnology co., ltd., dalian, china) according to the manufacturer's instructions and dissolved in 40 μl sterile diethylpyrocarbonate (depc)-treated water before being stored at − 70°c for further use. for the reverse transcription (rt) reaction, 5 μl of template rna, 2 μl of 5× rt mix, and 3 μl of rnase-free water were added and mixed. the reaction mixture was incubated at 37°c for 15 min and then at 85°c for 1 min. pcr amplification and cloning of the s1 gene was performed as the previous report (zou et al., 2010) . the recombinant plasmids containing the target gene were sequenced by shanghai sanggong biological engineering technology & services co., ltd. (shanghai, china). nucleotide sequences of the s1 gene obtained from the ibv isolates were aligned using the editseq program in the lasergene package (dnastar inc., madison, wi, usa) and compared to the sequences of 53 other reference ibvs using the megalign program in the same package. for the 53 reference ibvs, 43 strains were isolated from china, 2 strains were isolated from the usa, 2 strains were from japan, and the other 6 strains were vaccine strains. a phylogenetic tree of the s1 gene was created using the neighbor-joining method in mega version 7.0.14. bootstrap values were determined from 1000 replicates of the original data. the s1 subunit sequences of 24 ibv field strains and 53 reference strains were aligned by megalign, and putative recombinant strains were selected by sequence homology analysis. in order to identify the assumed parent sequences, the s1 subunit sequences of suspected recombinant isolates were blasted against the genbank database of the national center for biotechnology information (ncbi). recombination analysis of the selected sequences was conducted with the aid of recombination detection program (rdp 4.72) and simplot version 3.5.1 software. potential recombination events were identified using the rdp, maxchi, and geneconv methods in rdp 4.72 to identify putative parental sequences with significance set at p values b0.05 and the sliding window size set as 30 bp. putative potential recombination events were further identified using the simplot version 3.5.1. nucleotide identities were calculated using the kimura 2-parameter method with a transition-transversion ratio of 2 in each window of 200 bp, and the window was successively extended in 20-bp increments. for the preparation of antisera against the ten ibvs, 8-week old rabbits (n = 4) were immunized with purified 10 4 eid 50 ibvs subcutaneously mixed with an equal volume of complete freund's adjuvant (sigma, missouri, usa) for the first injection, and with the same antigen emulsified in freund's incomplete adjuvant for the following two booster injections (two-week interval). rabbits were held in separate biosafety level 2 (bsl2) isolators in the laboratory animal center of sichuan agricultural university (ya'an, sichuan, china) with a libitum access to feed and water and maintained under uniform standard management conditions. approval for these animal studies was obtained from the sichuan provincial laboratory animal management committee [permit number: xyxk(sichuan) 2014-187] and the ethics and animal welfare committee of sichuan agricultural university. antisera from vaccinated animals were collected at 12 days after the final immunization and stored at −20°c. to determine the antigenic relatedness between the field ibv isolates and the vaccine viral strains, double-direction viral cross-neutralization (vn) tests were performed in chicken embryo kidney (cek) cells using constant viral titers and diluted serum. the tested strains came from six different genotypes and included seven ibv field isolates (sczy3, ck/ch/scdy/141030, ck/ch/scls/140104, ck/ch/cqkx/ 150203, ck/ch/scyb/140913, ck/ch/scmy/10i, ck/ch/scyb/141102) and the three most commonly used vaccine viral strains (h120, m41, and 4/91). before vn testing, ibv strains were adapted to cek cells by serial passaging. briefly, allantoic fluid containing the ibv strain was propagated in monolayer primary cek cells prepared from 18-to 20-day-old chicken embryos. infected cek cells were cultured in dulbecco's modified eagle's medium (gibco, grand island, ny, usa) supplemented with 2% fetal bovine serum (zhejiang tian-hang biological technology stock co., ltd., zhejiang, china) and incubated at 37°c with 5% co 2 . the supernatant was harvested 40 h post-inoculation and passaged blindly in cek cells until a characteristic cytopathic effect (cpe), such as syncytia, was observed. determination of the tcid 50 of the cek-adapted ibvs in cek cells was conducted per the method of reed and muench (1938) . for the vn test, equal volumes of 100 tcid 50 of the cek-adapted ibvs and serial two-fold dilutions of antisera were mixed and kept at 37°c for 1 h. next, 0.4 ml of the virus-antisera mixture was then transferred to cek cell cultures in 24-well plates (6 wells for each dilution). the plates were incubated for 72 h, and the 50% end-point neutralizing titers were calculated by the method of reed and muench (1938) . negative rabbit serum was also incubated with 100 tcid 50 of ibv to calculate its non-specific neutralizing titer to ibvs, and this neutralizing titer was used as a background value for further analysis. the vn end-point titers were used to calculate the antigenic relatedness values (arv, r) by the method of archetti & horsfall (1950) :avrðrþ ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi r1 â r2 p â 100%, where r1 represents the ratio of the heterologous neutralizing titer of virus 1 to the homologous titer of virus 1, and r2 represents the ratio of the heterologous titer of virus 2 to the homologous titer of virus 2. isolates where r b 50% were considered to be antigenically unrelated, isolates with 50% ≤ r b 100% were considered to be antigenically related, and isolates with r ≥ 100% were considered to be antigenically identical. a total 86 clinical samples, including trachea, lung, and kidney samples, were collected from dead or diseased chickens displaying respiratory symptoms and/or nephritis from chicken flocks located in southwestern china. this included the si-chuan, yun-nan, gui-zhou, and chong-qing areas. from these, 24 ibv strains were isolated. typical signs of ibv, including embryo dwarfing and death, were observed during the passaging of samples through embryos. rt-pcrs of the clinical samples showed that only a few samples exhibited co-infection of the h9 aiv (2/24, 8.3%) or the ndv (1/24, 4.2%). bacterial isolation showed that e. coli and salmonella were often found in the clinical samples (6/24, 25.0%). the case histories of local strains are listed in table 1 . 3.2. phylogenetic analysis of s1 gene s1 gene sequences from the 24 ibv isolates were determined and submitted to genbank under the accession numbers ku364603-364620, ku516832, kx129907-129909 and kx344067-344068. the full-length s1 subunit gene open reading frame (orf) ranged from 1605 to 1626 bp. phylogenetic analysis based on s1 nucleotide sequences of the 24 wild strains showed that 16 of the 24 isolates could be grouped into four genotypes: qx, j2, tw (twi and twii), and mass. nine field isolates (ck/ch/scdy/141030, ck/ch/gzgy/150203, ck/ch/scms/140618, ck/ch/scmy/130118, ck/ch/scyb/130321, ck/ ch/scmy/140120, ck/ch/sclz/121120, ck/ch/scem/120312, and ck/ ch/scya/121223) were included in the qx group, sharing 94.0-99.9% nucleotide identity with the s1 sequences of other qx-like ibvs from pandemics in recent years. four isolates (ck/ch/scms/130101, ck/ch/ scyb/141102, ck/ch/scms/150527, and ck/ch/ynkm/160426) belonged to the tw type. among these, ck/ch/scyb/141102, ck/ch/ scms/150527, and ck/ch/ynkm/160426 belonged to the twi type, sharing 94.1-98.9% nucleotide identity with those of other twi-type reference strains; ck/ch/scms/130101 grouped with twii-type sequences, sharing 98.8-99.1% nucleotide identity with twii reference strains. two field isolates (ck/ch/gzlsy/160106 and ck/ch/scdy/ 160426) clustered with mass-type reference strains, sharing 96.3-99.7% nucleotide identity with mass reference strains. one isolate (ck/ ch/scls/140104) clustered with the j2 group, exhibiting 99.7-99.9% nucleotide identity with j2 reference strains. the phylogenetic tree is shown in fig. 1. recombination events in the s1 gene were identified using the rdp 4.72 and simplot3.5.1 software. simplot results were similar with rdp (date not shown). among the 24 field isolates, a total of eight recombinant strains were found (fig. 2) and were mainly clustered into four groups, termed variant-1, variant-2, variant-3, and variant-4 ( fig. 1) in the phylogenetic tree. the variant-1 group contained two isolates (ck/ch/scmy/121225 and ck/ch/scbz/130423), variant-2 contained four isolates (ck/ch/scyb/140913, ck/ch/cqkx/150203, ck/ch/gzxf/ 151223, and ck/ch/scem/160318), and the variant-3 and variant-4 group contained only one isolate each ck/ch/sctq/120718 and ck/ ch/gzlxm/160106, respectively. in the variant-1 group, the nucleotide sequences located at nucleotide positions 1-915 of strains ck/ch/scmy/121225 and ck/ch/scbz/ 130423 showed high identity to the sequences of the qx strains ck/ ch/lhlj/110949 (98.0%) and ck/ch/lhb/100908 (99.6%), respectively. the nucleotide sequences located at 916-1620 nt of both strains, however, exhibited 99.0% identity with that of tw2296/95 (twii type). the breakpoint site in both strains was located at 915 nt in the s1 subunit gene. in the variant-2 group, nucleotide sequences located at 1-716 nt of the four isolates ck/ch/scyb/140913, ck/ch/cqkx/150203, ck/ch/ gzxf/151223, and ck/ch/scem/160318 exhibited 99.2-99.6% identity with qx-type strain lc2, while nucleotide sequences located at 717-1620 nt of the four isolates shared 98.3-98.6% identity with those of ck/ch/lsc/99i-type strain saibk. the breakpoint site sequence in these four ibvs was located at 760 nt in the s1 subunit gene. for the variant-3 strain ck/ch/sctq/120718, positions 1-738 nt exhibited 97.7% identity with those of ck/ch/lsc/99i-type strain ck/ch/ guangxi/hezhou/0903, while positions 739-1626 nt exhibited 95.0% identity with that of 4/91-type strain ck/ch/ljl/130925. the breakpoint site was at position 703 nt in the s1 subunit gene. finally, for the variant-4 strain ck/ch/gzlxm/160116, positions 1-1402 nt exhibited 99.4% identity with sequences from the vaccine strain h120, while positions 1403-1611 exhibited 98.1% identity with sequences from the qx-type strain ck/ch/gd/ly12. the breakpoint site was located at 1396 nt in the s1 subunit gene. further analysis the sequences near the breakpoint site, revealed a new potential, a-t rich hotspot sequence, atttt(t/a), which was found near the breakpoint site of the s1 subunit gene in all of the variant-1, variant-2, variant-3 and variant-4 groups (fig. 3) . the antigenicities of representative isolates from different genotypes were assessed by viral cross-neutralization assays (tables 2 and 3 ). ten ibvs, including seven field strains from different genotype backgrounds and three commonly used vaccine strains (h120, 4/91, and m41), were analyzed and grouped into four serotypes: massachusetts (mass hereafter), 793b, sczy3, and scyb. the commonly used vaccines h120 and 4/ 91 had no cross-neutralization titers and were used as reference serotypes. four ibvsincluding the vaccine strain h120, field isolates ck/ ch/scdy/141030 (qx) and ck/ch/scls/140104 (j2), and the standard virulent strain m41, commonly used in inactivated vaccine production (table 4) . many ibvs, including six field strains and one vaccine strain (4/91) were included in different serotypes: ck/ch/scdy/141030 (qx) was included in both the mass and sczy3 serotypes, ck/ch/scls/140104 and m41 were included in all serotypes except scyb, ck/ch/scms/130101 and ck/ch/cqkx/150203 (variant-2) were included in both 793b and sczy3 serotypes, ck/ch/scyb/141102 was included in all serotypes except mass, ck/ch/scyb/140913 and 4/91 were included in both 793b and scyb serotypes. only h120 and the sczy3 standard were included in a single serotype (table 4 ). in the unidirectional neutralization assay of vaccine-associated strains, immune sera against h120 could only neutralize three ibvs from three different genotypes, while immune sera against 4/91 were able to neutralize six ibvs from five different genotypes. although h120 and m41 belong to the same serotype, immune sera against m41 neutralized ibvs from six different genotypes, and the neutralizing titers were higher than those of h120 in most cases. the neutralization abilities of antisera against 4/91 and m41 were higher than that against h120 (table 2 ). in terms of the unidirectional neutralization assays of the seven field strains, immune sera of ck/ch/scdy/141030, ck/ch/scls/140104, and ck/ch/cqkx/150203 neutralized all ten of the analyzed strains. immune sera of ck/ch/scyb/140913 and ck/ch/scyb/140913 neutralized all of the analyzed strains except h120. immune sera of ck/ch/scms/ 130101 and ck/ch/scyb/141102 could neutralize all strains with the exception of one field strain. the neutralization abilities of antisera against most of the field strains were almost identical to that of 4/91, which was higher than that of vaccine strain h120 (table 2) . for cross-neutralization assay, most analyzed ibvs displayed bidirectional neutralization activity, but several ibvs displayed unidirectional neutralization activity. for example, immune sera against ck/ch/ cqkx/150203, ck/ch/scmy/130101, and ck/ch/scyb/141102 neutralized h120 well, but immune sera against h120 did not react well with these three field strains (table 2) . although most strains belonging to the same genotype also belonged to the same serotype (e.g. h120 and m41, sczy3 and ck/ch/ scdy/141030) some ibvs in the same genotype exhibited low antigenic relatedness values, such as ck/ch/scyb/140913 and ck/ch/cqkx/ 150203 from the variant-2 group. in contrast, ibvs from different genotype groups sometimes also exhibited high antigenic relatedness values (r ≥ 100%), such as h120 (mass) and ck/ch/scls/140104 (j2); 4/91 (4/ fig. 1 . phylogenetic analysis of the s1 gene from 24 wild strains (filled triangles) and 53 reference strains of infectious bronchitis virus (ibv), starting at the aug translation initiation codon and ending at the cleavage recognition motifs. the phylogenetic tree was constructed using mega version 7.0.14 with the neighbor-joining method and 1000 bootstrap replicates. 91) and ck/ch/cqkx/150203 (variant-2); and 4/91 (4/91) and ck/ch/ scms/130101 (tw) ( table 4) . in recent years, outbreaks of ib have been reported frequently in southwestern china zou et al., 2010) . although the mortality rate of a single infection has been low, it could increase as a consequence of secondary infections or co-infection with e. coli, aiv, and ndv (dwars et al., 2009; hassan et al., 2016) . in our study, rates of co-infection of ibv and bacteria accounted for 25.0% of total cases. control of co-infections or secondary infections with other pathogens are very important for the prevention and control of ib (sid et al., 2015; smith et al., 1985) . the molecular characteristics of ibvs vary around the world. over the past ten years, epidemics of different genotypes have been observed in different countries or areas, such as the variant-2 (is/1494) and mass types in asia (chen et al., 2015; patel et al., 2015; promkuntod et al., 2015; seger et al., 2016; xu et al., 2016b; zou et al., 2010) ; the mass and 793b types in the middle east (ganapathy et al., 2015; hosseini et al., 2015; najafi et al., 2016) ; the mass, qx, and italy-2 types in africa (fellahi et al., 2015a; fellahi et al., 2015b; knoetze et al., 2014) ; the 4/ 91, qx, and italy-2 types in europe (kiss et al., 2015; krapez et al., 2011) ; and the mass type in south america (balestrin et al., 2014) and north america (mondal et al., 2013) . the major genotype circulating around the world, however, is the qx type. in our study, 24 ibvs were isolated from h120-and 4/91-vaccinated chickens. phylogenetic analysis of the s1 gene showed that those 24 ibvs could be primarily grouped into four genotypes, with qx-type strains (9/22) dominating. many scholars have recently prepared qx vaccine strains for use as a candidate vaccine in china (huo et al., 2016; zhao et al., 2015) , but there is still no official approval for field application of a qx-like vaccine in china. considering the possibility of recombination between vaccine strains or between vaccine strain and field strain to generate variants, field application of new attenuated live vaccine such as qx-like vaccine should be carefully assessed before implementation. in contrast to qx-type epidemics, epidemics of the tw type (twi and twii) and variant-2 type have been increasing in recent years (mo et al., 2013; xu et al., 2016a) , a fact that was also confirmed by this study. most scholars classify variant-2 strains into the ck/ch/lsc/ 99itype or saibk type, but our rdp and simplot analysis showed that variant-2 strains are the result of a recombination event between a qx-type and a ck/ch/lsc/99i-type (saibk-type) strain. both the twi and twii type were first identified in taiwan, and the isolation of twi-type strains in china has increased in recent years (fu-yan, 2013; zhang et al., 2016) , while twii-type strains were first identified in china in 2012 . in our study, we not only identified twii-type strains among our samples, but we also discovered recombinant ibvs originating from qx-type and twii-type strains. how twii and associated recombinant strains appeared in flocks from southwestern china remains unknown. measures should be taken to prevent the evolution of new variants and recombinant variants, such as strengthening supervision of the poultry trade, limiting the number and type of live vaccine strains, and increasing the biosecurity level of chicken farms. the s1 glycoprotein carries most of the neutralizing epitopes in the ibv genome, and the s1 subunit gene is highly variable. gene mutations may be introduced into the viral genome by viral rna-dependent rna polymerases, which display incomplete proofreading capabilities (denison et al., 2011 (denison et al., 2011 , and gene recombination can occur via a genomic template switching mechanism (lai, 1992) . the homology sequences between the donor and acceptor genome are usually required for the copy-choice of homologous recombination, and the rna secondary structure such as hairpin structure could also influence the recombination process (lai, 1992; nagy et al., 1998) . mutation and recombination of the s1 gene may lead to the emergence of new variants, genotypes, or serotypes . in this study, we found that mutations in the s1 subunit gene were mainly located in the three hyper-variable regions (hvr) (data not shown), which is consistent with our previous study (zou et al., 2010) . for the recombination occurred in the s1 gene, previous studies have demonstrated that most crossover occur at a relatively conserved sequence near the hvr (wang et al., 1994) . however, this was not the case for three of the four putative recombination events detected in this study. the breakpoint site in the variant-1 group was located at a conserved sequence in the hvr, while that in the variant-3 group was located in a variable region outside of the hvr. recombination hotspots are generally believed to be located adjacent to putative breakpoint sites, ct(t/ g)aacaa, cttttg, and cttttg(c/t) are usually considered to be potential hotspot sequences. in this study, the hotspot sequences above were not observed near the breakpoint sites, but a potentially new, a-t rich hotspot sequences, atttt(t/a), was observed near the breakpoint sites of the s1 subunit gene. sequence analysis of recombinant ibvs in other reports (mo et al., 2013; thor et al., 2011) showed that atttt(t/a) was also observed near the breakpoint sites in some of the strains (date not shown). previous report had shown that aurich sequence could be the important recombination-promoting signals of brome mosaic virus (bmv) (shapka and nagy, 2004) . to determine the antigenic relatedness between field strains and vaccine strains in southwestern china, viral cross-neutralization tests were performed. as most ibv isolates do not produce significant cpes in cek cells, ibvs should be allowed to adapt to cek cells before neutralization assays. in this study, h120, 4/91, and m41 were used as vaccine controls, as they represent the most commonly used vaccine strains. results of the cross-neutralization assay showed that all of the analyzed strains could be grouped into four serotypes: mass, 793b, sczy3, and scyb. the cross-neutralization ability of 4/91 was higher than that of h120, which may explain why the immunogenicity of 4/91 is higher than that of h120 in field applications. however, the antigenic relatedness between some field strains and 4/91 was also low, which may explain immune failure in flocks vaccinated with 4/91. sczy3 and scyb were two serotypes that differed from that of the h120 and 4/91 vaccine, and the cross-neutralization abilities of strains in the sczy3 serotype were higher than those of strains in the scyb serotype. representative strains in the sczy3 serotype may therefore be more suitable for vaccine development than those in the scyb serotype. in general, there is a correlation between s1 gene homology and the level of cross-protection between strains, with strains in the same serotype usually sharing n95% amino acid identity and strains from different serotypes sharing b 85% amino acid identity (cavanagh, 2005) . however, in this study, some strains with high amino acid identity (99.4%), such as ck/ch/scyb/140913 and ck/ch/cqkx/150203, exhibited low antigenic relatedness, corroborating a previous study that showed that strains with low amino acid identity could also display high antigenic relatedness (sjaak de wit et al., 2011) . it is unclear which amino acids play key roles in determining the serotype of ibvs, and genetic analysis could not be used to evaluate antigenic differences between ibvs. furthermore, we found that some ibv strains, such as ck/ch/scdy/ 141030, could be grouped into more than one serotype, similar to the results of other reports (cowen and hitchner, 1975; winterfield and fadly, 1972) . this phenomenon may be explained by the existence of common vn epitopes present in different ibv genes. for example, in ck/ch/scdy/141030 and sczy3, two common vn epitopes (87ppqgmaw93 and 412iqtrtep418) (zou et al., 2015) were observed in the s1 genes of both strains. although the five vn epitopes in the hvrs were quite different between strains ck/ch/scdy/141030 and h120, other common vn epitopes may be located in the s2 and n proteins of these two strains, as there are reports that the s2 and n proteins may induce neutralizing reactions (ignjatovic and sapats, 2005; koch et al., 1990) . in conclusion, we have demonstrated that the genetic and antigenic characteristics of ibvs isolated from southwestern china have undergone some changes in recent years. our results provide a reference for the prevention and control of ib in southwestern china. strains with r ≥ 50% were classified as the same serotype, and strains with r b 50% were classified as different serotypes. isolation, propagation, identification and comparative pathogenicity of five egyptian field strains of eimeria tenella from broiler chickens in five different provinces in egypt persistent antigenic 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viral replicase molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in iran isolation and molecular characterization of nephropathic infectious bronchitis virus isolates of gujarat state analysis of the s1 gene of the avian infectious bronchitis virus (ibv) reveals changes in the ibv genetic groups circulating in southern thailand genotypes and serotypes of avian infectious bronchitis viruses isolated during a simple method of estimating fifty percent endpoints phylogenetic characterization of newcastle disease virus isolated in the mainland of china during genotyping of infectious bronchitis viruses from broiler farms in iraq during the au-rich rna recombination hot spot sequence of brome mosaic virus is functional in tombusviruses: implications for the mechanism of rna recombination co-infection with multiple respiratory pathogens contributes to increased mortality rates in algerian poultry flocks infectious bronchitis virus variants: a review of the history, current situation and control measures the experimental infection of chickens with mixtures of infectious bronchitis virus and escherichia coli recombination in avian gamma-coronavirus infectious bronchitis virus comparative analysis of oncogenic genes revealed unique evolutionary features of field marek's disease virus prevalent in recent years in china incorporation of spike and membrane glycoproteins into coronavirus virions relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus some characteristics of isolates of infectious bronchitis virus from commercial vaccines characterization and analysis of an infectious bronchitis virus strain isolated from southern china in 2013 emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks serotype shift of a 793/b genotype infectious bronchitis coronavirus by natural recombination molecular detection and smoothing spline clustering of the ibv strains detected in china during safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine genetic analysis revealed lx4 genotype strains of avian infectious bronchitis virus became predominant in recent years in sichuan area two novel neutralizing antigenic epitopes of the s1 subunit protein of a qx-like avian infectious bronchitis virus strain sczy3 as revealed using a phage display peptide library this work was financially supported by the program for chang-jiang scholars and innovative research team in university "pcsirt" (grant no. irt0848). key: cord-300104-855iw9wi authors: hennion, ruth m.; hill, gillian title: the preparation of chicken kidney cell cultures for virus propagation date: 2014-12-18 journal: coronaviruses doi: 10.1007/978-1-4939-2438-7_6 sha: doc_id: 300104 cord_uid: 855iw9wi chicken kidney (ck) cell cultures have historically proved useful for the assay of a number of viruses including coronaviruses. a technique for the preparation of such cell cultures, using a combination of manual and trypsin disaggregation of kidneys dissected from 2to 3-week-old birds is described. this technique routinely gives high cell yield together with high viability and the resultant adherent primary cultures can be used for virus growth and plaque formation. techniques for the preparation of monolayer cultures from adult kidney cells suitable for the growth and quantitation of viruses have been available for many years; dulbecco and vogt [ 1 ] described the preparation of monkey kidney cultures in 1953 and youngner [ 2 ] published a modifi cation of the process in 1954. maassab in 1959 [ 3 ] describes the preparation of chicken kidney monolayer cultures from 4-to 5-day-old chicks, the cultures being used for studies with some human viruses and churchill [ 4 ] reports the use of chicken kidney tissue cultures derived from 3 to 8 week old chickens in the study of avian viruses including infectious bronchitis virus (ibv). the technique for the production of kidney cell monolayer cultures from young birds, as described here, is adapted from those published by dulbecco and vogt [ 1 ] , and youngner [ 2 ] for monkey kidney cells. whilst titration of ibv in ck cells gives lower titers than those obtained in embryonated eggs [ 5 ] or tracheal organ cultures [ 6 ], the ability of ck cells to support the growth of many strains of ibv is well proven. following adaptation in embryonated eggs, the beaudette strain of ibv produced characteristic cytopathic effects (cpe) on fi rst passage in ck cells, whilst the massachusetts strain produced cpe in the second ck passage [ 4 ] . cpe consists of syncytia formation which occurs at 6 h post inoculation with the beaudette strain [ 7 ] . the syncytia may contain as many as 20-40 or more nuclei and they quickly round up and detach from the culture surface. growth curves of ibv in ck cells show a lag phase of 2-4 h and maximum virus yield in 18-20 h [ 5 ] . the ability of ck cells to support the growth of ibv has been utilized in wide ranging studies including the assessment of ph stability of a series of ibv strains [ 8 ], the identifi cation of the presence of a leader sequence on ibv mrna a [ 9 ], the demonstration that the spike protein of ibv is a determinant of cell tropism [ 10 ], the induction of protective immunity with recombinant ibv beaudette [ 11 ] through to the identifi cation of novel zippered er and associated spherules induced by ibv [ 12 ]. week old chicken(s) from specifi c pathogen free (spf) fl ock killed by cervical dislocation ( see note 1 ). 13. growth medium: emem, 10 % nbbs, 2.95 g/l tpb, 10 mm hepes, 100 u/ml penicillin and 100 u/ml streptomycin. the number of kidney cells obtained from each bird will vary with the age and the strain of the birds used. we have found that the average cell yield from a 2 week old rhode island red bird is approximately 2.0 × 10 8 cells. 1. aseptically prepare disaggregation mix by addition of 7.5 ml of trypsin to 80 ml of edta and warm to 37 °c (trypsin/ edta). 2. add 50-100 ml nbbs to suitable sterile fl ask ( see note 5 ). 3. spray work area with ims, protect with clean paper towels and collect sterile instruments and glassware so that they are close to hand. 4. spray the back of the birds and under the wings with ims to clean and dampen the feathers and lay the bird, dorsal side uppermost, on the paper towels. 5. insert the blade of a large, robust pair of scissors just below where a wing attaches to the body and sever across the body, through the spinal cord to where the second wing attaches, taking care to avoid piercing the gut. 6. from the ends of this fi rst cut, once again taking care to avoid piercing the gut and your own hand, cut along each side of the body towards the legs and through the top of each leg. 7. carefully fold back the cut section of the bird to reveal the internal organs. move the intestines, which should have remained attached to this retracted section, to reveal the kidneys. 8. remove the kidneys using a small pair of scissors and forceps and place in a 250 ml beaker of pbsa. 9. repeat steps 4 -8 for every bird. 10. when all the kidneys required have been removed from the birds, agitate them in the beaker and discard the pbsa. repeat this process until the wash pbsa looks clear ( see note 6 ). 11. tip the drained kidneys into a large glass petri dish and using two scalpels shred and mince the kidneys into very small pieces removing as much clotted blood, connective tissue, and kidney core as possible. 12. transfer the minced tissue into a tightly capped bottle (for example a 150 ml medical fl at) and wash with approximately 80 ml of pbsa until the supernatant runs clear, allowing the tissue fragments to settle for 1 min in between the washes and discarding the pbsa washes ( see note 6 ). 13. add 50-80 ml trypsin/edta to drained tissue and shake moderately hard for 2 min. allow the tissue to settle and discard the supernatant ( see note 6 ). 14. add another volume of trypsin/edta and shake for 4 min. allow the tissue to settle and this time pour the supernatant into the conical fl ask containing nbbs. gently swirl the fl ask to distribute the isolated cells in the nbbs. 15. repeat step 14 until no more tissue remains ( see note 7 ). step 14 through the metal gauze fi lter supported in a funnel placed in a fresh conical fl ask ( see note 8 ). decant fi ltered cells into centrifuge tube(s) and centrifuge at approximately 300 × g for 10 min to pellet the cells. 1. warm growth medium to 37 °c. 2. working in a microbiological safety cabinet (class 2), carefully discard the supernatant from the centrifuge tubes and resuspend the pelleted cells in growth medium, triturating at least fi ve times. 3. using a 50 ml syringe pass the cell suspension through swinnex fi lter 1 then through swinnex fi lter 2 collecting the fi ltrates from fi lter 1 and 2 in a fresh fl ask each time ( see note 8 ). 4. measure and record the volume. take 0.1 ml of cell suspension add to 0.9 ml of trypan blue and count the viable cells ( see note 9 ). 5. dilute cell suspension in growth medium to the cell concentration required, seed culture fl asks and place in incubator until intact monolayer forms ( see note 10 ). 1. this should be done immediately prior to the removal of the kidneys to minimize the buildup of blood clots. 2. wire mesh is obtainable from locker wire weavers, www. wiremesh.co.uk . 3. as one batch of serum may not support the growth of ck cells as well as another, we recommend batch testing of serum prior to purchase and use. 5. the volume of nbbs, which is used to inactivate the trypsin/ edta, will depend on the number of birds. use at least 50 ml for up to 6 birds and increase the volume by 10 ml for each extra bird. the fl ask volume should be at least 500 ml. 6. whilst some kidney cells may be lost in this process, it is an effective way of removing many of the red blood cells that are still present at this stage of the preparation. 7. this may require 6-8 repeats. 8. this process helps to remove some of the larger aggregates of cells that remain in the preparation at this stage. removing the aggregates makes the cell counting process more accurate and the resultant monolayers more evenly dispersed. 9. there will still be a number of red blood cells at this stage. these should be excluded from the cell count. the red blood cells can be distinguished from other kidney cells by their size and shape. 10. for plaque assays and viral growth seed fl asks at approximately 0.3 × 10 6 /cm 2 and incubate for 72 h prior to virus introduction. plaque formation and isolation of pure lines with poliomyelitis viruses monolayer tissue cultures. 1. preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells the propagation of multiple viruses in chick kidney cultures the use of chicken kidney tissue culture in the study of the avian viruses of newcastle disease, infectious laryngo tracheitis and infectious bronchitis key: cord-271897-9oqzsd70 authors: domanska-blicharz, katarzyna; lisowska, anna; sajewicz-krukowska, joanna title: molecular epidemiology of infectious bronchitis virus in poland from 1980 to 2017 date: 2020-01-07 journal: infect genet evol doi: 10.1016/j.meegid.2020.104177 sha: doc_id: 271897 cord_uid: 9oqzsd70 the presence of infectious bronchitis virus (ibv) was identified for the first time in the poultry population in poland at the end of the 1960s. from this time a few waves of epidemics caused by different ibv variants spread across the country. in order to gain more insight into the molecular epidemiology of ibv in poland, in the present study the s1 coding region of 34 ibv isolates and nearly whole genome of 10 strains collected over a period of 38 years was characterized. phylogenetic analysis showed that these strains belonged to five recently established ibv lineages: gi-1, gi-12, gi-13, gi-19 and gi-23. additionally, two strains from 1989 and 1997 formed a separate branch of the phylogenetic tree categorized as unique early polish variants, and one strain was revealed to be the recombinant of these and gi-1 lineage viruses. irrespective of year of isolation and s1-dependent genotype, the genome sequences of polish ibv strains showed the presence of six genes and 13 orfs: 5′utr-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′utr, however their individual genes and putative proteins had different lengths. the phylogenetic analyses performed on the genome of ten polish ibv strains revealed that they cluster into different groups. the polish gi-1, gi-19 and gi-23 strains cluster with other similar viruses of these lineages, with the exception of the two strains from 1989 and 1997 which are different. it seems that in poland in the 1980s and 1990s ibv strains with a unique genome backbone circulated in the field, which were then replaced by other strains belonging to other ibv lineages with a genome backbone specific to these lineages. the recombination analysis showed that some polish strains resulted from a recombination event involving different ibv lineages, most frequently gi-13 and gi-19. infectious bronchitis virus (ibv) is the etiological agent of a highly contagious disease of chickens known as infectious bronchitis, but the virus can replicate in epithelial cells of different organs, also affecting the urogenital or digestive tracts beside the respiratory tract (cavanagh, 2005 (cavanagh, , 2007 . together with genetically similar viruses isolated from other domesticated galliformes, ibv belongs to the igacovirus subgenus within the gammacoronavirus genus (nidovirales order, cornidovirinae suborder, coronaviridae family, orthocoronavirinae subfamily). the nonavian sw1 gammacoronavirus isolated from beluga whales was recently assigned to the separate cegacovirus subgenus (dong et al., 2007; king et al., 2018) . the virus genome is an approximately 27 kb long single-stranded, positive-sense rna consisting of several open reading frames (orfs). two thirds of the genome in the 5′ end are occupied by two overlapping orfs encoding viral rna-dependent rna polymerase. the 1a and 1b orfs encode 15 non-structural polypeptides (nsp2-16) which are associated with rna replication and transcription. in the 3′ end are genes that among other products encode the four major structural proteins: spike (s), envelope (e), matrix (m), and nucleocapsid (n). the s glycoprotein is post-translationally cleaved into s1 and s2 subunits of about 534 and 627 amino acids during viral maturation. the s2 subunit anchors the spike into the virus membrane whereas s1 forms the extracellular part of the spike and plays a major role in tissue tropism and induction of protective immunity (cavanagh and gelb, 2008) . ibv undergoes many genetic changes generated both by recombinations and mutations such as substitutions, deletions and insertions, which could lead to the emergence of new variants. among factors that create favorable conditions for such events are characteristic features of coronaviruses in the genome structure (large singlestranded rna) and virus biology (minimal proofreading activity of viral polymerase) and modern poultry-rearing habits and immunological pressure caused by the worldwide use of vaccines (ovchinnikova et al., 2011; woo et al., 2009) . mutations within the s1 gene particularly result in new geno-or serotypes, and currently there are many such types around the world (de wit et al., 2011) . their number, diversity and naming and the plurality of methods used for their determination for years have caused much confusion. to avoid it, new classification rules based on the whole s1 gene phylogeny (about 1600 nt) and new nomenclature have been proposed. this system distinguished and named 32 lineages, aggregating into 6 genotypes (gi to gvi) (valastro et al., 2016) . however, in the last three years, two more lineages (gi-28 and 29) and even one more genotype (gvii) have been described in china jiang et al., 2017; ma et al., 2019) . in poland, the first suspicion of ib was based on clinical observations, as respiratory symptoms incurable with antibiotics in some flocks and/or misshapen eggs from commercial flocks came to notice. laboratory confirmation of ibv infection was obtained at the end of the 1960s. between 1964 and 1965, 4165 sera from two hundred ten chicken flocks at the age of 6-18 months were examined in an agar gel precipitation test and only 13% of them were positive although 79% of flocks contained birds with positive serum (karczewski and cakala, 1967) . outbreaks of ib with respiratory signs and a drop in egg production and egg quality in non-vaccinated breeding and particularly laying chicken flocks were recorded in the mid-1980s (bugajak et al., 1997) . since the mid-1990s, outbreaks of ib-nephritis have been reported in broiler flocks (minta et al., 2000) . a multiplex-pcr testing strains isolated between 1997 and 1998 revealed that the most of them belonged to the 793b type (capua et al., 1999) . the emergence of qx ibv was detected in 2004 (domanska-blicharz et al., 2006; domanska-blicharz et al., 2007) . more recently, the next variant of ibv called var2 which had been circulating only in the middle-east region for the previous 20 years was also detected in poland . in this study we attempted to molecularly characterize the field ibv strains detected in poland during the period between 1980 and 2017. strain determination was accomplished by phylogenetic analysis of the full s1 coding region sequences against reference strains representing all genotypes and lineages recently described (valastro et al., 2016) . additionally, we also analyzed the complete genome sequences of ten polish ib viruses. recombination analysis was also performed using the obtained sequences of these strains. thirty four field ibv strains isolated between 1980 and 2017 in poland were included in the study. these strains originated from poultry experiencing clinical forms of the disease as respiratory or enteric symptoms, nephritis, or problems with egg production. epidemiological information of the studied isolates is summarised in table 1 . the samples were named to fulfill the previously described criteria, but to make it easier to follow the results of the analysis, in subsequent parts of the text they were shortened to the individual symbol given in the laboratory and the year of identification (ducatez, 2016) . the earliest virus materials from the 1980s were available in the form of a lyophylizate of allantoic fluids from commercial chicken eggs. after propagation in spf embryos, materials from the 1990s were in the form of allantoic fluids stored deep frozen. field materials delivered to the department of poultry diseases for diagnostic purposes between 2004 and 2017 were isolated in specific pathogen-free (spf) chicken eggs as described previously (gelb and jackwood, 1998) . virus genome presence confirmation and genotype determination preceded the spf egg isolation. materials from 1980 to 1998 as referred to above were also refreshed using the virus isolation method on spf embryonating eggs. harvested allantoic fluids were processed using an rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's recommended procedure for rna extraction, and isolated rna was stored at −70°c until analysis. the rt-pcrs were conducted on the one-step model using the one step rt-pcr kit (qiagen, hilden, germany) according to the manufacturer's instructions. various combinations of primer pairs described recently as well as additional primers specifically constructed for some strains (appendix a supplementary material) were applied for amplification and sequencing of the whole s1 coding region (binns et al., 1986; boursnell et al., 1987; dolz et al., 2006 dolz et al., , 2008 lisowska et al., 2017; worthington et al., 2008) . the reactions were run according to the recommended protocol for the kit with different annealing temperatures depending on the melting temperature of the primer pair used. amplified pcr products were visualized by electrophoresis on a 2% agarose gel stained with ethidium bromide and then purified using a qiaquick gel extraction kit (qiagen, hilden, germany). typically, for the s1 coding region of polish ibv strains, 3-7 pcr products were sequenced in both directions using sanger sequencing technology by genomed (warsaw, poland). the complete genomes of these ibv strains were generated using illumina miseq technology (illumina, san diego, usa) in several laboratories. the five ibv strains 78/1989, 79/1989, 548/2004, g195/2012 and g103/2016 were processed in the department of microbiology of the swedish national veterinary institute (sva, uppsala, sweden), four subsequent virus strains 80/1989, 162/ 1997, 255/1997 and g225/2017 were analyzed in the department of omics analysis in our institute, and one ibv strain g074/2009 was sequenced by genomed (warsaw, poland). analyses in these organizations were made according to the standard procedure. briefly, rna extracted directly from the allantoid fluid was retrotranscribed into dna using a superscript iv first-strand cdna synthesis kit (invitrogen, waltham, usa) and the second strand was synthesized with the addition of klenow polymerase (new england biolabs, ipswich, usa). a 300 bp-long paired-end dna library was prepared using a nextera xt sample preparation kit (illumina, san diego, usa) and sequencing was performed using a miseq reagent kit v3 (illumina, san diego, usa). sequences of s1 coding region fragments obtained by sanger sequencing were trimmed based on quality and assembled into consensus sequences using geneious v11.1.3 (biomatters, auckland, new zealand). sequences of polish viruses were searched with blast (basic local alignment search tool) to find these ones with the highest similarity and include them in the phylogenetic analyses. then the full s1 sequences were aligned with 199 sequences representing 32 lineages in their 6 ibv genotype groupings and 26 unique variants as valastro et al. recommended (valastro et al., 2016) using clustal w. additionally, eight sequences representing the two newly identified gi-28 and gi-29 lineages and one gvii-1 genotype were included in the analysis. sequencing data from miseq technology obtained from the sva (uppsala, sweden) and genomed were processed with the clc genomics workbench (qiagen, hilden, germany). the reads obtained in the department of omics analysis of our institute were assembled into contigs with the spades assembler using the website at http://spades.bioinf. spbau.ru (bankevich et al., 2012) . for phylogeny of the complete genomes of the polish ibv strains, a preliminary analysis was carried out using all 362 gammacoronaviruses available in the niaid virus pathogen database and analysis resource (vipr) through the website at http://www.viprbrc.org/ (pickett et al., 2012) . next, 56 strains were selected for further analysis, taking into account their clustering in the vipr analysis. alignments of nucleotide sequences were performed using the multiple alignment using fast fourier transform (mafft) method in geneious software, v11.1.3 (biomatters, auckland, new zealand). the alignments were then exported to the mega program, v7.0.26 (tamura et al., 2013) . maximum likelihood (ml) phylogenetic analyses of the s1 coding region and of the complete genome were then conducted using the best-fitting nucleotide substitution models (the lowest bayesian information criterion (bic) scores in each analysis were for the general time reversible (gtr) model and a discrete gamma distribution (+g) with five rate categories, assuming that a certain fraction of sites are evolutionarily invariable (g + i)). bootstrap analyses of the resultant trees were performed using 1000 replicates. to detect any recombination events in the analyzed sequences, rdp4 software v4.97 was used (martin et al., 2017) . the full s1 coding region sequences of 241 ibv strains were screened to check if unusual clusters formed by polish ibv strains are viruses representing real new ibv lineage or recombinants. ten full genomes of polish ibv strains were also analyzed for recombination events using the complete genomes of 56 representative viruses selected for analysis as described above. the rdp4 analysis was accomplished using different available methods with their default parameters, however recombination events were only considered proven if detected by at least seven programs (rdp, geneconv, bootscan, maxchi, chimaera, siscan and 3seq) and the p-value was calculated at below 1.0 × 10e−30. full s1 sequences of the 34 analyzed polish ibv isolates as well as complete genomes of ten of them were submitted to the genbank database and accession numbers were assigned as given in table 1 . the nearly full genome sequences of ten ibv strains were obtained with the 5′ and 3′utr fragments incomplete. in all genomes the analysis predicted 6 genes consisting of 13 open reading frames (orfs) with a typical order for ibv of 5′utr-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′utr, but with their individual genes and putative proteins having different lengths ( table 2 ). the orfs/proteins with a constant conservative amount of nt and amino acids (aa) were 3a (174 nt/57 aa), 5b (249 nt/82 aa), and n (1230 nt/409 aa). the next orfs/proteins of conservative length were 4b, 4c and 5a counting 285 nt/94 aa, 171 nt/ 56 aa and 198 nt/65 aa respectively in 8 strains, whereas in two ibvs each of these structures was different (longer or shorter by one nt codon/aa). the difference in orf/protein length of accessory 3b protein was also slight as it fell within 192-195 nt/63-64 aa. the most diverse in terms of length was the orf coding e protein, ranging from 282 to 285 nt (93-94 aa) in polish gi-13 and gi-23 ibv strains to 330-333 nt (109-110 aa) in polish gi-19 ibvs. a similar relationship was also observed in the case of the orf encoding m protein, which was the shortest (627-681 nt/223-226 aa) in ibv strains of gi-13 and g-23 lineages and the longest (777 nt/258 aa) in ibvs of gi-19 lineage. the orf of the s protein was of varying lengths from 3462 nt/1153 aa to 3510 nt/1169 aa and did not show any dependence for length on identified ibv lineages. the molecular relatedness extents of the compete genome and the individual orfs between polish and selected ibv strains were 81-100% (appendix b1-b16 supplementary material). genotyping based on phylogenetic analysis of full s1 coding region sequences of 34 polish ibv strains from the years 1980-2017 grouped (caption on next page) k. domanska-blicharz, et al. infection, genetics and evolution 80 (2020) 104177 them into six groups: five distinct, previously known lineages and an additional new one (fig. 1) . five isolates comprised of four early ones from the 1980s and one identified in 2011 were assigned to the gi-1 lineage. one strain identified in 2017 affiliated to the gi-12 lineage. the group of gi-13 lineage contained eight ibv strains: two from the late 1990s isolated between 1997 and 1998, two identified between 2004 and 2005, and four strains isolated after 2010. the gi-19 lineage comprised ten strains detected between 2004 and 2014 and the group of gi-23 lineage held eight isolates detected between 2015 and 2017. two isolates, 162/1997 and 80/1989, were in the separate cluster designated early polish on the phylogenetic tree. sequence analysis revealed that five polish gi-1 strains shared nucleotide identities of 88.4-100% and formed two clades. in one clade were the dutch h120, north american, south african, indian and four polish isolates but the strain 81/1989 formed a distinct branch in this gi-1 subtree with low nucleotide identity of 86.2-88.1% to the rest of this group. the analysis of the s1 coding region sequences of eight polish isolates of gi-13 lineage showed that four strains formed a common branch, sharing 98.8-99.4% nt identity with the 7/91 strain from the united kingdom, whereas two earlier strains from 2004 and 2005 which were similar to each other with 99.3% identity form a sister group with the more recent viruses and had 97.9-98.2% nt similarity with the 7/91 strain. the earliest polish gi-13 strains from 1997 and 1998 were visibly different and had nt identity of 93.9-96.1% to the rest of the g-13 ibv strains. one of them, strain 58/1998, occupied positions close to the israeli variant 1 strain from 1996 with identity of 96.1%, and its similarity to the moroccan gi-13 lineage prototype g strain from 1983 was 93.9%. the similarity of the other, strain 255/ 1997, was 92.3% and 94% to strains from israel and morocco, respectively. the gi-12 lineage contained only one polish strain, g326/ 2017, which shared 98.7% nt identity with the dutch d274 virus. eight polish strains were in the gi-23 lineage and the similarity of their s1 coding region sequences was between 97.2 and 99.5%. their identity with the pathogenic israeli is/1494/06 strain from 2006 showed as from 98.5 to 99.5%. the subtree of gi-19 lineage contained polish qx strains in two branches, of which one contained nine strains with nt identity of 98.2-99.6% to the european qx prototype dutch l-1449 k/ 04 ibv. one strain, g019/2014, with similarity to the previous ones of 96.3-97.5%, constituted the offshoot branch. the two polish strains 80/ 1989 and 162/1997, isolated at an interval of 8 years from each other, formed a separate branch in the phylogenetic tree and they shared 85.1% nt identity. the phylogenetic analysis of the 66 analyzed full ibv genomes showed that ten polish ibv strains grouped into four phylogenetic groups (fig. 2) . two early strains, 78/1989 and 79/1989, clustered together with massachusetts-like strains (mass 41, peafowl/gd/kq6/ 2003 and ses 15ab-01) showing the highest nt identity of 90.5% with the sequence of the prototype gi-1 lineage beaudette strain. the two most recent strains, g103/2016 and g225/2017, clustered with ibvs of gi-23 lineage. the sequence identities of g103/2016 and g225/2017 to the previously described polish gammacov/ck/poland/g052/2016 strain were 99.8 and 95.5%, respectively. the three polish strains 548/ 2004, g074/2009 and g195/2012 were in the same cluster as other qx strains from europe and africa and were distantly related to chinese qx ibvs analyzed in this study (sdzb0808 and p100). they had nucleotide similarity to each other of 92.5-93.3% and were located in two subclades. a polish strain from 2009 clustered together with swe/ 0658946/10, the first described full-genome ibv strain of qx type in europe, with similarity of 93.7%. the other two polish qx strains were 93.3% similar to each other and formed a common branch on the phylogenetic tree. three early polish strains 80/1989, 162/1997 and 255/1997 were in a separate branch on the phylogenetic tree and showed nucleotide similarity with each other in the range of 90.5-92.5%. recombination analysis of all 241 aligned full s1 sequences was performed to assess the existence of possible recombinants among the analyzed polish ibv strains, especially those with less obvious membership to the lineage, i.e. 80/1989, 81/1998 and 162/1997 . our analysis identified only one s1 coding region which resulted from recombination events and it belongs to the 81/1989 strain; this event having taken place was supported by seven different methods with a very good global ka p-value of 1.277e−34. we confirmed this recombination breakpoint with phylogenetic trees. the region from 1 to 651 nt of the 81/1989 ibv strain clustered together with 80/1989 and 162/1997 isolates, the viruses which formed the separate early polish cluster on the full s1 coding region phylogenetic tree (fig. 3a) . in turn, the region from 652 to 1585 nt clustered with viruses belonging to gi-1 lineages (strains 78/1989, 79/1989, ibv438 india 2012 and h120) (fig. 3b) . the relevant s1 coding region fragments of the other ibv strains analyzed in this study grouped in the same way as they did in the phylogenetic analysis of the entire, intact s1 coding region. to check if any of the analyzed genomes of polish ibv strains result from recombination events, their sequences were thoroughly examined using the rdp4 program. our analysis revealed many such events. however, we selected five of them identified in six strains and they were supported with seven different methods (rdp, geneconv, bootscan, maxchi, chimaera, siscan and 3seq) and a very good global ka p-value below 1.0 x 10e−30 (table 3) the retrospective phylogenetic analysis of ibv included field strains collected over a period of 38 years, between 1980 and 2017. we investigated the s1 coding region of 34 ibv strains and the whole genome of ten strains. polish ibv strains showed different molecular features of the s1 coding region allowing their genotype or lineage to be determined, and their appearance in time reflects the history of ibv epidemics in europe (de wit et al., 2011) . the plot showing the timeline of various ibv lineages's detection and introduction of different vaccines to poultry population in poland is given in fig. 4 . the first identified ibv isolates in europe belonged to the mass type. in the netherlands they were diagnosed in the middle of the 1950s and one of them was even attenuated for vaccine development purposes (bijlenga et al., 2004) . the first ibv material available in our laboratory originates from 1980 and its s1 sequence displayed the features of gi-1 lineage, although no information was provided about the disease symptoms observed in the chicken flock where it was identified. later on, especially in the middle of the 1980s, numerous cases of a drop in egg production were recorded. the problem was so serious that polish veterinary authorities decided to allow the first ib vaccine introduction, but only for immunization of commercial layer flocks. the health problems in layers were significantly mitigated, but at the end of the 1980s respiratory fig. 1 . phylogenetic tree of the s1 gene of 207 reference and 34 polish ibv strains (bold underlined letters). the tree was constructed using mega 7 using the maximum likelihood method based on the gtr + g + i model and 1000 bootstrap replicates (bootstrap values shown on the tree). to make the tree clearer visually, branches with ibv lineages only distantly correlated with studied polish strains are collapsed. problems and mortality manifested in broiler chicken flocks, and so the vaccination of chickens of this production type was also started (minta et al., 1990) . we thoroughly examined four virus strains from that time, 78/1989 78/ , 79/1989 78/ , 80/1989 78/ and 81/1989 78/ . two of them, strains 78/ 1989 78/ and 79/1989 , have the s1 structure typical of the gi-1 lineage. similarly, their entire genomes revealed the highest identity to masslike strains such as h120 or beaudette. these two viruses came from broiler chickens on farms in the silesia region separated by only a few kilometers. the two other viruses, 80/1989 and 81/1989, were identified in broilers delivered to the laboratory near the same period (in june 1989) but from farms about 30 km from the previous ones. however, our investigation revealed a distinction between them. phylogenetic analysis of the full s1 coding region showed that strain 80/ 1989 forms a separate branch on the tree designated as early polish and was classified as a unique variant of ibv within the gi genotype. deep analysis of the 81/1989 strain strongly suggests that its s1 coding region was created as the result of a recombination event between the mass-like strains and the unique early polish variants circulating in the field at that time. it should be emphasized that the identified recombination breakpoint (651 nt) was in the intermediate region between highly variable regions (hvrs) 1 and 2 and hvr3 previously described as the most frequent locations of variations between ib viruses, and moreover, it exactly matches the breakpoint (550 and 652 nt) of recombinants between viruses of gi-19 and gi-22 lineages (valastro et al., 2016) . the introduction of vaccines based on the mass-like strains for chicken immunization significantly reduced the economic losses caused by ib. this state of ib control lasted for about 10 years until 1997, when ib disease inducing kidney damage appeared, caused by 793b-like ibv strains. the first case of nephritis was in 3-week-old broilers in the south of poland. the birds showed signs of severe enteritis and the observed gross lesions were congested tracheas and lungs and swollen and pale kidneys with the presence of urine. in subsequent months, further broilers with nephritis were provided for diagnostic purposes and the diseased flocks from which they came were located in all regions of poland; most of them had not been vaccinated against ib, but some had been immunized with mass-like vaccine in the first days of life. the strains identified at that time, 255/1997 and 58/1998, have an s1 sequence similar to ibv strains of gi-13 lineage. surprisingly, one of the first isolates known to cause nephritis, strain 162/1997 together with strain 80/1989 inflicting respiratory disorders, were located in the early polish branch of the phylogenetic tree with well supported uniformity to others (bootstrap value of 74) (hillis and bull, 1993) . the next polish gi-13 strains identified between 2004 and 2015 revealed the highest nt similarity to the s1 sequence of the 4/91 strain contained in the most commonly used vaccine in poland at that time (adzhar et al., 1997) . the next epidemic wave of ib in poland was caused by qx strains. the first report of disease induced by this virus type was published in 2006, but our studies showed the presence of the virus in poland in 2004 (domanska-blicharz et al., 2006) . it was from this year that qx strains were first identified in holland, germany, belgium and france, and next year they became dominant in some of these countries phylogenetic tree of the s1 gene fragment between potential recombination breakpoints 1 and 651 (a) and 652 and 1585 (b) among 241 ibvs included in the analysis. sequences of polish ibv strains are marked with bold underlined letters and recombinants with black dots. the tree was constructed using mega 7 using the maximum likelihood method based on the gtr + g + i model and 1000 bootstrap replicates (bootstrap values shown on the tree). to make the tree clearer visually, branches with ibv lineages only distantly correlated with studied polish strains are collapsed. (worthington et al., 2008) . the analysis of the migration history of gi-19 strains suggests that most european ones came from a single introduction from china, which then spread in european countries, evolving in them separately, since they tend to cluster by country. however, the genetic variability of gi-19 ibvs sometimes identified within countries suggests subsequent introduction of the virus in epidemic waves (franzo et al., 2017) . the division of polish gi-19 strains into four clusters could reflect a separate introduction or epidemic wave of this virus variant into the country. the last large epidemic wave of ib was caused by gi-23 (var2) strains. the first strain of this lineage was identified in december 2015 and in the following months it was the most common virus type detected in field samples delivered to our laboratory for diagnostic purposes apart from strains of 793b . the s1 coding region of most polish gi-23 strains is in the same phylogenetic cluster, except for strain g269/2017, which constitutes a separate one and could result from a separate virus introduction or from its intensive evolution. the single polish g326/2017 virus strain of gi-12 lineage analyzed in our study was identified in a 6-week-old broiler flock vaccinated with poulvac ib primer so it is highly probable that the identified strain originated from vaccine virus. although ball et al. (ball et al., 2017) showed that after vaccination of 1-day-old broilers with this vaccine only rna of the mass strain was detected in tissues and swabs and explained it through the higher replication potential of the mass virus. it cannot be ruled out that, as some other ibv strains are, the d274 virus is deposited in the body of chickens (possibly in the cecal tonsils) and after some time it is shed with cloaca (alexander and gough, 1978; naqi et al., 2003) . it should be noted that the ibv strains discussed here in detail are those that caused the greatest losses in polish poultry farming. during this period, strains of other genotypes and lineages also circulated in the field but their detection or type determination was not possible using available methods. the comprehensive studies of polish ibv isolates from 1998 to 1999 using serological and molecular tests conducted in cooperation with italian researchers showed that the 793b type was a major component of the ibv population in poland during this period, but serologically the presence of 624/i isolates was also identified and one isolate even showed no serological cross-reaction in an hi test nor amplification in rt-pcr (capua et al., 1999) . recently, the 624/i and q1 types were determined to affiliate to the gi-16 lineage, which has been present in europe (italy) since 1963 and persists until now (franzo et al., 2018) . in the period 2011-2013 numerous cases of d1466 ibv (gii-1 lineage) were detected, however, in subsequent years (2014) (2015) , the number of d1466-positive samples dropped to 3.1%, and currently we do not detect these viruses at all (domanska-blicharz et al., 2012; domanska-blicharz et al., 2017) . the complete genome sequences of all polish field ibv strains showed the presence of six genes and 13 orfs in the order previously reported, irrespective of the year of first isolation (abolnik, 2015; gomaa et al., 2008; hewson et al., 2011) . most accessory proteins are conservative in their lengths, in contrast to the structural ones which differ by even as much as 35 aa (m protein of gi-13/23 and gi-19). an interesting observation is clustering of polish ibvs based on the complete genome sequences. the earliest strains 78/1989 and 79/1989 belonging to the gi-1 lineage cluster with other representatives of this lineage such as the beaudette and dutch h120 strains, which could indicate the common origin of mass-like viruses. the viruses from the next epidemic wave are 162/1997 and 255/1997, and they were on the separate branch of the phylogenetic tree together with the strain 80/ 1989. the 80/1989 virus was located on the s1 coding region phylogenetic tree with the 162/1997 strain in the separate ibv branch of early polish ibv. on the other hand, the third virus of this separate group, 255/97, found its place in the gi-13 lineage on the phylogenic tree based on the s1 coding region. thorough analysis using rdp4 software revealed that the s gene of this virus was acquired from ibv k. domanska-blicharz, et al. infection, genetics and evolution 80 (2020) 104177 strains of gi-13 lineage during a recombination event. the results suggest that in the late 1980s and 1990s two ibv variants circulated in the polish poultry population: gi-1 and unique early polish ones. these viruses differ not only in the s1 coding region, which is the basis for the differentiation of lineages, but also in the remaining part of the genome. the viruses with such a genome backbone recombined with other viruses that were donors of the s1 coding region. grouping on a phylogenetic tree based on the complete genome of the other five polish strains was as expected. three g-19 strains, 548/2004, g074/2009 and g195/2012, took positions among other qx-like viruses from europe (sweden and italy) and africa (south africa and sudan) (abolnik, 2015; abro et al., 2012; ducatez et al., 2009; naguib et al., 2016) . in turn, two strains of gi-23 lineage, the viruses g103/2016 and g225/2017, were in the branch with the previously characterized polish g052/2016 and iranian is-1494 strains of gi-23 lineage isolated in 2015. it seems that in poland in the 1980s and 1990s ibv strains with a unique genome backbone circulated in the field, which were then replaced by strains belonging to other ibv lineages with a genome backbone specific to these lineages. in addition to the aforementioned recombination, five such events were also identified in polish ibv strains. three strains of the gi-19 lineage had orf1a and orf1b which revealed a high frequency of recombination events with 4/91 and sdzb0808-like strains (qx type strains from china from 2008). two strains of the gi-23 lineage exhibited recombination with the italy/90254/2005-type ibv, and a similar recombination pattern was also previously indicated . in conclusion, phylogenetic analysis performed on the s1 coding region of polish ibv strains collected during a 38-year period showed that these strains belonged to five recently established ibv lineages: gi-1, gi-12, gi-13, gi-19 and gi-23. additionally, two strains formed a separate branch of the phylogenetic tree described as unique early polish variants and one strain revealed itself to be the recombinant of gi-1 lineage viruses and these unique early polish variants. the phylogenetic analyses performed on the complete genome of ten polish ibv strains showed that they cluster into different groups. polish gi-1, gi-19 and gi-23 strains cluster with other similar viruses of these lineages, with the exception of the strains from 1989 to 1997 which are different. the recombination analysis showed that polish strains are a mosaic of different parental viruses most likely resulting from recombination events involving different ibv lineages, most frequently gi-13 and gi-19. it should be also stressed that the major epidemics of ib in poland appeared every 8-11 years: gi-1 in 1988, gi-13 in 1997 , gi-19 in 2004 and gi-23 in 2015 . these subsequent ibv lineages 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from sudan reveals distinct spots of recombination establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens molecular characterization of infectious bronchitis virus isolates from russia and neighbouring countries: identification of intertypic recombination in the s1 gene vipr: an open bioinformatics database and analysis resource for virology research mega6: molecular evolutionary genetics analysis version 6.0 s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification infectious bronchitis virus variants: a review of the history, current situation and control measures coronavirus diversity, phylogeny and interspecies jumping a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from the authors wish to acknowledge dr. siamak zohari and karin ullman (department of microbiology, national veterinary institute -sva, uppsala, sweden) and dr. ewelina iwan and arkadiusz bomba (department of omics analysis, national veterinary research institute, puławy, poland) for their support while conducting ngs. we also acknowledge justyna opolska for her help in molecular diagnostic tests.an ethical statement is not required as samples from animals were delivered to our laboratory by the owners or veterinarians for diagnostic purposes. chickens on the farms were under the supervision of appropriate persons, who took different samples as part of their routine work.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2020.104177. key: cord-276755-ctzrgqe7 authors: emmott, edward; smith, catriona; emmett, stevan r.; dove, brian k.; hiscox, julian a. title: elucidation of the avian nucleolar proteome by quantitative proteomics using silac and changes in cells infected with the coronavirus infectious bronchitis virus date: 2010-09-08 journal: proteomics doi: 10.1002/pmic.201000139 sha: doc_id: 276755 cord_uid: ctzrgqe7 the nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. the nucleolus is composed of complex protein/protein and protein/rna interactions. it is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. stable isotope labeling with amino acids in cell culture coupled to lc‐ms/ms with bioinformatic analysis using ingenuity pathway analysis was used to investigate whether the nucleolar proteome altered in virus‐infected cells. in this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand rna virus, avian coronavirus infectious bronchitis virus. data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. these demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus‐infected cells. the nucleolus is a subnuclear structure formed from complex protein-protein and protein-nucleic acid interactions [1] . the primary function of the nucleolus is in ribosome biogenesis, regulation of the cell cycle and the response to cell stress [2] . the nucleolus may be formed around hub proteins [1] , depletion of which can result in alterations in nucleolar architecture [3] and possibly function. the roles the nucleolus play outside its traditional function in ribosome biogenesis have only recently come to light, due in a large part to ms-based approaches to elucidating the nucleolar proteome. the first studies of the human nucleolar proteome revealed 271 and 213 proteins, respectively [4, 5] . the most recent edition of the nucleolar proteome database (nopdb 3.0) identified over 4500 proteins as being resident within the nucleolus under certain conditions; it estimates at least 80% coverage [6] . it is noteworthy that the majority of studies of the nucleolar proteome have been carried out on human hela cells, whereas the only other species investigated in depth is arabidopsis thaliana where 217 proteins with a large degree of conservation between the plant and the human proteomes were found [7] . the nucleolus is also a target for cellular and infectious diseases [8] . many viral proteins localize to this structure. these range from proteins encoded by rna viruses with purely cytoplasmic replication strategies [9, 10] to dna viruses with nuclear replication strategies [11] . the nucleolus can change structure in virus-infected cells [12] and this may correspond with a change in proteome [13] . a recent quantitative analysis of the nucleolar proteome isolated from hela cells infected with the nuclear replicating dna virus adenovirus identified 351 proteins, of which 24 proteins showed at least a twofold change in abundance, compared with nucleoli from mock-infected cells [14] . the precise interactions of viruses with the nucleolus are largely unknown [13] , particularly puzzling is the case of the rna viruses that have cytoplasmic replication strategies [9] . for example, in one such virus, avian infectious bronchitis virus (ibv), the virally encoded nucleocapsid (n) protein localized to the nucleolus in a cell cycle-dependent manner [15] and contained appropriate targeting motifs [16] [17] [18] . elucidation of the avian nucleolar proteome would provide a useful data set. similarly, how the nucleolus responds to rna virus infection would provide a useful comparator with the recent analysis of the nucleolar proteome in cells infected with a dna virus [14] . this would help determine whether any general principles can be established, despite different replication strategies. one of the difficulties in studying the avian nucleolus is the lack of appropriate and extensive immunological reagents. in this study, stable isotope labeling with amino acids in cell culture (silac) coupled to lc-ms/ms was used to generate a preliminary map of the avian nucleolar proteome in the presence and absence of ibv infection and was conducted by dundee cell products. the cell type used in this study was df-1 cells, a chicken fibroblast cell line, which we (and others) have used to study ibv e.g. [17] . cells were infected at a multiplicity of infection of 1 as described previously [19] . nucleolar fractions were prepared 18 h postinfection as described previously [20] and validated by detecting appropriate marker proteins (data not shown). quantification was performed with maxquant version 1.0.7.4 [21] , and was based on 2-d centroid of the isotope clusters within each silac pair. the generation of peak list, silac-and extracted ion current-based quantitation, calculated posterior error probability and false discovery rate based on the search engine results, peptide to protein group assembly, and data filtration and presentation was carried out using maxquant. the derived peak list was searched with the mascot search engine (version 2.1.04; matrix science, london, uk) against a concatenated database combining 80 412 proteins from international protein index human protein database version 3.6 (forward database), and the reversed sequences of all proteins (reverse database). the complete methodology has been described previously [20, 22] . raw data sets used to generate supporting infor-mation tables 1 and 2 (described below) can be found on the proteomics identifications (pride) database [23] and were uploaded using the pride database convertor tool [24] . cellular proteins were initially assigned by comparing with the avian genome database, and identified 835 proteins (supporting information table 1 ). however, this database had poor annotation compared with the human genome, and many of the proteins were unassigned or uncharacterized. therefore, the human database was used for protein assignment from peptide identification. in addition, candidate proteins were then examined to determine whether they were previously identified in the human nucleolar proteome database, which contains some 4500 proteins with an estimated 80% coverage. only if both of these criteria were met was the protein then assigned as being present in the avian nucleolar proteome (supporting information table 2 ). these resulted in a final list of 378 cellular proteins being identified and of these 260 proteins being identified and quantified. one viral protein, the n protein, was identified in the nucleolus with 59.4% coverage (fig. 1 ). this had not been previously observed in the nucleolus of ibvinfected df1 cells, possibly because of the difficulties associated with the indirect immunofluorescence detection of nucleolar proteins [25] . previous overexpression of an egfptagged n protein (in the absence of ibv infection) in df1 cells demonstrated the potential cytoplasmic and nucleolar localization of this protein [17] . no other ibv proteins were detected in the nucleolar fraction, and this analysis demonstrated the nucleolar localization of n protein in the context of ibv-infected cells. several proteins were selected from the silac lc-ms/ ms analysis and their abundance and localization investigated in mock and ibv-infected cells using indirect immunofluorescence confocal microscopy (as described in [18, 19] ) in order to validate the quantitative proteomic approach (supporting information fig. 1 ). selection of these proteins depended primarily on available antibody but also to demonstrate the validity of the silac lc-ms/ms analysis for several different cases: proteins identified by multiple peptides showing either no significant change (hsp70, 1.38fold increase, identified by 14 peptides) or a significant change (hsp90, 4.30-fold increase, five peptides), to a protein identified by a single peptide and large increase (shroom3 $50-fold increase). in ibv-infected cells, there was no apparent change in hsp70, whereas hsp90 appeared to have increased punctate localization in the nucleolus, nucleus and cytoplasm. in ibv-infected cells, the nucleolar localization of shroom3 appeared to increase. therefore, in general, the indirect immunofluorescence confocal microscopy analysis reflected the observations of the silac lc-ms/ms analysis. ingenuity pathway analysis was used to investigate the data sets to group together any proteins that shared similar functions in order to build an overview of the avian nucleolar proteome (fig. 2) and potential roles of proteins in infectious and respiratory disease. predominant functions in the nucleolus included 54 proteins identified as being involved in protein synthesis (p-value, 1.55 â 10 à11 -1.54â 10 à2 ), 37 proteins involved in rna post-transcriptional modification (p-value, 4.63 â 10 à13 -1.94 â 10 à2 ), 38 proteins involved in molecular transport (p-value, 5.70 â 10 à7 -1.94â 10 à2 ), 25 proteins involved in nucleic acid metabolism (p-value, 1.05 â 10 à6 -2.75 â 10 à2 ) and 20 proteins involved in dna replication, recombination and repair (p-value, 1.11 â 10 à6 -2.75 â 10 à2 ). major linked networked functions include those in protein synthesis, gene expression, rna post-transcriptional modification, dna replication, recombination and repair (supporting information fig. 2) , cellular signaling (supporting information fig. 3) , where induction of interferon has been established experimentally in ibv infection [26] and also the network of cell growth and proliferation linked to protein synthesis (fig. 3a) . for quantitative analysis, the previous investigations using silac and lc-ms/ms have applied ratio cutoffs ranging from nearly 1.3-to 2.0-fold. a previous study that investigated changes on nuclear proteins in rsv-infected cells using 2-d gel electrophoresis coupled to maldi-tof used a ratio cut off of 2.0-fold for comparison. the more recent investigation of the nucleolar proteome in adenovirus-infected cells compared with mock-infected cells using silac coupled to lc-ms/ms as well 2-d gel electrophoresis used a cutoff of twofold to access significant differences. in our study, a 2.0-fold cutoff was also used as a basis for investigating potential proteome changes using ingenuity pathway analysis in order to provide comparison with the previous studies. selection of a twofold difference gave 179 proteins that showed differences in abundance between nucleoli isolated from ibv-infected cells from nucleoli isolated from mock-infected cells. despite this, not all nucleolar proteins changed in ibv-infected df-1 cells. for example, the nucleolar proteins nucleolin and nop56 and nop58 were not significantly increased in ibv-infected cells with 1.8-fold, 1.2-and 1.5-fold differences, respectively. analysis of the relationship of identified nucleolar proteins by ingenuity pathway analysis suggested perturbations in the nucleolar proteome could have potential effects on cell metabolism. for example, several proteins were directly linked to regulation and/or binding by cyclin a and retinoblastoma protein (fig. 3a) . also several molecules which are involved in g2/m-phase transition were increased approximately four to fivefold in abundance in the nucleolar proteome in ibv-infected cells including three components of the 14-3-3 complex. although the interaction between the ibv and the cell cycle has been characterized in several different nonavian cell lines [19, 27, 28] , the potential interaction between the ibv and the host cell in nonavian cell lines has been reported to be different to what occurs in avian table 3 . cells [29] . therefore, to investigate whether the cell cycle was altered in ibv-infected avian cells and to validate network pathway analysis, dual label flow cytometry was used to accurately compare the proportion of cells in different stages of the cell cycle between mock and infected cells [19, 30] . the experiment was conducted in triplicate, on three occasions and the data indicated that in ibv-infected cells there was a significantly greater proportion of cells in the g2/m stage compared with mock-infected cells (fig. 3b) . these data sets (both raw e.g. supporting information table 1 ) and processed (supporting information table 2 ) provide the first solution of the avian nucleolar proteome. the use of silac allowed the perturbation of the nucleolar proteome in ibv-infected cells to be examined and compared with mock-infected cells. the data indicated that, in general, there was an increase in nucleolar proteins in ibv-infected cells that was validated by indirect immunofluorescence confocal microscopy. this technique was independent of purification of proteins from the nucleolus and thus provided distinct validation of lc-ms/ms data. functional predictions such as cell cycle aberrations could be made in terms of bioinformatic analysis of the nucleolar proteome and this was validated using an appropriate assay. overall, the study demonstrates how silac coupled to lc-ms/ms can be used to study changes in the proteome of a cellular compartment (in this case, the nucleolus) in virusinfected cells. raw data sets used to generate supporting information tables 1 and 2 have been uploaded to the pride database. nucleolar targetting: the hub of the matter the multifunctional nucleolus depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in hela cells directed proteomic analysis of the human nucleolus functional proteomic analysis of human nucleolus nopdb: nucleolar proteome database -2008 update proteomic analysis of the arabidopsis nucleolus suggests novel nucleolar functions old and new faces of the nucleolus. workshop on the nucleolus and disease rna viruses: hijacking the dynamic nucleolus the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication brief review: the nucleolus -a gateway to viral infection? changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus nucleolar proteomics and viral infection proteomics analysis of the nucleolus in adenovirus-infected cells cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein viral nucleolar localisation signals determine dynamic trafficking within the nucleolus cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication quantitative proteomics using stable isotope labeling with amino acids in cell culture (silac) reveals changes in the cytoplasmic, nuclear and nucleolar proteomes in vero cells infected with the coronavirus infectious bronchitis virus maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus pride converter: making proteomics data-sharing easy a guide to the proteomics identifications database proteomics data repository a higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies chicken interferon type i inhibits infectious bronchitis virus replication and associated respiratory illness cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53 infection of hela cells by avian infectious bronchitis virus is dependent on cell status differential localization and turnover of infectious bronchitis virus 3b protein in mammalian versus avian cells the cell cycle and virus infection this study was supported by the award of a leverhulme trust research fellowship grant to jah. ee is funded by a bbsrc doctoral training grant awarded to the astbury centre for structural molecular biology. dr paul ajuh at dundee cell products ltd is thanked for help with interpretation of ms/ms data sets.the authors have declared no conflict of interest. key: cord-304322-k9egxskw authors: promkuntod, n.; thongmee, s.; yoidam, s. title: analysis of the s1 gene of the avian infectious bronchitis virus (ibv) reveals changes in the ibv genetic groups circulating in southern thailand date: 2015-06-30 journal: research in veterinary science doi: 10.1016/j.rvsc.2015.05.002 sha: doc_id: 304322 cord_uid: k9egxskw abstract the new variants of the avian infectious bronchitis virus (ibv) produce a range of symptoms and cause global economic losses to the poultry industry. we investigated the s1 glycoprotein of 24 recent ibv isolates from chickens and demonstrated that two predominant genetic groups were circulating in southern thailand between 2008 and 2013. seven ibv variants, isolated from 2008 to 2009, were clustered in the thailand tha001 group i while 15 ibv variants, isolated from 2009 to 2013, were classified into the qx-like group ii. moreover, a single isolate from a broiler was categorized into the massachusetts-type, and an isolate from a layer belonged to the 4/91 type virus. interestingly, both the ibv groups i and ii were isolated from native chickens (62.5%) and caused a range of symptoms. our results indicate that the qx-like viruses were predominant after 2009, replacing the tha001 type viruses. furthermore, native chickens may contribute to the epidemiology of ib. infectious bronchitis (ib), caused by the gammacoronavirus avian infectious bronchitis virus (ibv), is a major and highly contagious disease of the poultry industry. ib is frequently involved in upper respiratory disease, nephritis, and enteritis. furthermore, ib frequently results in poor fertility and low egg production in layers. recently, it appears that ib broadly affects other tissues, for example, the proventriculus (reviewed in cook et al., 2012; de wit et al., 2011) . moreover, super infection and other notable bacterial co-infections can lead in an increase in the severity of ib (bakaletz, 1995) . ibv is a member of a genus gammacoronavirus. ibv genome consists of a single-stranded rna that encodes four structural proteins: envelope (e) glycoprotein, membrane (m) glycoprotein, nucleocapsid (n) protein, and spike (s) glycoprotein. the s glycoprotein of ibv, which forms large club-shaped projections, is a dimer or trimer (cavanagh, 2007) . the s protein is formed by two non-covalently bound polypeptides, s1 and s2 (stern and sefton, 1982) . the function of the s1 protein is to attach to host cell receptor molecules. the s1 protein includes epitopes and determinants for virus neutralizing antibodies that provide protective immunity. thus, s1 gene analysis has been widely used to differentiate ibv genotypes and serotypes (cavanagh, 2007; ignjatovic and galli, 1994; ladman et al., 2006) . it is well known that two major forces drive coronavirus evolution: mutations due to the high error rates of the viral rna polymerase, and recombination of the s1 sequences. thereby, these generate new strains or genotypes of ibv worldwide yu et al., 2001) . the first ib outbreak in thailand was reported between 1953 and 1954 (chindavanig, 1962) . despite several available commercial vaccines, for example, m41, h120, ma5, connecticut, 4/91 and the locally produced dld vaccine, which have been widely used for many years, ib outbreaks continue to occur and seem to be endemic. the commonly circulating ibv strains in thailand can be clustered into the following: an indigenous thailand tha001 group, an unnamed unique group, a qx-like group, and a massachusetts group (antarasena et al., 1990 (antarasena et al., , 2008 pohuang et al., 2009 pohuang et al., , 2011 . in this study we investigated the situation and distribution of ibv strains in southern thailand by collecting isolates from different bird species, with confirmed infections, from different regions between 2008 and 2013. we then differentiated them using sequence analysis and determined their relationships with vaccine and reference strains. suspected ib cases from chicken flocks, raised in the southern area of thailand, were submitted to an authorized laboratory between 2008 and 2013. most birds were village chickens exhibiting mild to moderate symptoms associated with respiratory, enteric, and kidney signs and depression with low morbidity and mortality. of the total cases submitted, 24 samples were ibv positive. coinfections or secondary bacterial infections were not found in any diseased birds. virus isolation and propagation were achieved by inoculating 0.2 ml of 10% (w/v) pooled and homogenized suspected tissues into the allantoic cavity of 9-to 11-day old antibodyfree embryonated chicken eggs (ece), which came from healthy flocks screened for key poultry diseases (guy, 2008) . forty-eight to 72 hours after inoculation, the allantoic fluid and chorioallantoic membrane (cam) were harvested. following three to five blind passages, indirect fluorescent antibody test (ifa) was used to identify the causative virus. briefly, frozen infected cams were acetonefixed, following this the slides were incubated with a nucleocapsid protein-specific mouse monoclonal antibody (clone ch/ibv 48.4; prionics, the netherlands), for 30 min at 37°c. after washing three times with pbs, the slides were incubated with fitc-conjugated rabbit anti-mouse immunoglobulin (dako, denmark) for 30 min at 37°c to detect antibody binding. next, the sections were washed in a similar manner and examined using epifluorescence microscopy at 100-200× magnifications. the allantoic fluids harvested from the inoculated ece that were ibv positive, during ifa testing on cam, were kept at −80°c until use. viral genomic rna was extracted from the allantoic fluid using a viral rna extraction kit (omega bio-tek, usa). a single-step rt-pcr was performed using a commercial kit (qiagen, usa), according to the manufacturer's protocol to amplify a 298-bp cdna fragment of the 3′utr gene that confirmed positive ibv strains (utr 1 − 5′-gctctaactctatactagcctat-3′ and utr 2 + 5′-aaggaag ataggcatgtagctt-3′) (adzhar et al., 1996) . next, the viral rna extracts were phylogenetically characterized using the specific s1 glycoprotein gene primer set, xce 1 + 5′-cactggtaatttttcagatgg-3′ and xce 3 − 5′-cagattgcttacaaccacc-3′, to amplify a 385 bp fragment of the s1 gene between nucleotides 729 and 1111 (amino acids 243-370) (adzhar et al., 1996; cavanagh et al., 2002) . the amplified rt-pcr products were purified using a qiaquick pcr purification kit (qiagen, usa). the purified products were sequenced using a commercial sequencing service (solgent assa service, korea). the partial s1 nucleotide and deduced amino acid sequences obtained from the thai ibv isolates were compared with ibv sequences deposited in the genbank database using the national center of biotechnology information (ncbi) blast search (http://www.ncbi .nlm.nih.gov/blast/), and included the reference ibv vaccine strains commonly used in thailand. sequence identities by blast were included in the alignment and phylogenetic construction. bioedit version 7.0.5.2 (www.mbio.ncsu.edu) was used to perform multiple sequence alignments and determine the nucleotide and amino acid identities. aligned sequences were used for phylogenetic analysis using the neighbor-joining method available in mega software version 6.0 (tamura et al., 2013) . the bootstrap values were determined from 1000 replicates of the original data to designate confidence levels to branches. the phylogenetic analysis of 24 purified pcr products revealed that the local isolates could be clustered into two predominant groups. group i comprised seven isolates, correlating to the previously published tha001. group ii, hosting a larger branch, consisted of 15 isolates. this group belonged to qx-like viruses, correlating to the previous isolate reported by pohuang et al. (2011) . in addition, two single isolates were detected, one of which was related to the massachusetts-type vaccine, while the other was related to the 4/91 strain (fig. 1) . the group i viruses were isolated in 2008-2009, and were geographically disseminated in the southernmost areas and southwestern coast. the isolates in group ii, found in 2009-2013, were distributed mostly in the central part of the southern region, ranging from the west to the east coast. a total of 62.5% of the viruses in group ii were isolated from native chickens associated with a range of symptoms. moreover, single isolates of the massachusetts-like (mass-like) and the 4/91-like viruses were isolated in 2012 and 2013, respectively (fig. 2) . this indicates that the ibv infection is endemic and has regularly circulated within this region. furthermore, occasional co-circulation was present in some areas. the nucleotide and amino acid identities were then determined within and between phylogenetic groups. the isolates in group i were highly related at the nucleotide and amino acid levels with 92.2-98% and 89.2-97.5% identities, respectively. the isolates of the qx-like group ii shared 89.3-97.7% nucleotide identity and 85-95.8% amino acid identity to each other. finally, we determined the phylogenetic relationships of the local isolates both with vaccine strains commercially used in thailand and with the ibv sequences available in genbank from other geographical origins. during this analysis, we found that the single isolates b/sk3589/55 and l/nk3251/56 fell within the mass-type (ma5 vaccine) and the 4/91 groups, respectively (fig. 1) . ibv is a major pathogen in both vaccinated and unvaccinated flocks of commercial broilers and layers, as well as in village/free range birds. the reasons for this are flock management system, mismatched vaccination, and emergence and reemergence of new variant strains. extensive antigenic variation, caused by genetic mutation in the hypervariable region of the s1 glycoprotein, is a feature of ibv and new variants thereof . moreover, the s1 protein is associated with virus-neutralizing epitopes, thereby inducing a strain-specific neutralizing antibody (de wit et al., 2011; hofstad, 1981) . in thailand, commercially available ibv vaccines are applied in intensive poultry farming. aside from commercial poultry, a limited number of backyard chickens are vaccinated. this is, at least in part, why ibvs are still prevalent. a better understanding of the genetic diversity of the ibv s1 gene from diverse geographies is essential for effective control and prevention of ib. selection of proper vaccines is an ultimate goal for minimizing economic loss. as previously mentioned, ibv has been reported to circulate in southern thailand. a major outbreak occurred in 2007, in which diseases were mainly associated with respiratory and kidney symptoms (antarasena et al., 2008) . our data indicated that the qx-like viruses appear to be the predominant viruses currently circulating in southern thailand, replacing the tha001-type viruses. in this study, in addition to the two predominant ibv groups detected, only the single isolates were related to vaccine strains. to determine whether they are directly vaccine-derived or mutated vaccine viruses that have been circulating for some time in birds, full-length gene sequence data, and the analyses of mutation rate and recombination events are required. we also observed a range of subclinical signs in the infected village-birds diagnosed with tha001 and qx-like ibv infections. birds showed mild respiratory symptoms and depression. these clinical signs differed from the nephropathogenic form of ibv-infected chickens previously reported by antarasena et al. (2008) . they were also different from the clinical forms detected in chickens infected with the novel qx ibv that caused severe proventriculitis, nephritis, and decreased egg production (yudong et al., 1998) . however, most native chickens are not vaccinated or they are raised in poor farming condition. therefore, they most likely play an important role in the spread of ibv. in summary, isolation and typing of the ibv field isolates is necessary for studying ib epidemiology. although the partial s1 sequence can be used to obtain preliminary genotype information of the isolates and determine whether available vaccines are expected to be protective, future studies must incorporate the entire s1 sequences and, preferably, whole genome sequences to further investigate evolution and recombination of the virus. fig. 1 . phylogenetic relationship of local isolates, vaccine and global ibv strains. analysis was based on partial s1 nucleotide sequences determined using mega 6.0 with the clustal w method. the isolates sequenced in this study are illustrated with black triangles, black squares, black diamonds and black circles. universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction avian infectious bronchitis in the southern part of thailand s1 glycoprotein gene analysis of avian infectious bronchitis virus strains isolated in southern thailand viral potentiation of bacterial superinfection of the respiratory tract coronavirus avian infectious bronchitis virus coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys studies on the attenuated on infectious bronchitis virus the long view: 40 years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures isolation and propagation of coronaviruses in embryonated eggs cross-immunity in chickens using seven isolates of avian infectious bronchitis virus the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens molecular evolution and emergence of avian gammacoronaviruses infectious bronchitis virus s1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand sequence analysis of s1 genes of infectious bronchitis virus isolated in thailand during 2008-2009: identification of natural recombination in the field isolates coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins mega6: molecular evolutionary genetics analysis version 6.0 molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia isolation and identification of glandular stomach type ibv (qx ibv) in chickens geographic distribution of field isolates of ibv in southern thailand between this work was supported by department of livestock development, ministry of agriculture and cooperatives, thailand (project 85/2013). the authors would like to acknowledge director of veterinary research and development center (southern region) for research support and staff members of virology laboratory for excellent technical assistance. key: cord-292428-j3wdlpp6 authors: zhou, j.‐y.; zhang, d.‐y.; ye, j.‐x.; cheng, l.‐q. title: characterization of an avian infectious bronchitis virus isolated in china from chickens with nephritis date: 2004-06-30 journal: j vet med b infect dis vet public health doi: 10.1111/j.1439-0450.2004.00744.x sha: doc_id: 292428 cord_uid: j3wdlpp6 one ibv isolate, sc021202, was isolated from the kidneys of the infected young chickens by inoculating embryonated eggs, and its morphology, physiochemical and haemagglutonating properties were detected. virulence of the isolate sc021202 was determined with specific pathogen‐free (spf) chicken inoculation. nucleotide acid sequence of s1 gene of the isolate sc021202 was further sequenced and analysed. the physiochemical and morphological properties of the isolate sc021202 were in accordance to that of typical infectious bronchitis virus (ibv). in a pathogenicity experiment, the clinical signs and related gross lesions resembling those of field outbreak were reproduced and the virus isolate sc021202 was re‐isolated from the kidneys of the infected chicken. sequence data demonstrated that the full length of the amplified s1 gene of the isolate sc021202 was composed of 1931 nucleotides, coding a polypeptide of 543 amino acid residues. compared with ibv strains from genbank, the nucleotide and deduced amino acid sequence of s1 gene of the isolate sc021202 shared 60.0–91.4% and 49.1–88.9% identities, respectively. a nucleotide fragment of ′ctttttaattatactaacgga′ was inserted at nucleotide site 208 in the s1 gene of the isolate. these results indicated that ibv isolate sc021202 was a new variant ibv isolate and responsible for field outbreak of nephritis. infectious bronchitis virus (ibv) is the prototype of the family coronaviridae, which is the causative agent of infectious bronchitis (ib) and results in a highly contagious disease of great economic importance to the poultry industry (calnek, 1997) . ibv has a single-stranded positive sense rna genome that is 27.6 kb in size (boursnell et al., 1987) . genome of ibv directs the synthesis of six major single-stranded polyadenylated rna species in infected chicken embryo kidney cells. these rnas include the intracellular form of the genome (rna f) and five smaller rna species (rnas a, b, c, d and e) (stern and kennedy, 1980) . mrna a, c and e encode three major structural proteins: the spike protein (s), the membrane (m) and the nucleocapsid protein (n), respectively. the s protein of ibv is cleaved post-translationally into n-terminal s1 and c-terminal s2 proteins (cavanagh and davis, 1986) . ibv exists as many serotypes that can be identified by virus neutralization (vn) test (el-houadfi et al., 1986; ambali and jones, 1990; gelb et al., 1997) . it is known that the s1 subunit was involved with infectivity and haemagglutinin activity and carries serotype-specific sequences (cavanagh and davis, 1986; cavanagh et al., 1986a,b) and antigenic epitopes inducing virus neutralizing antibody. the different serotypes, subtypes or variants of ibv was thought to be generated by nucleotide point mutations, insertions, deletions or rna recombinations of s1 genes (kusters et al., 1987; wang et al., 1993 wang et al., , 1994 jia et al., 1995) , which were responsible for outbreaks of ib in the vaccinated chicken flocks. in addition to serotype changes, the genetic variation may result in changes of the tissue tropism and pathogenicity of the virus, which lead to the generation of new ibv pathotypes. since classical ib was reported in 1930s (calnek, 1997) , enterotropic ibv strains (el-houadfi et al., 1986; ambali and jones, 1990) , nephropathogenic ibv strains (albassam et al., 1986; fulton et al., 1993; zhou et al., 1998; li and yang, 2001) , proventriculus pathogenic strains (zhu et al., 1998; zhou, 2000) , and the strain 4/91 (parsons et al., 1992) associated with a deep muscle myopathy have been found. in november 2002, a disease outbreak with kidney swelling has been occurred in young chicken flocks in sichuan, china. in this study, a coronaviral isolate associated with nephritis was isolated from the kidney tissue of the infected chickens. the symptom and pathological changes similar to the field outbreaks were reproduced in specific pathogen-free (spf) chickens and the virus isolate were molecularly characterized. chicken embryos and chickens spf white leghorn embryonated chicken eggs and chickens were purchased from beijing merial vital laboratory animal technology co., ltd (beijing, china). embryonated eggs were used for isolation of field isolates and re-isolation attempt of ibv from renal tissues of the experimental infected chickens. spf chickens were used for pathogenicity experiment. the isolate sc021202 was isolated from kidney of h120vaccinated 26-day-old young chicken flock experiencing an outbreak of nephritis, which resulted in 62% mortality rate. the cardinal clinical signs of naturally infected chickens included depression, weakness, slight respiratory signs and excretion of pale water-like egesta. at necropsy, the prominent lesion was swollen kidney with severe urate deposition. to isolate the virus, tissue sample of kidney from the diseased chickens was homogenized (25% w/v) and the suspension was subjected to freeze-thawing for three times. this was followed by centrifugation at 7800 g at 4°c for 15 min. the suspension was filtered through 0.45 lm membrane (acrodisc ò syringe filters, pall life sciences, new york, usa) and passaged in 10-day-old spf-embryonated eggs by intra-allantoic route for virus isolation. ten-day-old spf-embryonated eggs were inoculated with a dose of 0.4 ml of the suspension via the allantoic route and were incubated for 168 h at 37°c. the harvested allantoic fluid was used as inoculum for further serial passage (0.1 ml per embryo). egg lethal dose (eld) 50 of the virus isolate was detected using the embryonated eggs. the infected allantoic fluid was then negatively stained with 2% sodium phosphotungstate and examined by transmission electron microscopy. the allantoic fluid containing the isolate sc021202 was exposed to 0.25% trypsinase for 3 h, 0.5 and 1.0% trypsinase for 1 h at 37°c, 20% ether, repeated freeze-thawing, acidic (ph 3.0) and alkaline (ph 12.0) conditions. ten-day-old spfembryonated eggs were inoculated with such treated isolate samples (0.1 ml per embryo, 5 · 10 6.5 eld 50 /ml) by intraallantoic route, and the chicken embryo lesions induced by the isolate sc021202 were checked during subsequent incubation. haemagglutinating property of the virus isolate was detected as described previously by alexander and chettle (1977) . briefly, the virus was first concentrated 100-fold by centrifugation at 30 000 g for 60 min. the concentrated virus was treated with the phospholipase-c type i (sigma, st louis, mo, usa) for 2 h at 37°c with a final enzyme concentration of 1 unit/ml. one per cent chicken erythrocytes were used for haemagglutination test (ha). normal saline and allantoic fluid without virions were used as negative control. fourteen-day-old spf chickens were randomly divided into three groups with 10 birds in each group. each bird in groups 1 and 2 was inoculated with 0.2 ml of the allantoic fluid containing the isolate sc021202 (5 · 10 6.5 eld 50 /ml) and the normal allantoic fluid via trachea, respectively. the third group was used as the negative control. all chickens were reared in the isolater. the infected birds were observed for clinical signs and disease course, and were later killed by intravenous inoculation of barbiturate for observation of gross lesions, sampling of kidney, trachea and lung. these tissue samples were processed as described previously for virus re-isolation and histopathological observation. both the purified sc021202 isolate were used as the materials for rt-pcr from the allantoic fluid of the inoculated embryonated eggs with the original material of field-infected animals and experimentally infected animals. primers for amplification of s1 gene of the isolate sc021202 was designed with reference to the nucleotide sequence of ibv-beaudette strain (m94356) with a few modifications. s1-forward (5¢-ca-aagcttgaaaactgaacaaaagaca-3¢) and s1reverse (5¢-ttggatccataactaacataagggcaa-3¢) primers flanked a 1.9-kb sequence containing the whole s1 gene and part of s2 gene the genomic rna of the isolate sc021202 was extracted as described by zhou and he (2000) . rt-pcr was carried out as described by zhou et al. (2003) . briefly, first strand cdna corresponding to the s1 gene was obtained by reverse transcription using reverse transcription system (promega, madison, wi, usa). pcr was performed by 35 cycles of denaturation at 94°c for 1 min, annealing at 53°c for 2 min, and polymerization at 72°c for 2 min. the final polymerization step was conducted at 72°c for 10 min. the pcr products were analysed on a 1% agarose gel. the amplified cdna fragments containing the entire s1 gene of sc021202 were purified, and transformed to escherichia coli top 10 strain. five clones were sequenced in an automated dna sequencer (abi prism 377, abi perkin-elmer, foster city, ca, usa). the alignment and phylogenetic analysis of the deduced amino acid sequences of s1 gene were performed with the jotun hein algorithm and clustal method by dnastar (dnastar, madison, wi, usa). genbank accession numbers of ibv strains used in this study were shown in table 1 . embryos were inoculated with the renal suspension containing the virus isolate sc021202 and the allantoic fluids of the dying embryos were harvested for further passage in embryos. in the first passage, the embryo mortality inoculated with 0.4 ml of the kidney suspension was 100% (6/6) 70-84 h after inoculation. in following passage, 100% embryo mortality was observed with a dose of 0.1 ml of the infected allantoic fluid and death time of embryos moved to 30-48 h after inoculation. the gross lesions of the infected embryos were haemorrhage on the legs, mottled necrosis of the liver, swelling of the kidneys, dwarfing and curling (fig. 1) . in electron microscopy, the size of the virus isolate sc021202 was 80-130 nm in diameter. the virions were coronal, pear-shaped and covered with an envelope connected to pedunculate projections (fig. 2) . the virus isolate sc021202 lost its infectivity to chicken embryos after it was exposed to 45°c for 90 min or 56°c for 15 min, ether for 20 min, acidic condition (ph 3) or alkaline conditions (ph 12) for 3 h at room temperature (25°c). meanwhile, the virus isolate sc021202 still remained to the infectivity to chicken embryo, exposed to the repeated freezethawing five to 10 times, 0.5 and 1% trypsin for 1 h, and 0.25% trypsinase for 3 h at 37°c. one per cent chicken erythrocytes were used for ha. normal saline and allantoic fluid without virions was used as negative control. the infected allantoic fluid could not directly agglutinate 1% chicken erythrocytes while it could do that after being treated with the phospholipase-c type i. all the young chickens inoculated with a dose of 0.2 ml of the virus isolate (5 · 10 6.5 eld 50 /ml) exhibited respiratory signs 3 days after inoculation. respiratory signs disappeared 24 h later and birds took much more water while less feeds. other signs such as ruffled feather and depression were also observed. birds excreted pale water-like egesta and then died continuously 14 days after inoculation. lastly, the infected chickens appeared 50% mortality. necropsy revealed that kidney of dead birds was swollen and exhibited severe urate deposition. in microscopy, kidneys of birds died early showed severe granular degeneration in epithelia of distal or proximal convoluted; swollen or broken epithelia desquamated from basement membrane formed protein or cellular casts in renal tubule. in kidneys of birds died later, histological properties included the focal proliferation of lymphocytes around degenerated renal tubule, or epithelial atrophy of renal tubules, or dilatation of lumen of proximal or distal convoluted tubule. local bronchitis and bronchopneumonia of dead birds were observed. after chicken embryos were inoculated with a dose of 0.4 ml of the renal suspension of the experimentally infected chickens, the infected embryo showed the similar lesions with that inoculated with the original material of the field-infected chickens. the viral genomic rna obtained from the purified sc021202 was used as template for rt-pcr. the pcr products of s1 gene were approximately 1.93 kb in size, which was consistent with expected s1 gene (fig. 3) . the full length of the amplified s1 gene of the isolate sc021202 was composed of 1931 nucleotides and coded a polypeptide of 543 amino acid residues (genbank accession number: ay237817). meanwhile, s1 gene of the isolate sc021202 re-isolated from the experimentally infected chickens shared 100% nucleotide and deduced amino acid identities with the original material of the field-infected chickens, indicating that there existed no nucleotide variation of s1 gene from the virus isolate sc021202. compared with those of other ibv strains from genbank, the nucleotide and deduced amino acid sequences of the isolate sc021202 shared 61.9-91.4% and 49.2-88.9% identity with them, respectively. the deduced amino acid sequence of s1 gene of the isolate sc021202 was most similar to that of nephropathogenic ibv isolates j (88.9%),jx/99/01 (86.9%), tj/96/02 (82.8%), z (82.8%) and a preventriculus-origin ibv isolate qxibv (82.3%) which were all isolated recently from the diseased flocks in china, and showed lower identities to other ibv strains ga0470/98 (49.2%), de072 (49.3%), n1/88 (57.7%), gray (75.6%), 6/82 (75.7%), holte (76.3%), ark99 (76.6%), d3896 (76.7%), cv-56b (77.1%), jmk (77.6%), b1648 (77.9%), florida 1288 (78.3%), zj971 (78.3%), d41 (78.5%), beaudette (78.7%), h52 (78.7%), h120 (78.9%), m41 (78.9%), sd/97/01 (78.9%), jl/97/01 (79.1%), connecticut (79.5%) and australian t (81.5%) (data not shown). in addition, an insertion of a 21-nt fragment (ctttttaatta-tactaacgga) within the s1 gene of the virus isolates sc021202 and j at nucleotide site 208 was the most remarkable variations compared with s1 gene of other ibv strains published. as shown in fig. 4 , the predicted amino acid of s1 protein polypeptide of the isolate sc021202 showed close phylogenetic position to ibv isolate j, z, jx/99/01, tj/96/02 and qxibv isolated from diseased flocks in china recently. the isolate sc021202 recovered from kidney of the ibvaccinated young layer flock was shown to produce dwarfing fig. 3 . rt-pcr products of s1 protein gene of the isolate sc021202. m, dna marker; a, 1.93 kb nucleotide fragment of s1 gene. fig. 4 . phylogenetic tree constructed on the basis of the predicted amino acid sequence of s1 protein of the isolate sc021202. in the infected embryos and showed similar characteristics of coronavirus ibv such as size in diameter, morphology, physichemical and haemagglutinatiing properties. the clinical manifestations, gross and microscopic lesions of the infected chickens were the same as those infected with nephropathogenic ibvs (calnek, 1997; zhou et al., 1998) , and sc021202 was re-isolated from the damaged kidneys of the infected chickens. rt-pcr products of s1 gene of the isolate sc021202 were obtained using specific primers of ibv. sequence of s1 gene of the isolate sc021202 also showed high identity with the published certain ibv isolates. these results indicated that the characteristics of the virus isolate sc021202 was consistent with that of known ibv strains as well as the causative agent of chicken nephritis that was responsible for the field outbreaks recently. clinical ib case was first reported in 1970s in guangdong province, china as a typical respiratory disease. since 1988, many nephropathogenic ib cases have been reported in china, and nephropathogenic ib has become a familiar infectious disease in chicken farms. unfortunately, up to now, serotypes of the different tissue tropism of field ibv isolates were not identified using virus neutralization test or monoclonal antibody. nephropathogenic ibv usually damages kidneys and reproductive tract of chicken in addition to the slight respiratory signs, and then causes a high mortality rate in young flocks. although the control measures of ib were widely vaccination of live attenuated ibv vaccine (mass serotype) such as h52, h120 and ma5 strains in china, but the chicken flocks vaccinated with live attenuated ibv vaccine usually fail to present full protection to field virulent ibv challenge, and the vaccinated chicken flocks were often subjected to the attack of nephropathogenic ibv. in our study, the virus isolate sc021202 also caused 62% natural mortality of chicken flock vaccinated with live attenuated vaccine (h120 strain) and experiencing ibv infection, indicating that mass serotype of live attenuated ibv vaccine might be very poor in protection of chickens subjected to nephropathogenic ibv. rt-pcr of s1 gene is a fast and precise method to identify ibv, which can not only identify ibv but also discriminate different strains or isolates. it can even help predict some of its biological characteristics. the predicted amino acid sequence of s1 gene of the ibv isolate sc021202 exhibited high identities to nephropathogenic ibv isolates j (88.9%), jx/99/01 (86.9%), tj/96/02 (82.8%), z(82.8%) and a preventriculus-origin ibv isolate qxibv (82.3%) isolated recently from the diseased chicken flocks in china mainland. however, it shared lower identities with other published ibv strains, indicating that nephropathogenic ibvs in china were closely related to each other (fig. 4) . moreover, nephropathogenic ibvs in china also showed relatively high similarity with t strain while low similarity with gray, holte or b1648 which were all classical nephropathogenic ibvs, indicating that the origin of nephropathogenic ibv in china might have something to do with t serotype strains. a lot of point mutation and an insertion of ¢ctttttaattatactaacgga¢ fragment were found in s1 gene of the isolate sc021202 and j compared with other ibv strains. nephropathogenic ibv isolate j was isolated in 1998 and was defined to be a new variant ibv isolate by restriction fragment length polymorphism and sequence analyses of s1 gene (zhou et al., 1998; he et al., 2000; zhou and he, 2000) . although the s1 gene of sc021202 shared a relatively high identity with isolate j including the same insertion mutation, but a lot of point mutation had occurred in s1 gene of the isolate sc021202 compared with that of the isolate j. these characteristics of isolate sc021202 showed that it is a new variant ibv isolate and might directly evolved from other field ibv strains which was currently epidemic in china. nevertheless, it must be pointed that genotype doesn't always agree with serotype and more work still need to be done to clarify the precise serotype of currently epidemic ibvs. as nephropathogenic ib are epidemic in many provinces in china currently and poor protection was presented by traditional vaccine, it is strongly suggested that nephropathogenic ibv strains should be screened for vaccine using. reported nephropathogenic ibv strains were not identical in virulence and ability to cause renal lesion (winterfield and albassam, 1984; chandra, 1987) . the virulence of the isolate sc021202 was relatively strong and damaged mainly kidney of chickens although slight lesions in respiratory tract were also observed. as shown in fig. 4 , the tissue tropism of ibvs didn't always agree with their similarity of s1 gene. for example, the preventriculus-origon isolate qxibv shared 89.9% similarity in s1 protein with nephropathogenic isolate z while it shared only 76.4% similarity in s1 protein with the preventriculusorigin isolate zj971 (zhou, 2000) . the molecular basis for ibv tissue tropism is not known. nevertheless, based on the s1 sequence data, amino acid residues located between 99 and 127 sites may play a role in the different pathogenesis of these viruses (kwon and jackwood, 1995; sapats et al., 1996) . it is known that 5¢ hypervariable regions of the s1 protein (residues 56-69 and 117-131) contribute to serotyping or neutralizing activities (koch et al., 1990) and this region shares a similarity between serotypes and genotypes of ibv (kusters et al., 1989; wang and huang, 2000) . comparison of the 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genetic variations in the s1 gene of infectious bronchitis virus nephropathogenicity of infectious bronchitis virus structural analysis of the s1 gene of proventriculuspathogenic infectious bronchitis virus (zj971 isolate) and its structural proteins cloning and sequence analysis of s1 gene of isolate j of avian nephropathogenic infectious bronchitis isolation and identification of chicken's infectious bronchitis nephritis virus expression of immunogenic s1 glycoprotein of infectious bronchitis virus in transgenic potatoes coronavirus ibv strain with h95 variant isolated from chickens suffered from avian infectious proventriculus disease this work was financially supported by grants from the natural science foundation of china (grant no. 39970030), zhejiang provincial natural science foundation (grant no. 399411) and zhejiang provincial commission of science and technology (grant no. 991102030). key: cord-291754-1zxztadu authors: zhao, ye; cheng, jinlong; xu, gang; thiel, volker; zhang, guozhong title: successful establishment of a reverse genetic system for qx-type infectious bronchitis virus and technical improvement of the rescue procedure date: 2019-10-15 journal: virus res doi: 10.1016/j.virusres.2019.197726 sha: doc_id: 291754 cord_uid: 1zxztadu in this study, a pathogenic avian infectious bronchitis virus (ibv) qx-type strain yn was successfully rescued by vaccinia virus based reverse genetic technology. ten fragments contiguously spanning the complete ibv genome were amplified and cloned into the vaccinia virus genome by homologous recombination. the full-length genomic cdna was transcribed in vitro, and its transcript was transfected into bhk-21/n cells that could stably express ibv n protein. at 48 h post transfection, the culture medium was harvested and inoculated into 10-day-old specific-pathogen-free embryonated chicken eggs to replicate the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ryn virus will not require any cell culture adaptations. after only one in ovo passage, the recombinant yn virus (ryn) was successfully recovered and confirmed to possess the introduced silent marker mutation in its genome. biological characteristics of ryn such as the eid(50), tcid(50), replication in ovo, and replication kinetcs in vitro were tested and all were similar to its parental strain yn. our findings demonstrate the successful construction of highly-pathogenic qx-type ibv using a modified rescue procedure, allowing for future studies of the molecular biology and pathogenicity of ibv field strains. avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (ibv) that affects poultry production worldwide (cavanagh, 2007) . ibv belongs to the gammacoronavirus genus, in the coronavirinae subfamily, of the coronaviridae family, in the order nidovirales. the ibv genome is approximately 27.6 kb in size. all coronaviruses share a similar genome organization with gene 1 located at the 5ʹ end of the genome. as the replicase gene, it has been proved to be a determinant of pathogenicity of ibv (armesto et al., 2009) . the structural genes cluster at the 3ʹ end and encode the spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins (cavanagh, 2005) . ibv also possesses two accessory genes, gene 3 and gene 5, which have been shown to be dispensable for virus replication in cell culture youn et al., 2005; hodgson et al., 2006; bentley et al., 2013) . however, it seems that the deletion of accessory genes 3a, 3b, 5a or 5b from ibv could induce an attenuated phenotype both in vitro and in vivo (laconi et al., 2018; van beurden et al., 2018) . although ibv has been controlled by routine vaccination for a long time, it remains a significant threat to the global poultry industry due to the existence of many serotypes (capua et al., 1999) . the high evolution and recombination rates of ibv result in new serotype viruses emerging in the field and often causing great losses since current vaccines only provide partial protection against new strains (cavanagh et al., 1992; de wit et al., 2011) . specifically, a new ibv variant has been circulating in china since 1998. this virus has been identified as the qx strain and has been primarily associated with various renal pathologies (wang et al., 1998) . phylogenetic analyses showed that the ibv isolates that clustered with qx were mainly chinese isolates (zhao et al., 2016) . these results further indicated that ibv isolates prevalent in china were evolutionarily distant from massachusetts (mass)-type strains (terregino et al., 2008; abro et al., 2012; yan et al., 2017) . understanding the pathogenic mechanism and molecular characteristics of circulating strains could significantly facilitate the development of vaccines that provide efficient protection against the highly variable ibv. since the initial development of a reverse genetic system for rna (taniguchi et al., 1978) , this technique has proven to be a powerful method for investigating the molecular biology of rna viruses and for studying the role of individual genes in pathogenesis. however, despite these successes, not all reverse-genetic systems of positivestranded rna viruses can be easily established. the large genome of the coronavirus poses a great obstacle to the construction of reverse genetic systems based on full-length cdna. cloning techniques using traditional plasmid dna cloning vectors cannot guarantee stability of large fragments of coronavirus cdna, and many studies have found that certain coronavirus gene sequences can have toxic effects in e. coli or cannot be cloned into conventional plasmid vectors or passaged in prokaryotic systems (gonzalez et al., 2001) . therefore, infectious fulllength clones of coronaviruses have only been established at the beginning of the 21 st century (casais et al., 2001) . subsequently, reverse genetics was successfully applied to study the role of accessory genes in viral replication youn et al., 2005; laconi et al., 2018) , the potential of ibv as a vaccine vector (britton et al., 2012; bentley et al., 2013) , the virulence determinants of the beaudette and m41 strains (fang et al., 2007; maria et al., 2009; keep et al., 2018) , and the relationship between the s gene and tissue tropism of the virus (casais et al., 2003; britton et al., 2005; promkuntod et al., 2013; shan et al., 2018) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on vero cell-adapted beaudette strain, which is non-pathogenic in chickens (geilhausen et al., 1973) , or the mass-type strains, which are genetic quite distant from the prevalent qx-type strains. neither of these systems therefore served as an ideal virus model to study the replication and pathogenic features of qx-type strains. therefore, the application of reverse genetics techniques to qx-type strains may allow for modification of the viral genome and may provide a powerful tool for novel vaccine development. we previously isolated and characterized a virulent ibv strain from yun nan province, china, designated yn strain. this isolate is genetically similar to most of the prevalent qx-type ibv strains found in china, albeit with increased pathogenicity compared with previously characterized strains (feng et al., 2012) . in this study, we describe the establishment of a reverse genetic system of the virulent yn strain. we also developed a modified rescue strategy, which combines full length rna electroporation with in ovo virus amplification, thus avoiding cellculture adaption and providing improved rescue efficiency of ibv. these advances will enable us to modify specific gene regions and investigate their influence on gamma coronavirus replication and pathogenicity. ibv strain yn was isolated and propagated in the allantoic cavities of 10-day-old specific-pathogen-free (spf) embryonated chicken eggs (ece) (feng et al., 2012) . the complete genome of yn has been sequenced and submitted to the genbank database under accession no. jf893452. vaccinia virus vnoti/tk, cv-1, and d980r, and bhk-21 cells were described previously hertzig et al., 2004) . vaccinia virus vnoti/tk and vaccinia virus recombinants were propagated, titrated, and purified on monkey kidney fibroblasts (cv-1) by standard procedures . all cells were maintained in minimum essential medium containing 10% fetal bovine serum (fbs, gibco, grand island, ny, usa), 100 u/ml penicillin, and 100 mg/ml streptomycin. human embryonic kidney (hek) 293 t cells (american type culture collection) were cultured in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut, usa) supplemented with 10% fbs, 100 u/ml penicillin, and 100 mg/ml streptomycin. viral rna was extracted from yn-infected allantoic fluid with a genmark rna purification kit (genmark, tai-chung, taiwan) according to the manufacturer's directions. reverse transcription was performed with the primescript™ rt master mix (takara, tokyo, japan). vaccinia y. zhao, et al. virus research 272 (2019) 197726 viral dna was extracted from strain vnoti/tk according to a previously described method (bridgen, 2013) . primers used for amplifying the yn strain complete sequence (genbank, accession number jf893452) were designed according to the intrinsic restriction enzyme sites in the genome. silent mutations were introduced into the genome by primers if no restriction sites were present, and also served as molecular markers of the recombinant viruses. each dna fragment was amplified from cdna templates by pcr using the cloneamp hifi pcr premix (takara). pcr primer pairs used to amplify genomic regions are listed in table 1 . nucleotide (nt) changes and restriction sites have been presented as boxes and italicized typeface in table 1 , respectively. pcr amplification of cdna fragments was performed under the following conditions: denaturation at 98℃ for 1 min, then 29 cycles of 98℃ for 10 s, 55°c -59℃ for 10 s, and 72℃ for 30-60 s depending on the size of the products (10 s/kb), and a final extension at 72℃ for 2 min. the pcr products were purified using a nucleospin® gel and a pcr clean-up kit (macherey-nagel, düren, germany) and cloned into pgpt-1 vector, which contains a dominant selective marker gene, escherichia coli guanine phosphoribosyltransferase (ecogpt) (mulligan and berg, 1981) , under the control of the vaccinia virus p7.5 k early/late promoter. in order to construct the recombinant vaccinia virus containing complete ibv genome through vaccinia virus-mediated homologous recombination, the first step was to construct flanking region belonging to the vaccinia virus genome into the pgpt-1 plasmid. the left flank vvt7 was 557 bp in size, an xbai and an bamhi restriction site were added at the 5ʹand 3ʹ end for the ligation into pgpt-1, and an additional type iis restriction site bambi and five nucleotides 'actta' (shown in red in table 1 ) from ibv 5′utr beginning was added at the 3ʹ end of vvt7 for the further ligation with ibv fragements. between the vaccinia dna sequence and bsmbi site was a bacteriophage t7 rna polymerase promotor and an additional g nucleotide for the initiation of the in vitro transcription reaction. the pcr product vvt7 was digested with xbai and bamhi to ligated into pgpt-1 plasmid, next, we constructed the ibv segment 1 f (corresponding to nt 1 to 3285 in the yn genome) by rt-pcr. upstream of the segment 1 f 5′-end was a bsmbi restriction site, downstream of the segment 1 f 3′-end was a bamhi site, thus to allow insertion of the 1 f cdna downstream of vvt7 in pgpt-1 by restriction endonuclease digestion and t4-ligation. on the other side of pgpt-1, a 362 bp vaccinia virus flanking region vv-3′ was cloned into the plasmid following digestion with xhoi and ecori, and an additional noti restriction site was added before the ecori site to stop in vitro transcription after the poly(a) tail. the obtained plasmid, pgpt-1 f, was integrated into the vaccinia virus genome by homologous recombination under the positive selection pressure of mycophenolic acid (mpa) in the presence of xanthine and hypoxanthine, to which only recombinant vaccinia viruses with the ecogpt gene show resistance (falkner and moss, 1990) . ecogpt-selected viruses were purified and sequenced to ensure that ibv segment 1 f and the ecogpt gene had been inserted into the correct position in the vaccinia genome, and verified virus was termed vv-yn-1 f. next, to generate a complete 3ʹ untranslated region (utr) with a 25-bp poly(a) tail in strain yn, a dna was synthesized and cloned in the puc57 plasmid containing a 500 bp fragment 1 f (ibv-yn nts 2785-3285) plus 304 nts of the ibv-yn 3ʹutr sequence (27,378-27,657) followed by a 25-bp poly(a) tail and 508 bp of vaccinia virus flanking sequence. we also incorporated several restriction sites for further modification in the order: 5ʹ-saci-1 f (2785-3285)-bamhi-sali-3ʹutr (27,378-27,657)-poly(a) (25 bp)-noti-vv (508 bp)-xhoi-3ʹ. this fragment was digested with saci and xhoi and ligated into the digested pgpt-1 plasmid to replace the ecogpt gene. next fragment 1r corresponding to the ibv-yn genomic region from nt 3286 to 6559 containing bamhi and sali sites was amplified and inserted between 1 f (2785-3285) and the 3ʹutr (27, 657) in pgpt-1 (fig. 1a) . the homologous recombination between pgpt-1r-3ʹutr and vv-yn-1 f was performed by exerting negative selection pressure on d980r cells due to the presence of 6-thioguanine (hertzig et al., 2004) . after successful construction of the ibv-yn fragment 1 f1r containing the 5ʹ-t7 promoter and the 3ʹ-synthetic poly(a) tail in the vaccinia virus genome, the other eight remaining fragments (2 f, 2r, 3, 4 f, 4r, 5 f, 5r, 6) were inserted into the vaccinia virus genome by vaccinia virus-mediated homologous recombination using the method described above. the individual recombination steps are summarized in fig. 1b . the final recombinant vaccinia virus was purified and sequenced by deep sequencing. the vaccinia virus possessing the complete ibv-yn genome was amplified on a large scale in bhk-21 cells and designated vvibv-ryn. the recombinant vaccinia virus vvibv-ryn was then used to construct recombinant vvibv, vvibv yn-5a/egfp, by two rounds of vaccinia virus-mediated homologous recombination. this virus consisted of direct replacement of ibv gene 5a sequence, beginning at the orf 5a initiation codon, and ending at the orf 5a termination codon in order to maintain the genomic sequence upstream of the 5b gene. the introduced reporter gene egfp, replacing orf 5a, was expressed utilising the existing gene 5 trs. first, plasmid pgpt△5a was constructed to encode ibv-yn nt 25038-25539 and 25733-26629 upstream and downstream of the gpt gene. recombination of vvibv-ryn with plasmid pgpt△5a resulted in the isolation of the recombinant vaccinia virus vvibv-gpt△5a. vvibv-gpt△5a was used for recombination with the plasmid pibv-gfp, encoding ibv-yn nt 25038-25539 upstream and ibv-yn nt 25733-26629 downstream of the gfp sequence. the resulting recombinant vaccinia virus vvibv yn-5a/egfp was obtained after gpt negative selection. to improve the rescue of recombinant ibv a cell line stably expressing the ibv n protein (bhk-21/n) was generated using a lentivirus-based system. the construction procedure is shown in fig. 2 . briefly, the complete n gene of ibv-yn was amplified by rt-pcr. the primers used were as follows: ibv-n-bamhi-f: ttaaggatc cgccac catg gcaagcagtaaggcat; ibv-n-not i-r: aaa agc ggc cgc tca aag ttc att ttc acc aag tg. two restriction sites, bamhi and noti, were introduced into the pcr product to digest and insert it into a plasmid with the hsv-tk promoter just upstream of the insertion site, and the obtained n gene plus tk promoter plasmid was designated ptk-n. then, the n gene plus tk promoter was amplified using the ptk-n plasmid as template with primers: tk-n-f-ecori: aacatacgctctcc atcaaaacaaaa; tk-n-r-bamhi: gatgcaatttcct cattttattag gaaag g. the psmpuw-sv40-puro plasmid was cut with enzymes ecori and bamhi and the fragment containing the n gene plus the tk promoter was inserted by an in-fusion reaction. after the n gene and tk promoter were inserted into the plasmid expression system psmpuw-sv40-puro (cell biolabs, san diego, ca, usa) ( fig. 2a) , the plasmid was cotransfected along with pcmv-dr8.74 and pmd2g-vsvg (cell biolabs, san diego, ca, usa)into 293 t cell lines to package the lentiviral particles. lentiviral vector production was performed according to standard protocols. briefly, subconfluent 293 t cells in a t25 flask were cotransfected with 5 μg of psmpuw-sv40-puro-n, 2 μg of pcmv-dr8.74, and 2 μg of pmd2g-vsvg using the transfection reagent xtremegene 9 (sigma-aldrich, st. louis, mo, usa). after 6 h, the medium was changed and the lentiviruses were harvested after 72 h and filtered through a 0.45 μm filter. stable transduction of the lentiviruses was carried out as follows: 10 6 of bhk-21 cells were seeded in a 6-well plate and transduced at day 2 with 10 6 of the lentiviral vector. selection was performed from day 3 using 2 μg/ml puromycin until the parental cells from a parallel experiment died (fig. 2b ). the expression of n protein in the bhk-21/n cell line was detected by immunofluorescence analysis (ifa). bhk-21/n cells were cultured in 6-well plates for 48 h at 37℃ and 5% co 2 , the medium was then y. zhao, et al. virus research 272 (2019) 197726 removed, and cells were fixed with 80% (v/v) cold acetone for 15 min in an ice bath. next, 500 μl of chicken polyclonal anti-ibv positive serum diluted 1:200 in pbs was added and the reaction was incubated for 1 h at 37℃. the cells were washed, stained with anti-chicken fitc-labelled conjugate (sigma) for 1 h at 37℃, and directly examined by fluorescence microscopy (olympus, tokyo, japan). parental bhk-21 cells were treated in the same way as a control. to compare the rescue efficiency of our lentivirus transduced bhk-21/n cell line with commonly used bhk-21 cells, we further constructed a recombinant vvibv strain (vvibv yn-5a/egfp) expressing the reporter genes enhanced green fluorescent protein (egfp), in which gene 5a was replaced with reporter genes egfp. 10 μg of yn-5a/egfp full-length rna was used for electroporation of 10 7 bhk-21/n cells or bhk-21 cells respectively. afte 48 h incubation at 37°c, the green fluorescence in both electroporated cell lines were observed under fluorescence microscopy. 2.6. in vitro transcription, transfection, and generation of the rescued virus ryn ibv-yn full-length rna transcript was generated in vitro using purified genomic dna isolated from recombinant vaccinia virus (vvibv-ryn) containing the 27.9-kb full-length ibv cdna, that was digested with noti and in vitro transcribed by the ribomax™ large scale rna production system-t7 kit (promega, madison, wi, usa) according to the manufacturer's instructions in brief, 10 μg of ibv-ryn full-length rna was used for electroporation of 10 7 bhk-21/n cells as described previously (eriksson et al., 2008) . the transfected bhk-21/n cells were incubated at 37°c for 48 h in dmem supplemented with 10% fbs. after 48 h, the virus-containing medium was collected and inoculated into six 10-day-old spf embryonated chicken eggs (ece). after incubation in ece at 37°c for 48 h, allantoic fluid from three ece was harvested for further rt-pcr detection. the other three eggs were kept at 37°c until 144 h post-inoculation to check for embryo curling caused by ibv. the allantoic fluid that cause embryo curling and rt-pcr detection positive was further passaged in ece for 10 times to get the stable virus stock (p10 virus stock) that reproducibly induced embryo curling. this stock was then used for further analyses, such as determination of the 50% egg infective dose (eid 50 ), the 50% tissue culture infective dose (tcid 50 ) as well as replication in vitro and in ovo. to differentiate the rescued viruses from the parental viruses, viral rnas were extracted from allantoic fluid (ryn and parental yn) as described above. rt-pcr was performed with selected primer pairs designed around the silent molecular markers that had been introduced into the ryn genome (5ʹ-gtgtgctatgtaagg tggtg-3ʹ, 5ʹ-ggagc atttttaactcgtagg-3ʹ), and the pcr product was digested to verify the existence of the sali silent nucleotide change at position 6555-6560. the pcr product was also sequenced for further confirmation. to determine eid 50 of ryn, serial 10-fold dilutions (10 −1 -10 -8 ) of the amplified virus were inoculated into 10-day-old spf ece via the allantoic sac route. for each dilution, 0.1 ml of virus solution was injected into each egg and five eggs were used for each dilution. inoculated eggs were incubated for 144 h at 37℃ to check for classical embryo curling caused by ibv. eggs that died within 24 h of inoculation were discarded. eggs that died between 24-144 h were considered as infected. the viral titer was also assessed by determining tcid 50 . for this procedure, chicken embryo kidney (cek) cells were prepared and seeded into 24-well plates two days before virus infection as previously described (hennion and hill, 2015) . serial 10-fold dilutions (10 −1 -10 -6 ) of the amplified virus were added onto 80% confluent cek cells, and each dilution of virus was inoculated into four replicate wells on 24well plates. after 48 h incubation at 37℃, 5% co 2 , the 24-well plates were assessed for ibv infection by an ifa assay as described above. the eid 50 and tcid 50 calculations were based on the reed and muench method (reed and muench, 1938) . to compare the in ovo replication of the rescued virus ryn and its parental strain yn in ece, 200 μl pbs containing 10 2.0 eid 50 of ryn or yn virus were inoculated into the allantoic cavities of 10-day old eces, and 200 μl allantoic fluid of five eggs from each group were harvested at the time points of 12, 24, 36, 48, 60 and 72 h post inoculation (hpi) and pooled for the real-time pcr detection assay. in brief, the total rna of 200 μl allantoic fluid were extracted using the viral rna isolation kit (foregene co., ltd, chengdu, china) according to the manufacturer's instructions. reverse transcription was conducted at 37 ℃ for 15 min, with 3 μg of total rna using the primescript™ rt reagent kit (takara bio inc, beijing, china). the cdna samples were submitted for sybr green i real-time rt-qpcr to detect the viral load for ibv n gene as described before (zhao et al., 2015) . all quantitative pcr reactions were carried out in triplicate and repeated at least twice, and the expression of ibv was calculated according to a standard curve. to compare the in vitro replication of the rescued virus ryn and its parental strain yn on cek cells, 200 μl pbs containing 10 3.0 tcid 50 of ryn or yn virus were inoculated onto the cek cells in 24-well plates, and 200 μl supernatants from three wells from each group were harvested at the time points of 6, 12, 24, 36, 48, and 60 hpi for a real-time pcr detection assay for ibv n gene as described above. the virus copy numbers for each time point were detected in triplicate and calculated according to a standard curve. unless otherwise stated, all data are represented as the mean values ± standard derivations obtained from experiments performed at least in triplicate. data were analyzed using graphpad prism 6 (graphpad software, san diego, ca, usa). the statistical significance of differences between groups was verified using one-way anova followed by bonferroni's multiple comparison tests. for all tests, the following notations are used to indicate significant differences between ryn and yn virus: * p < 0.05; ** p < 0.01. to construct a cdna clone covering the complete ibv-yn genome we generated ten overlapping dna fragments by rt-pcr from ibv-yn rna and chemical dna synthesis. the fragment corresponding to the ibv-yn 5′-end was engineered to contain a promoter for the t7 rna polymerase that was placed immediately upstream of the ibv-yn 5′terminal a nucleotide. the fragment corresponding to the ibv-yn 3′end contained a polya sequence of 25 nts followed by a noti restriction endonuclease recognition site. this strategy has been successful for other full-length coronavirus cdna clones (casais et al., 2001; van den worm et al., 2012; tekes, 2016) . it allows to generate an in vitro transcribed rna containing one additional g nucleotide at the 5′-end (required as transcription start site for the t7 rna polymerase) and a polya tail at the 3′-end (fig. 1a) . the full-length cdna was sequentially assembled into the vaccinia virus genome by six rounds of homogenous recombination using the e. coli guanine phosphoribosyltransferase as selection marker for positive or negative selection as appropriate (fig. 1b) . the integrity of the resulting vaccinia viruses was assessed by rt-pcr and sequencing analysis of the recombined regions after each recombination step. the final recombinant vaccinia virus, vvibv-ryn, containing the complete ibv-yn genome was deep sequenced and the result showed 100% identity with the parental virus genome, with the exception of three silent nucleotide substitutions, t3281a, a3284c, and y. zhao, et al. virus research 272 (2019) 197726 t6557c, which are diagnostic for the recombinant ibv genomic cdna (fig. 1a) . as demonstrated previously, the coronavirus n protein greatly facilitates the rescue of recombinant coronaviruses (yount et al., 2000; casais et al., 2001) . we therefore constructed a lentivirus-transduced bhk-21/n cell line that stably expresses the ibv-yn n protein. the complete n gene of yn strain was amplified by rt-pcr and cloned downstream of the hsv-tk promoter. this expression cassette was then inserted into the lentivirus vector psmpuw-sv40-puro and the resulting plasmid was used to generate lentiviral vector particles ( fig. 2a, b) . following lentivirus vector transduction and puromycin selection the resulting bhk-21/n cells were assessed for n protein expression. as shown in fig. 2c , the expression of ibv n protein was readily detected by ifa, whereas no specific fluorescence was observed in parental bhk-21 cells. expression of reporter genes by ibv has been previously described and used to facilitate the studies of virus replication and transcription in target cells (bentley et al., 2013) . in this study we replaced the accessory 5a gene by genes encoding the egfp, resulting in the recombinant viruses vvibv yn-5a/egfp (fig. 3a) . after electroporation of 10 μg of yn-5a/egfp full-length rna into 10 7 bhk-21/n cells or bhk-21 cells and incubation at 37℃ for 48 h, green fluorescence were observed in both electroporated cell lines under fluorescence microscopy, confirming the expression of gfp (fig. 3b ). however, we were able to detect cells displaying green fluorescence in bhk-21 cell lines, but only a very low number; the numbers of cells displaying green fluorescence in bhk-21/n cell lines were obviously higher than in bhk-21 cell lines, indicating a higher rescue efficiency by using the bhk-21/n cell lines. the recovery of infectious ibv-yn from cloned cdna was performed using a protocol that was successfully used for the recovery of recombinant hcov-229e . briefly, vvibv-ryn dna purified from virus particles was cleaved with noti and used as a template for in vitro transcription with the t7 rna polymerase in the presence of a cap analog. the rna transcripts (10 μg) of the ibv cdna were electroporated into 10 7 bhk-21/n cells, and the bhk-21/n cell supernatant was harvested after incubation at 37 ℃ in 5% co 2 for 48 h. the supernatant was subsequently inoculated into the allantoic cavities of ece for replication of the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ibv-ryn replicate efficiently in ovo. after 48 h incubation in the allantoic cavities, the recovery of ibv-ryn was confirmed by rt-pcr amplifying and sequencing the genomic region that contained the molecular markers to distinguish ibv-ryn from ibv-yn. as shown in fig. 4a, b , the presence of a silent mutation that gave rise to a sali restriction site at position t6557c was verified by restriction enzyme digestion as well as sequence analysis. this analysis showed that the pcr product of ibv-ryn can be digested into two segments corresponding to the mutation introduced into the genome, whereas the pcr product of parental yn strain was resistant to cleavage by sali (fig. 4a) . accordingly, the t6557c mutation was confirmed by sequencing analysis (fig. 4b) . collectively, these results demonstrate the successful rescue of the pathogenic ibv strain yn from cloned cdna by using electroporation of full-length ibv in vitro transcripts into n-protein expressing cells and subsequent virus amplification in the allantoic cavities of ece. ece that have been inoculated with supernatants of ibv-ryn rnaelectroporated bhk-21/n cells were also assessed for chicken embryo development, and as shown in fig. 5a , after only two passages in ece, typical embryo lesions such as curling, stunting, and dwarfing were observed. a similar phenotype was also observed when ece were inoculated with wild-type ibv-yn. to compare replication of ibv-ryn with the parental strain ibv-yn in ece, we first produced virus stocks that reproducibly induced embryo curling by passaging the viruses 10 times in ece. 100 μl of collected p10 ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted (10 −1 -10 -8 ) and inoculated into 10-day-old spf ece. five eggs were used for each dilution. after 144 h incubation at 37 ℃, inoculated eggs were evaluated for classical embryo curling caused by ibv and the eid 50 titer was calculated according to the reed and muench method (reed and muench, 1938) . ibv-yn and ibv-ryn were pathogenic to chicken embryos up to a dilution of 10 6 , and the eid 50 y. zhao, et al. virus research 272 (2019) 197726 titer of ibv-ryn (10 7.17 /100 μl) was comparable to that of its parental virus ibv-yn, (10 6.83 /100 μl). we also assessed the virus titers of the same virus stocks in cek cells to determine the tcid 50 . 100 μl of collected p10 ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted (10 −1 -10 -6 ) and added onto 80% confluent cek cells. four replicate wells on 24-well plates were used for each dilution. after 48 h incubation at 37℃, 5% co 2 , the 24-well plates were assessed for ibv infection by an ifa assay as described above. titers were determined by observing infected cells under a fluorescence microscope and calculating the tcid 50 per 0.1 ml. the tested tcid 50 titer of ryn was almost equal to that of yn at 10 3.33 and 10 3.00 , respectively. finally, we assessed growth kinetics of ibv-ryn and ibv-yn in eces and cek cells. for the replication profile comparison in ece, 200 μl pbs containing 10 2.0 eid 50 of ibv-ryn or ibv-yn were inoculated into the allantoic cavities of 10-day old eces, and the allantoic fluid of five eggs from each group were harvested at the time points of 12, 24, 36, 48, 60 and 72 hpi and pooled for the real-time pcr detection assay (zhao et al., 2015) . as shown in fig. 5b , the recombinant ibv-ryn replicated in eces with similar growth kinetics as the parental ibv-yn virus. although the ibv-ryn had a relatively low replication speed at the first 24 h, both viruses could reach the peak titers after 36 h and stay at a high level as long as 72 h. to assess the replication kinetics in cek cells, 200 μl pbs containing 10 3.0 tcid 50 of ibv-ryn or ibv-yn were used to infect cek cells in 24well plates, and the supernatants from three wells from each group y. zhao, et al. virus research 272 (2019) 197726 were harvested at the time points of 6, 12, 24, 36, 48, and 60 hpi. viral load was determined by a real-time pcr detection assay quantifying ibv n gene as previously described (zhao et al., 2015) . as shown in fig. 5c , growth kinetics and peak titers of the recombinant ibv-ryn were indistinguishable from those of ibv-yn (fig. 5c) . collectively, these results show that the recombinant ibv-ryn has the same in ovo and in vitro phenotype as the parental ibv-yn. in 2000, the first full-length infectious clone of coronavirus was successfully rescued using the bac system (almazán et al., 2000) . the advantage of the bac system is that it can accommodate larger foreign genes or dna fragments, and methods for modification and screening are well established (almazán et al., 2015) . nevertheless, for certain coronaviruses stability of full-length bac clones remained a problem. an alternative system was described in the same year by yount and colleagues (yount et al., 2000) . they assembled a series of smaller subclones into an intact full-length tgev cdna by in vitro ligation (yount et al., 2000) . viral mrna was obtained by in vitro transcription, and then electroporated into bhk21 cells to successfully rescue recombinant tgev. notably this study also revealed that expression of the n protein facilitates the rescue of recombinant coronaviruses. one year later a third reverse genetic system for coronaviruses was described that is based on the use of vaccinia virus as eukaryotic cloning vector. this system has been first described for the generation of recombinant hcov-229e and was subsequently applied to the generation of recombinant mouse hepatitis virus (mhv; coley et al., 2005) , ibv (casais et al., 2001) , feline infectious peritonitis virus (fipv; tekes et al., 2008) , and sars coronavirus (van den worm et al., 2012). as a large dna virus, vaccinia virus has the following advantages: 1) vaccinia virus itself can accommodate large fragments of foreign genes (at least the size of coronavirus genomes), and the virus' own infectivity and replication ability are not affected. 2) the inserted foreign dna can be stably maintained and amplified in the vaccnia virus genome without the need to rely on prokaryotic cloning systems. 3) vaccinia virus can be modified by homologous recombination, making it simple and easy to introduce mutations, insertions or deletions into cloned coronavirus genomes. several studies have employed the reverse genetic system of ibv to study pathogenicity, tissue tropism, and functionality of accessory genes, as well as to explore the potential for a vector-based vaccine of ibv (casais et al., 2003 armesto et al., 2009; bentley et al., 2013) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on the non-pathogenic beaudette strain (casais et al., 2001) or the mass-type attenuated vaccine strains (zhou et al., 2013; beurden et al., 2017) . while according to previous researches, the prevalent serotype of ibv circulating in china and many other countries is qx type (monne et al., 2008; zhao et al., 2016) , and the qx-type strains typically share < 80% nucleotide identity with mass-type strains. therefore, the application of currently available mass-type ibv reverse genetics systems cannot provide full insight into the immunogenicity and pathogenicity of qx-type strains. in this study, we successfully established a reverse genetic system based on a highly virulent qx-type (gi-19) strain yn isolated in china. we could show that the recombinant ibv-ryn replicates with the same kinetics, reaches the same peak titers, and displays comparable pathogenicity as the parental ibv-yn in eces. this indicates that this system will be a powerful tool to elucidate crucial viral and host factors that impact the high pathogenicity of qx-type ibv strains, and it may also serve as a starting point for the generation of novel vaccine candidates directed against ibv-qx-type strains. we also developed a modified rescue strategy by combining full length rna electroporation into a bhk-21/n cell line and subsequent virus amplification in ovo. this strategy was highly efficient for the rescue of field strains. by introduction of reporter gene egfp into the ibv-ryn genome, we could easily compare the rescue efficiency of bhk-21 and bhk-21/n cell lines. after electroporation with same amount ryn-egfp/5a full-length rna, bigger numbers of green fluorescent cells were observed in the bhk-21/n cell line than bhk-21 cell line, indicating the higher rescue efficiency of it. also, in our study, a remarkable difference of virus infectivity in ece (eid 50 could reach 10 6.0 -10 7.0 per 0.1 ml) was observed compared to cek cells (tcid 50 could only reach 10 3.0 per 0.1 ml) for ibv-yn, suggesting that ece are more susceptible to ibv infection and thus greatly facilitate the rescue of recombinant viruses. by combining electroporation of ibv rna into bhk-21/n cells and subsequent virus amplification in ece we could indeed reach a rescue efficiency of 100% and only one passage in ece was required for virus recovery, which makes the procedure for generating recombinant ibv field strains feasible and less labor intensive. in conclusion, we demonstrate the successful development of a vaccinia virus-based qx-type ibv reverse genetics system. this system allows for the generation of defined, genetically modified viruses to study ibv molecular biology and pathogenesis. we expect that the improved rescue procedure, involving rna transfection into bhk21/n cells and virus amplification in ece, is readily applicable to other ibv field strains, and may lead to the development of vaccines to prevent or control infections of newly emerging ibv field strains. the authors declare that they have no conflicts of interest. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus engineering the largest rna virus 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bronchitis in chickens reverse genetics of sars-related coronavirus using vaccinia virus-based recombination isolation and identification of glandular stomach type ibv (qx ibv) in chickens analysis of antigenicity and pathogenicity reveals major differences among qx-like infectious bronchitis viruses and other serotypes in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine evolution of infectious bronchitis virus in china over the past two decades establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 this study was supported by the national key research and development program of china (2017yfd0500700). we thank kate fox, dphil, from liwen bianji, edanz group china (www.liwenbianji. cn/ac), for editing the english text of a draft of this manuscript. key: cord-301720-majpfxqn authors: saadat, yousef; bozorgmehri fard, mohammad hassan; charkhkar, saied; hosseini, hossein; shaikhi, nariman; akbarpour, bijan title: molecular characterization of infectious bronchitis viruses isolated from broiler flocks in bushehr province, iran: 2014 2015 date: 2017-09-15 journal: vet res forum doi: nan sha: doc_id: 301720 cord_uid: majpfxqn the aim of this study was to provide information on the molecular characteristic and the phylogenic relationship of infectious bronchitis viruses (ibv) strains in bushehr province in comparison to other strains reported in the middle east. samples were collected from broiler flocks in bushehr province during 2014 2015. these flocks had respiratory problems such as gasping, sneezing and bronchial rales. a number of 135 tracheal swabs were taken from fifteen flocks (nine swabs per flock). each three swabs collected from each flock were pooled in one tube (finally, we had three tubes for each flock). the samples were subjected to reverse transcription polymerase chain reaction (rt-pcr). the pcr products of positive samples were analyzed by sequencing of a (392 bp) segment of the spike gene and the related results were compared with the other ibv sequences in genbank database. samples from twelve farms (80.0%) were found to be positive. the viruses from seven farms (46.6%) were identified as field viruses closely related to variant 2. the viruses from three farms (20.0%) were characterized as mass type and were related to vaccine strains. two different ib viruses (variant 2 and mass) were detected in samples from two farms (13.3%). the variant 2 genotype detected in bushehr had high similarity to variant 2 reported from the middle east. these variants displayed homologies ranging from 72.9% to 76.5%, and 78.8% to 80.0% with h120 and 4/91, respectively. it is necessary to design vaccination program of poultry farms using ibv strains circulating in the region. infectious bronchitis (ib) is an acute and highly contagious respiratory disease of chickens characterized by respiratory signs, and in young chickens by severe respiratory distress and a decrease in egg production in layers. 1 the chicken was considered the only natural host of infectious bronchitis virus (ibv) but recently pheasants has been introduced as the other natural host for ibv. 2 the disease is transmitted by the respiratory route, direct contact and indirectly through mechanical spread. 3 the virus belongs to coronaviridae, order nidovirales. the ibv and other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses. 4 its genome consists of about 27 kb and codes for four structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope (e) protein. 5, 6 the spike glycoprotein (s) is anchored in the viral envelope and is post-translationally cleaved into two proteins s1 and s2. 7 the s protein is very diverse in terms of both nucleotide sequence and deduced primary protein structure, especially in the upstream part of s1. 8 three hypervariable regions (hvrs) have been identified in the s1 subunit. [9] [10] [11] the s1 subunit induces neutralizing, serotype-specific, and haemagglutinationinhibiting antibodies. [12] [13] [14] [15] [16] [17] amino acid changes in the spike (s) glycoprotein lead to the generation of genetic variants. 18, 19 the high frequency of new ibv variants is a distinguished characteristic of this virus among other coronaviruses. 20 many ibv serotypes have been described probably due to the frequent point mutations that occur in rna viruses and also recombination events. therefore, the characterization of virus isolates which exists in the field is very important. 21 more than 50 serotypes of ibv have been identified and new variants continued to emerge despite the use of live attenuated and killed ibv vaccines. [22] [23] [24] the usage of live attenuated vaccines is the most important preventive measure of the disease, but antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. 18, 19 the ibv massachusetts (mass) type was first detected in iran by aghakhan et al. 25 in 1998, a virus similar to the european 793/b type was isolated in iran (iran/793b/19/08). 26 in recent years, new variants of ibv have been reported from different part of the country. [27] [28] [29] the aim of this study was to provide information on the molecular characteristic and the phylogenetic relationship of prevalent ibv genotypes circulating in chicken flocks in bushehr province, iran. sampling. samples were collected from broiler flocks in different regions of bushehr province as mentioned in table 1 during 2014-2015. these flocks showed respiratory problems such as gasping, sneezing and bronchial rales. a number of 135 tracheal swabs were taken from fifteen flocks (nine swabs per flock). each three swabs collected from each flock were pooled in one tube and submitted to veterinary diagnostic laboratory (tehran, iran). rna extraction viral rna was extracted from the directly pooled tracheal swabs in rlt buffer (qiagen, hilden, germany) and 10 μl 2-mercaptoethanol (merck, darmstadt, germany) per 1 ml buffer using rneasy mini kit (qiagen), according to the manufacturer's protocol. reverse transcription the reverse transcriptation (rt) reaction was performed using reveraid™ first strand cdna synthesis kit (thermo scientific, burlington, canada), according to the product manual. the resultant cdna was immediately used in a pcr or stored at -20 ˚c for later use. amplification of the spike gene nested reverse transcription polymerase chain reaction (rt-pcr) was performed using spike gene primers as described previously to amplify 392 bp fragment of the spike gene. 30 the first round of amplification (495 bp) was performed using sx1 (5ʹ-cacctagaggtttgt/cta/tgcat-´3) and sx2 (5ʹ-tccacctctataaacaccc/ttt-´3) primers. the pcr reaction was performed in 25 μl reaction mixture containing 1 μl dntp (10 mm), 0.50 μl of each primer (25 pmol μl -1 ), 1 μl mgcl2 (50 mm), 2.50 μl 10x pcr buffer, 0.20 μl taq dna polymerase, 2.50 μl cdna and 16.80 μl dh2o (all from sinaclon, tehran, iran). the amplification was performed using 35 thermal cycles including 94 ˚c for 30 sec, 58 ˚c for 30 sec, and 72 ˚c for 30 sec. the pcr product was used as template for the second round of amplification in which sx3 (5´-taatactggc/taattt ttcaga-´3), and sx4 (5ʹ´aatacagattgct tacaaccacc-3) primers were used. the pcr reaction was carried out under the above condition. agarose gel electrophoresis. the pcr products were electrophoresed on 2% agarose gel and visualized by staining with 0.50 μg ml -1 ethidium bromide by uv transilluminator (m-15; uvp, upland, usa). pcr product purification. the pcr products were purified using pcr purification kit (roche, mannheim, germany) according to kit's manufacture instructions. nucleotide sequencing, deduced amino acid analysis and phylogenetic tree. purified rt-pcr products were sequenced by abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, usa) in a forward direction using primer sx3 and in a reverse direction using primer rx4. nucleotide sequence of the pcr product (392 bp), which was submitted to ncbi, were compared with the ibv sequences in genbank database and sequence similarities were analyzed by blast. multiple sequence alignments were carried out with clustal w and phylogenetic tree was constructed with mega software (version 5; biodesign institute, tempe, usa) using the neighbor-joining tree method with 1000 bootstrap. 31 genbank accession number of ibv sequence. the partial s1 gene sequences of ibvs were submitted to the genbank database under accession numbers kx578825-kx578834. the rna was extracted and cdna was synthesized, and further a (392bp) segment of the s1 gene was amplified by nested rt-pcr (fig. 1) . samples from twelve farms (80%) found to be positive (table 1) . on the base gene sequences, phylogenetic tree was constructed from the nucleotide sequences of the s1 glycoprotein gene, revealing that the sequences of the recent iranian strains formed two main groups (fig. 2) . the first group was subdivided into two subgroups: one including kurdistan-sulaymania/12, egypt/beni suef/ 01, turkey/10rs-3161/2010, is/var2-06, variant_2, eg/ clevb-2/ibv/012, ir/bu/variant2/sh1229. 5 it is imperative to recognize the prevalent strain(s) of infectious bronchitis virus in a region or country, and to select the best vaccine strain and the vaccination program for controlling the disease. the major problem in the immunization against ibv is the presence of various ibv serotypes in the field against available vaccines which fig. 2 . the phylogenic tree for ibv strains detected in the current study and other related isolates in the gene bank middle east sequences using mega-5 program. analyses were based on s1 gene 392bp nucleotides. cannot induce proper immunity. the aim of the present study was to detect and identify the type of prevailing ibv strains in bushehr province. previous studies comparing conventional and nested rt-pcr methods indicated that nested rt-pcr was more sensitive for detection of ibv. [32] [33] [34] the implementation of nationwide genotyping of ibv strains is necessary to determine the distribution of virus genotypes and to develop and adopt suitable vaccination strategies. antigenic characterization of ibv isolates is important for selecting new and appropriate vaccines for the corresponding geographical regions. 35 new serotypes or variant strains may emerge due to only a few changes in the amino acid sequence of the s1protein. 3 therefore, the s1 gene of the isolates should be determined to differentiate field and vaccine isolates. 36 regarding the results obtained, 12 ibv isolates were identified. phylogenetic analysis based on s1gene nucleotide sequences showed that most of the iranian isolates belonged to two distinct groups. based on nucleotide sequencing of the s1 gene, a number of field isolates in the present study showed maximum similarity to variant 2 (is/1494/06 like strain). this is the first report of ibv variant 2 in the broiler flocks of bushehr province. these variants displayed homologies ranging from 72.90% to 76.50%, and 78.80% to 80% with h120 and 4/91, respectively ( table 2) . the second group included three strains which were closely related to massachusetts (mass) type strains. in the present study, ir/variant2 viruses (is/1494/06 like) were recognized as major dominant genotypes and the most important ibv type in bushehr province chicken flocks. 27 homayounimehr et al. reported ir/7/2011, r/8/2011, and ir/9/2011 isolates which appeared different from 37 najafi et al. reported variant 2-like viruses (is/1494/06 like) that were the most predominant ibv type in iranian chicken flocks. they shared the highest identity of 99.22% with is/1494/06, turkey/tr8, and eg/ clevb-2/ibv/012. these findings had high similarity with our results. 28 our findings are also in agreement with several other studies carried out in the middle east countries between 2004 and 2015. some iraqi researchers studied circulating viruses in broiler farm and showed that these strains belong to variant 2 (is/1494-lik) that had high nucleotide sequence identity with ibv isolates from iran, israel, egypt, turkey, and kurdistan. 38 the ib viruses in egypt, jordan, turkey and libya showed a close relationship to israeli variants. 28, [39] [40] [41] [42] [43] following the first report by meir et al., 44 the variant 2 has been reported from some middle east countries such as iran. [27] [28] [29] 42, 43 since these countries have close connections (e.g. through language, religion, relationship, holy places, sectarian war, economic exchange, immigration etc.), so these connections can play an important role in spreading of this variant. the first isolation of ibv in iranian chicken flocks was reported in 1994. 25 the present study is the first report on var2 is/1494/06 in bushehr province, in iran, confirming the presence of the var2 genotype. ma5, h120, and attenuated 4/91 ibv-based vaccination strategies have been applied to ib control on poultry farms in iran recently, 45, 46 and despite their use, diagnosis of ib in the vaccinated chickens is common. the results of this study may partially explain the failure of massachusetts-type vaccines and therefore necessitates revising the iranian vaccination strategy against infectious bronchitis. the low identity between most of iranian isolates with mass-type vaccine strain, the presence of variant 2, and other new genotypes 4/91 can be regarded as the causes of vaccination failure. moreover, secondary infections and immunosuppressive agents like infectious bursal disease virus and chicken anemia virus may also lead to vaccination failure and consequently ibv outbreaks among poultry flocks. these substantial reasons can result in immune failure, poor cross-protection between the field virus and vaccine strain, and the continual emergence of new variants. 28 genotypes found in sulaymania-kurdistan, iraq, included group a (very similar to iranian isolates), and group c (similar to is/1494 and egypt/beni-seuf/01 isolates). 47 these findings are in agreement with the present study. cross-protection between ibv strains depends on the amino acid similarity of s1. based on s1 glycoprotein amino acid sequence, iranian ibv's homology with h120 vaccine, massachusetts vaccine and 793/b vaccine ranges from 72.90% to 76.50%, from 71.80% to 75.30% and from 78.80% to 80.00%, respectively. these findings explain the poor vaccine performance in the field and show that the disease outbreaks were associated with ibv variants, which circumvent vaccination immunity. further, the findings emphasize the need for new control strategies of ibv in iran. in summary, the present study is the first report of ibv in bushehr, iran, illuminating the circulation of a variant of ibv genotype in chicken farms. heterogeneity with vaccine strains can explain a poor vaccination performance and disease outbreak in this area. the results emphasize the need for new control strategies and re-arrangement of preventative measure of ibv in bushehr, iran. one of a series of the veterinary medicine-large animal clinical sciences department, florida cooperative extension service avian infectious bronchitis virus detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand severe acute respiratory syndrome vaccine development experiences of vaccination 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of sul/01/09 avian infectious bronchitis virus indicates the emergence of a new genotype in the middle east genotyping of infectious bronchitis viruses from broiler farmsin iraq during 2014-2015 emergence of a novel genotype of avian infectious bronchitis virus in egypt al saad s. presence of infectious bronchitis virus strain ck/ch/ldl/97i in the middle east presence of is/1494/06 genotype-related infectious bronchitis virus in breeder and broiler flocks in turkey detection and molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 detection of variant infectious bronchitis viruses in broiler flocks in libya identification of a novel nephropathogenic infectious bronchitis virus in israel phylogenetic study of iranian infectious bronchitis virus isolates during 2010 -2011 using glycoprotein s1 gene a survey of the prevalence of infectious bronchitis virus type 4/91 in iran investigation and molecular characterization of avian infectious bronchitis virus in suspected broiler farms in slemani governorate key: cord-296167-np0b9a7o authors: mardani, karim; noormohammadi, amir h.; ignjatovic, jagoda; browning, glenn f. title: naturally occurring recombination between distant strains of infectious bronchitis virus date: 2010-06-24 journal: arch virol doi: 10.1007/s00705-010-0731-z sha: doc_id: 296167 cord_uid: np0b9a7o new variants of infectious bronchitis virus (ibv) have emerged in australia despite its geographical isolation and intensive vaccination programs. in the present study, the 3′ terminal 7.2 kb of the genome of a recently isolated variant of ibv (n1/03) was sequenced and compared with the sequences of classical and novel strains of ibv, the two main groups of these viruses in australia. the comparison revealed that recombination between classical and novel ibvs was responsible for the emergence of the new variant. it was concluded that novel ibvs, which have not been detected since 1993, and which are phylogenically more distant from classical ibvs than turkey coronaviruses, might still be circulating and contributing to the evolution of ibv in australia. infectious bronchitis virus (ibv), the prototype of the family coronaviridae, is an important pathogen in chickens and infects the respiratory tract, kidneys and oviduct, causing reduced performance, reduced egg quality and quantity, increased susceptibility to infection with other pathogens and increased mortality [4] . the genome of ibv is single-stranded, positive-sense rna of about 27.6 kb, encoding four structural proteins, including the spike glycoprotein (s), the membrane glycoprotein (m), the phosphorylated nucleocapsid protein (n) and the small membrane protein (e). new serotypes and genotypes of ibv emerge frequently in different parts of the world [1, 6, 9, 10, 15, 18, 30, 31] . a number of factors, including mutation and recombination, and the widespread use of live attenuated vaccines, play an important role in increasing the number of new genetic variants [3, 8, 15, 16, 28] . recombination and mutation are two major forces in coronavirus evolution, and polymerase jumping during coinfection may promote recombination events between coronaviruses [22] . molecular characterization using sequencing and phylogenetic analysis are effective methods for detection and characterization of recombination events among rna viruses [29] . n1/62, a nephropathogenic strain of ibv, was the first isolate of ibv obtained in australia, in 1962 [7] . since 1962, a number of distinct strains of ibv have been isolated, including a group of strains that were found to have no epitopes in common with other ibv strains on either their n or m proteins [12] . in a study on the pathogenicity of australian strains of ibv, a change was detected in the prevalence of ibv strains, from highly nephropathogenic strains in the 1960s and 1970s to strains predominantly pathogenic for the respiratory tract in the 1980s and early 1990s, indicating that ibv strains in australia have undergone a significant change since 1962 [13] . ibv strains in australia have been classified into two groups, classical and novel [21] , based on their genotypes. the novel ibvs are phylogenetically distant from the classical ibvs, and in fact, classical ibvs and turkey coronaviruses are more similar to each other than they are to the novel ibvs. recently, the isolation of a new variant of ibv in australia, from broiler farms located in new south wales, that had low s1 gene identity to those of classical and novel ibvs but n gene and 3 0 untranslated region sequences similar to those of classical ibvs, suggested the emergence of a new subgroup of ibvs in australia [14] . in this study, the new variant of ibv that circulated in nsw during 2002 and 2003 was further analysed by comparing the 3 0 terminal 7.2 kb of its genome (all the structural protein genes) with those of the other two ibv groups in australia. the ibv strain n1/03 used in this study has been described previously [14] . virus propagated in allantoic fluid was used for rna extraction. extraction of rna was performed using an rneasy kit (qiagen, hilden, germany) according to the manufacturer's instructions. approximately 50 ll of allantoic fluid was used for each extraction, and rna was eluted in 30 ll of elution buffer. the extracted rna was used as template in a reverse transcription reaction. for synthesis of cdna, 5 ll of extracted rna was denatured at 100°c for 1 min, cooled by placing on ice for 5 min, and then mixed with 20 ll of reaction buffer containing 10 ll of diethylpyrocarbonate-treated water, 0.5 lm oligo (dt) (promega, madison, wi, usa), 0.65 u of rnaguard (amersham pharmacia biotech, sydney, australia), 50 lm each of datp, dttp, dgtp and dctp, 5 ll of 59 reaction buffer (promega), and 50 u of moloney murine leukaemia virus reverse transcriptase (promega). the reaction mixture was incubated at 42°c for 1 h and subsequently incubated at 100°c for 5 min to inactivate the reverse transcriptase. the resultant cdna was immediately used or stored at -70°c for later use. for the amplification reaction, two primers, poly-f1 (gattgtgcatggtggacaatg) and utr-r1 (ctgt accctcgatcgtactc), binding to the 3 0 end of the polymerase gene (nucleotides 20,070-20,090, beaudette strain, genbank accession number nc_001451) and the 3 0 untranslated region (nucleotides 27,489-27,508, beaudette strain) of the ibv genome, respectively, were used to amplify a 7.2-kb fragment of the ibv genome that contains all of the genes for the structural proteins. the pcr reaction was carried out in 50-l mixtures containing 50 lm each of datp, dttp, dgtp and dctp, 0.5 lm of each primer, 5 ll of 10 9 high fidelity pcr buffer (invitrogen, carlsbad, ca, usa), 2 mm magnesium sulfate, 1.5 u platinum taq high fidelity dna polymerase (invitrogen) and 5 ll of cdna as template. amplification was performed using 35 cycles of incubation at 94°c for 30 s, 57°c for 30 s and 68°c for 8 min, with a final extension at 68°c for 10 min. the resultant pcr products were separated in a 0.8% agarose gel, and the gel was photographed. pcr product was purified using a pcr purification kit (ultra clean pcr clean-up, mo bio laboratories, solana beach, ca, usa) according to the manufacturer's instructions. the 3 0 7.2-kb pcr product from ibv strain n1/03 was cloned in the pgem-t plasmid using a pgem-t vector system cloning kit (promega). the cloned pcr product was sequenced using a big dye terminator v3.1 cycle sequencing kit (applied biosystems), and the reaction products were sent to the australian genomic research facility, walter and eliza hall institute of medical research, melbourne, for analysis on an abi prism 3100 genetic analyzer. sequences of different genes of n1/03 were aligned with the same genes from other australian ibvs and the beaudette strain (table 1) using clustalw [11] . evolutionary relationships between australian ibv genes were inferred using the neighbour-joining method [24] . the evolutionary distances were computed using the maximum composite likelihood method [26] and measured as the number of base substitutions per site. phylogenetic analyses were conducted in mega 4 [27] . the 3 0 -terminal 7.2 kb of the genomes of the representative classical and novel australian ibv strains were aligned with the sequences from the same region of the n1/03 isolate, and the multiple alignment results were introduced into simplot version 3.5.1 to identify likely recombination sites [19] . bootscanning analysis was performed in simplot to identify and map the putative recombinant ibv genome [25] . this program was also used to find phylogenetically informative sites, as described previously by robertson et al. [23] . statistical analysis of the distribution of phylogenetically informative sites was performed using minitab version 15 (minitab inc, us). a two-proportion comparison was performed, and a p value of less than 0.05 was considered significant. amplification, sequencing and analysis of the 3 0 7.2-kb region of n1/03 the 3 0 7.2 kb of the n1/03 genome was amplified, cloned and sequenced successfully and compared with the same region of a number of classical and novel strains. phylogenetic trees were constructed for each gene. analysis of the s gene sequences of australian ibv strains showed that, among classical strains, they differed by 3-12.5% and, among novel strains, by 3.2-16.4%. the classical and novel ibvs differed by 36.9-39.9%, indicating a major difference between the s genes of these two groups of ibvs. interestingly, the s1 gene sequence of strain n1/03 differed from those of both classical and novel ibvs by 29.2-32%. phylogenetic analysis of the s gene grouped n1/03 in a separate cluster from classical and novel ibvs (fig. 1) . e gene sequences of classical strains differed by 0-9.8%, and those of novel strains by 24-27%. the difference between classical and novel ibvs ranged from 66.5 to 78.8%. more interestingly, the e gene sequence of n1/03 was very similar to those of the classical strains (differences of 3.8-8.9%) and very different from those of novel ibvs (differences of 72.2 to 73.4%). in the e gene phylogenetic tree, n1/03 clustered with the classical ibvs (fig. 2) . m gene sequences of classical strains differed by 0-10.4%, and those of novel ibvs differed by 3-3.6%. the m gene sequences of classical and novel ibvs differed by 54.2-60.9%. the n1/03 m gene clustered with the classical strains (differences of 2.4-9.8%) (fig. 3) . the n gene of classical ibvs differed by 0.2-8.3%, and those of novel ibvs differed by 9.9-16.4%. the n gene sequences of classical and novel ibvs differed by 50.7-54.4%. the n gene of n1/03 was more similar to the n genes of the classical ibvs (differences of 2.4-7.7%) than of novel ibvs (differences of 54.3-55.6%) and clustered with the classical strains in phylogenetic analyses (fig. 4) . to identify the region likely to have been involved in recombination in n1/03, similarity plots and bootscan analysis were performed using strains vics and n1/88 as representatives of the two main groups of ibv in australia and the armidale strain as a query. in both the similarity plots and the bootscan graph, n1/03 had greater similarity to n1/88 (a novel ibv strain) in its s gene region (almost 3 kb), while the reminder of the 3 0 -terminal 7.2-kb region had greater similarity to vics (a classical ibv strain) (fig. 5, 6 ). data from phylogenetic analyses also suggested that n1/03 had sequence similarity to both main groups of australian ibvs. these findings suggested that there had been a recombination event with a crossover point at the end of the s gene (base number 3000) of n1/03 (fig. 5, 6) . to obtain a precise picture of this possible crossover point, the distribution of phylogenetically informative sites along the 3 0 -terminal 7.2-kb sequences of n1/88 and vics were examined. using simplot, 456 informative sites were found that could support one of three possible trees. for almost 90% of the s gene (3 kb), 365 informative sites were found, and 84% of these informative sites supported a tree clustering n1/03 with the novel ibv strain n1/88. in contrast, only 8% of the informative sites supported each of the other two possible trees in this region (p \ 0.0005). for the rest of sequences (3,000-7,200 bp), 95 informative sites were found, with 60% supporting the tree clustering n1/03 with vics and only 14% supporting the tree clustering n1/03 with n1/88 (p \ 0.05). this indicated that n1/03 clustered with the classical ibv strain vics throughout the 3 0 -terminal 4.2 kb of its genome and confirmed that a recombination event had occurred at the end of the s gene region (at around position 3000) and that n1/03 is a recombinant virus that emerged from recombination between these two groups of ibvs in australia. new ibv isolates have been reported in recent years in australia and have been classified as subgroup 3 viruses. the emergence of these new isolates was considered to be unexplained, as was the earlier emergence of subgroup 2, or novel viruses. n1/03 is one of these new viruses, which have been isolated from broiler farms using one of the four commercial ibv vaccines [14] . this study was undertaken to explore the mechanism behind the emergence of the subgroup 3 ibvs in australia. based on phylogenetic analysis of the complete 3 0 7.2-kb region of the genome, n1/03 was distinct from both classical and novel ibvs. however, the s gene sequence of fig. 4 evolutionary relationships between n genes of australian ibv strains inferred using the neighbour-joining method. the evolutionary distances were computed using the maximum composite likelihood method and are given in the number of base substitutions per site n1/03 was equally different from both the classical and novel ibvs, while the rest of the 3 0 -terminal 7.2-kb region of the genome clustered closely with those of the classical ibvs and was very different from those of the novel ibvs. the similarity of the n1/03 e, m and n genes to those of classical ibvs suggested that this isolate was closely related to the most commonly used australian ibv vaccine strains. as these strains have been used for a long time in australia, it is possible that they have contributed to the emergence of variant ibvs in the field. the s gene of n1/03 clustered more closely with the novel ibvs, and similarity plotting and bootscan analysis confirmed the close relationship between the s genes of n1/03 and the novel ibvs and suggested that the crossover event occurred near the 3 0 end of this gene. these analyses provide convincing evidence that recombination has occurred between classical and novel ibvs, leading to emergence of variant ibvs in the field. novel ibvs have not been isolated since 1992, but this finding suggests that novel ibvs are still circulating in the field in australia. the failure to detect them since 1992 may be a result of the relatively slow growth of these viruses [20] . these results confirm the hypothesis of ignjatovic et al. [14] that recombination may have played a role in the emergence of this new variant of ibv in australia. recombination between ibvs and its role in emergence of new ibv variants has been reported previously [2, 15, 17, 21] and may occur at multiple sites [15, 17] , although crossover events occur more frequently at the 3 0 end of the s gene [5, 15, 21] , as seen in this study. however, it is particularly notable here because of the very significant phylogenic distance between the novel and classical australian strains of ibv. indeed, the novel strains of ibv are more distinct from the classical strains than is turkey coronavirus, and they lack several of the smaller nonstructural genes found in classical strains. in conclusion, this study showed that the recombination was involved in the emergence of the new variant of ibv in australia and that a rapid and reliable technique is needed to determine whether the novel ibvs are still circulating in poultry farms. it would be appropriate to sequence and analyse the polymerase genes of classical, novel and new variant strains of ibv to obtain further information about the relationships between the different australian ibvs. isolation and characterization of new infectious bronchitis virus variants in hungary comparisons of envelope through 5b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes recent advances in avian virology severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan isolation of a new serotype of infectious bronchitis-like virus from chickens in england infectious avian nephrosis (uraemia) in australia a recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adaptedvaccine strain variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens a 'new' strain of infectious bronchitis virus infecting domestic fowl in great britain bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt a long-term study of australian infectious bronchitis viruses indicates a major antigenic change in recently isolated strains pathogenicity of australian strains of avian infectious bronchitis virus isolation of a variant infectious bronchitis virus in australia that further illustrates diversity among emerging strains a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china fulllength human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination infectious bronchitis viruses with a novel genomic organization genotypic and phenotypic characterization of the california 99 (cal99) variant of infectious bronchitis virus coronavirus replication and pathogenesis: implications for the recent outbreak of severe acute respiratory syndrome (sars), and the challenge for vaccine development recombination in aids viruses the neighbor-joining method: a new method for reconstructing phylogenetic trees identification of breakpoints in intergenotypic recombinants of hiv type 1 by bootscanning prospects for inferring very large phylogenies by using the neighbor-joining method mega4: molecular evolutionary genetics analysis (mega) software version 4.0 evidence of natural recombination within the s1 gene of infectious bronchitis virus evolutionary aspects of recombination in rna viruses characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens avian infectious bronchitis: characterization of new isolates from italy acknowledgments the senior author would like to thank the department of education, employment and workplace relations of australia for providing him with an endeavour research fellowship award, which enabled this work to be completed. he also thanks urmia university, iran, for granting him leave to go to australia to undertake this work. key: cord-285323-473d7zvg authors: jang, hyesun; koo, bon-sang; jeon, eun-ok; lee, hae-rim; lee, sang-myeong; mo, in-pil title: altered pro-inflammatory cytokine mrna levels in chickens infected with infectious bronchitis virus date: 2013-09-01 journal: poult sci doi: 10.3382/ps.2013-03116 sha: doc_id: 285323 cord_uid: 473d7zvg infectious bronchitis virus (ibv) replicates primarily in the respiratory tract and grows in various organs in chickens, with or without pathological effects. the diversity of this virus has been verified by sequence analysis of the s1 glycoprotein gene, but this method must be supplemented with further analysis for characterization of the agent. to increase our understanding of the pathogenesis of the disease caused by this virus, we investigated the response of chickens to 2 ibv with different genotypes, kiia and chvi. the clinical signs induced by the viruses were observed. in addition, the mrna levels of the pro-inflammatory cytokines, il-6, il-1β, and lipopolysaccharide-induced tumor necrosis factor-α factor and the serum levels of α(1)-acid glycoprotein, which is a major acute phase protein, were measured. the kiia genotype (kr/adl110002/2011) induced clinical signs accompanied by the excessive production of pro-inflammatory cytokines and a higher viral load. in chickens infected with this isolate, simultaneous peaks in the viral copy number and cytokine production were observed at 7 dpi in the trachea and 9 d postinoculation in the kidney. on the other hand, the chickens infected with the chvi genotype (kr/adl120003/2012) did not show a response other than a mild upregulation of cytokines at 1 d postinoculation, which appears to indicate the invasion of the virus. in summary, we confirmed a differential innate response following infection with distinct ibv. we hypothesize that an excessive innate response contributes to the scale of the pathophysiologic effect in chickens. infectious bronchitis (ib) is a common, highly contagious, and acute viral disease of poultry. the disease is caused by a coronavirus known as ib virus (ibv). the virus initiates infection via the respiratory tract and replicates in various types of epithelial cells, including those of the kidney, gonads, and alimentary tract (villegas, 1998; saif et al., 2011) . after the identification of the virus, a high diversity among isolates was reported, not only in antigenicity, but also in pathogenicity, and the nature of this virus motivated researchers to develop an efficient way to classify the isolates (hofstad, 1958; gelb et al., 1991; lee et al., 2008) . genotyping is a relatively recent method of viral classification that is preferred today because of its ability to rapidly detect new strains. it is based on sequence analysis of the s1 portion of the s protein gene, which encodes the virus-neutralizing epitope and is responsible for viral attachment to cells (ma et al., 2012) . at least 3 genotypes of ibv have been identified in korea by s1 sequence analysis (lim et al., 2012) . the kii type, one of the antigenic clusters of the korean isolates, has frequently been isolated in field outbreaks and is related to nephropathogenic chinese ibv strains (e.g., the ch/qxibv strain; lee et al., 2008) . the rapid detection of variant isolates is the strongest aspect of the s1 sequence analysis method. however, because the presumed tissue tropism from this system is not always in agreement with the actual tissue tropism (sapats et al., 1996) , a more detailed understanding of the immunopathogenesis of ib infections is required. such an understanding will enable scientists to be prepared for the emergence of variants with unusual tissue tropisms (raj and jones, 1997) . the study of immunopathogenesis has been used to verify disease progress in diverse viral infections. the hyesun jang ,* bon-sang koo ,* eun-ok jeon ,* hae-rim lee ,* sang-myeong lee , † and in-pil mo * 1 * preventive veterinary medicine, college of veterinary medicine, chungbuk national university 12, cheongju, chungbuk, republic of korea; and † cell biology & molecular immunology laboratory, division of biotechnology, college of environmental & bioresource sciences, chonbuk national university, iksan, jeonbuk, , republic of korea abstract infectious bronchitis virus (ibv) replicates primarily in the respiratory tract and grows in various organs in chickens, with or without pathological effects. the diversity of this virus has been verified by sequence analysis of the s1 glycoprotein gene, but this method must be supplemented with further analysis for characterization of the agent. to increase our understanding of the pathogenesis of the disease caused by this virus, we investigated the response of chickens to 2 ibv with different genotypes, kiia and chvi. the clinical signs induced by the viruses were observed. in addition, the mrna levels of the pro-inflammatory cytokines, il-6, il-1β, and lipopolysaccharide-induced tumor necrosis factor-α factor and the serum levels of α 1 -acid glycoprotein, which is a major acute phase protein, were measured. the kiia genotype (kr/adl110002/2011) induced clinical signs accompanied by the excessive production of pro-inflammatory cytokines and a higher viral load. in chickens infected with this isolate, simultaneous peaks in the viral copy number and cytokine production were observed at 7 dpi in the trachea and 9 d postinoculation in the kidney. on the other hand, the chickens infected with the chvi genotype (kr/ adl120003/2012) did not show a response other than a mild upregulation of cytokines at 1 d postinoculation, which appears to indicate the invasion of the virus. in summary, we confirmed a differential innate response following infection with distinct ibv. we hypothesize that an excessive innate response contributes to the scale of the pathophysiologic effect in chickens. correlation between immunopathogenesis and disease progress is widely accepted in diverse studies. in the case of severe acute respiratory diseases, such as sars, immunopathogenesis has been suggested to contribute to disease progression, providing the rationale for therapy with corticosteroids (muñoz-fernandez, 2004) . during h5n1 influenza infection, an excessive cytokine level in the blood and the innate immune response were suggested to play a role in disease progression peiris et al., 2004) . in poultry, a role for cytokines in disease progression was suggested in marek's disease, mycoplasmosis, salmonellosis, and coccidiosis (laurent et al., 2001; swaggerty et al., 2004; withanage et al., 2004; jarosinski et al., 2005; mohammed et al., 2007) . especially in ib infections, analyzing the correlation between immunopathogenesis and disease progression appears to be valid and effective, considering that the severity of ib infection depends significantly on host factors such as immune status, age, and breed. in a previous study, the renal disease induced by an ib isolate was reproduced at different scales according to the age of the infected chickens (dolz et al., 2012) . the discrepancy in pathogenicity among different genetic lines of specific pathogen-free (spf) chickens following the same nephropathogenic strain has also been documented (otsuki et al., 1990; ignjatovic et al., 2003) . however, little is known about immunopathogenesis during ib infection in chickens (asif et al., 2007) . one report investigated the overexpression of il-6 induced by the t strain and its role in exaggerated pathogenicity in s-line chickens (asif et al., 2007) . this report demonstrated that the overexpression of il-6 by the host may be responsible for the pathological sequelae, but it remains to be verified whether similar dysregulation could be induced by distinct pathotypes, including nonpathogenic strains (asif et al., 2007) . in this study, we observed changes in the transcriptional levels of 3 pro-inflammatory cytokines that are known to be involved in the innate immune response in chickens (hong et al., 2006; davison et al., 2008) after inoculation with 2 ib isolates. because these 2 isolates are distinct in genotype, we expected to confirm that the scale of change in the cytokines in the trachea and kidney would be correlated with pathogenicity. from this observation, we expected to suggest a strategy to reduce loss from ib infection and to provide helpful information about the immunopathogenesis of ib infection (cook and huggins, 1986) . among ibv isolated from chickens showing significant clinical signs, 2 field isolates were chosen for this study (table 1) . briefly, the isolate of the kiia genotype (kr/adl110002/2011) was isolated from kidneys of 3-wk-old broilers suffering from severe nephritis, respiratory signs, and increased mortality (1% per day). according to s1 sequence analysis, this isolate belongs to the kiia type, which is closely related to the qx-ib strain. the isolate of the chvi genotype (kr/ adl120003/2012) was derived from 38-wk-old layers with a slight decrease in egg production. this genotype (chvi) does not belong to major clusters of korean ib isolates but appears to be derived from a chinese strain (liu et al., 2006) . the virus stock for the challenge study was produced by inoculating field isolates into embryonated spf chicken eggs via the allantoic cavity and collecting the infectious allantoic fluid 72 h postinoculation. reverse-transcription (rt) pcr targeting the ibv s1 gene was carried out, and each 630-bp s1 amplicon was sequenced for genotyping by the direct sequencing method. the median embryo infective dose (eid 50 ) was determined by the reed and munch formula. the isolates were shown to be free from contamination by other avian pathogens by inoculating 3-wk-old spf chicks and testing their sera at least 2 wk later using the procedures for the accreditation of spf flocks. in this preliminary experiment, the chickens inoculated with the isolate kr/adl110002/2011 showed severe nephritis after prolonged infection (>14 d), whereas no significant clinical symptoms were observed in chickens inoculated with the same dose of the isolate kr/adl120003/2012. during the experiment, the chickens were raised in a hepa-filtered isolation system. all procedures, including euthanasia, followed the guidelines of the institute of laboratory animal resources (confirmation number: cbnua-446-12-01). forty-five 3-wk-old spf chickens (valo biomedia north america, adel, ia) were used and were divided into 3 groups of 15: an unchallenged control group (group 1), a group challenged with an isolate of the kiia genotype (kr/adl110002/2011, group 2), and a group challenged with an isolate of the chvi genotype (kr/adl120003/2012, group 3; table 1 ). the chickens were inoculated with an eye drop of 0.2 ml of 10 5 eid 50 of the ib isolates or 0.2 ml of sterilized pbs. at 1, 3, 5, 7, and 9 d postinoculation (dpi), 3 birds in each group were bled before euthanasia, followed by necropsy and sample collection. the blood was kept at room temperature for approximately 3 h, and the serum was separated and stored at −20°c. during necropsy, tissues of the trachea and kidney were collected and immediately treated with trizol reagent (invitrogen, carlsbad, ca) for total rna isolation; tissue samples were taken from the upper one-third of the trachea by the horizontal long axis and the upper left lobe of the kidney. total rna was isolated from tissues obtained from necropsy using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions. homogenized tissue samples were treated with trizol reagent followed by phenol-chloroform phase separation. the rna was precipitated using 2-propanol, washed with 75% ethanol, dried, and resuspended in 100 μl of diethylpyrocarbonate-treated water. the rna was quantified by measuring the absorbance at 260 nm on a spectrophotometer, and the purity was assessed using the 260:280 nm ratio. the viral copy number was determined by semiquantitative rt-pcr. semiquantitative rt-pcr was conducted as previously described, with slight modifications (liu et al., 2008) . briefly, rna samples and primers targeting the n gene of the ib virus (table 2) were added to maxime rt-pcr premix tubes (intron biotechnology, seongnam-si, gyeonggi-do, korea) and brought to 20 μl with sterilized distilled water. the rt-pcr reaction was performed as follows: one cycle of 30 min at 45°c and 5 min at 94°c, followed by 35 cycles of 94°c for 30 s, 52°c for 30 s, and 72°c for 1 min. upon completion of the last cycle, the reaction mixtures were incubated at 72°c for 5 min. then, 10 μl of each product was separated by 2% agarose gel electrophoresis. the band intensity of the pcr products was calculated using the image j 1.37 v program (http://rsb.info.nih. gov/ij). to generate a standard curve, 10 5 eid 50 of viral stock was serially diluted 10-fold, and rna was extracted from each diluent. the rt-pcr, agarose gel electrophoresis, and image analysis were conducted as described above, and the results were plotted against the dilution factor. for comparison, the corresponding value of a 10 5 dilution of the viral stock on the x-axis was normalized to 1, and the relative viral load for each sample was calculated according to the standard curve ( figure 1 ). the cdna synthesis from the rna samples was performed using the primescriptrt reagent kit (perfect real time, takara biotechnology co., ltd., dalian, china) according to the manufacturer's recommendations. briefly, 10 μl of reaction mixture (primescript buffer, primescript rt enzyme mix i, 25 pmol of oligo(dt) primer, and 0.01 μg of total rna) was incubated at 37°c for 15 min and 85°c for 5 s. then, qrt-pcr was performed using a smart cycler (cepheid, sunnyvale, ca). all reactions were performed in 3 independent samples derived from 3 chickens in 25-μl reaction mixtures containing 12.5 μl of 2 × sybr premix ex taq ii (tlirnasehplus, takara), 9.5 μl of distilled water, 2 μl of cdna template, and 0.1 μm of each primer ( table 2 ). the pcr conditions were the same for each targeted gene: 30 s at 95°c, followed f: 5′-tgtgtatgtgcagcaacccgtagt-3′ ay765397 r: 5′-ggcattgcaatttggacagaagt-3′ by 40 cycles of 95°c for 5 s and 62°c for 80 s. cycling was terminated after 40 cycles, and dissociation curves of the products were generated by increasing the temperature incrementally from 55 to 100°c as the final step of the real-time pcr. to calculate the fold-change over the control chickens treated with pbs, the ratio of the concentration of the target gene to a reference gene (gapdh) in samples from pbs-inoculated controls (calibrator) was normalized. the normalized ratio was then compared with the ratio of the concentration of the target gene to the reference gene in individual samples from ib-isolate inoculated chickens. the mann-whitney u test (spss 12.0 k for windows) was used to determine significant differences between fold change values. the sd was calculated using the fold-change values of 3 replicates for each gene measured. alpha-1 acid glycoprotein (agp), an acute phase protein, was measured in serum using a commercially available elisa kit (chicken α 1-acid glycoprotein (ogchi) elisa kit, cusabio biotech. co. ltd., wuhan, hubei, china). the elisa was conducted according to the manufacturer's instructions. to determine the agp level in the serum, a standard curve was generated, and a regression assay was conducted. to de-termine significant differences, a 2-tailed independent student t-test was used (spss 12.0 k for windows, spss inc., chicago, il). until 7 dpi, no clinical sign was observed in chickens in the unchallenged control group (group 1) or the challenged groups (group 2 and 3). however, in chickens of group 2 (kiia genotype), apparent clinical signs were began to be observed; at 8 dpi, all 3 chickens infected with ibv of kiia genotype sat crouched while the water consumption increased 200% (p < 0.05). the mortality (1 out of 3 birds) was observed only in group 2 at 9 dpi. during necropsy of chickens of group 2, however, we could not observe gross lesions related with these symptoms in kidneys or other organs. in the microscopic examination of chickens of group 2 (kiia genotype), acute response lesions, such as the loss of cilia, acute necrosis of tracheal epithelial cells, and multifocal areas of acute tubular necrosis without any cellular infiltration in the kidney, were observed ( figure 2 ). on the other hand, no mortality or clinical sign observed in chickens of group 2, such as increase in water consumption and depression, was observed in chickens of group 3 (chvi genotype). the n-gene of ibv was amplified by rt-pcr, and the band intensity was rated on a scale of 1 to 10,000 ( figure 3) . until 3 dpi, the viral gene was not detected by rt-pcr. in the tracheae of chickens in group 2 (kiia genotype), the viral copy number was first detected at 5 dpi (1,859.9 ± 642.9) , and the peak (2,954.7 ± 1,021.4) was observed at 7 dpi. in the kidneys of chickens in the same group, the viral copy number was first detected at 7 dpi (97.24 ± 21.3), and the peak copy number was observed at 9 dpi (3,626.1 ± 724.8 ). in group 3 (chvi), the viral gene was first observed at 7 dpi, whereas the viral copy number remained at approximately 100 in the trachea and kidney. these results indicate that the copy number was higher in the chickens from group 2, and their viral replication peaked in the trachea and the kidney at 7 and 9 dpi, respectively. the changes in 3 pro-inflammatory levels are illustrated in figure 4 . at 1 dpi, a significant increase in il-6 and il-1β transcription levels was observed in the . the rt-pcr products were separated on a 2% agarose gel, and the signals were calculated using imagej software (http://rsb. info.nih.gov/ij). a: a standard curve was constructed by amplifying the 10-fold diluted viral stock. lane m is a 100-bp ladder. b: amplification products from different concentrations of the n gene of ibv. the size of the amplification product was 390 bp. estimated viral copy numbers were assumed as 1, 10, 100, and 1,000. kidney and trachea of group 2 (kiia), whereas in group 3 (chvi), an increase was observed only in the kidney il-6 level (p = 0.049; figure 4a ). in the case of lipopolysaccharide-induced tumor necrosis factor (tnf)-α factor (litaf) in the kidney, the arithmetic mean of triplicates in the challenged groups was higher compared with the control group, but a significant difference was not observed among the 3 groups. at 3 dpi, the levels returned to the range of the unchallenged control ( figure 4b) , and the levels of il-6 in the trachea were suppressed. the litaf transcription level in the tracheae of both challenged groups was significantly decreased compared with the control group (p = 0.049) at 3 dpi. at 5 dpi, the mrna levels of il-6 and litaf were higher in the kidneys from group 2 (kiia genotype; figure 4c ), whereas no obvious change was observed in the tracheae at 5 dpi. at 7 dpi, the litaf levels were significantly increased in the tracheae of both challenged groups ( figure 4d ). at the same time, the il-6 levels of the tracheae appeared to be higher; however, a significant difference was not observed. at 9 dpi, the levels of all of 3 pro-inflammatory cytokines were substantially increased in the kidneys in chickens from group 2. considering the viral copy numbers (figure 3 ) and the transcription profiles at 7 and 9 dpi, the peak viral copy number occurred simultaneously with peak cytokine production in each organ. table 3 shows the serum agp level after inoculation with ib isolates. in the unchallenged control group, the agp level was in the range of 37 to 45 μg/ml. the agp level of both challenged groups was compared with the unchallenged group. no significant difference was observed among all 3 groups at 1 dpi. at 3 dpi, the level of agp in chickens from group 2 (kiia genotype) had increased to approximately 44 μg/ml (p = 0.02; table 3 ). at 5 dpi, the agp levels in group 2 (kiia genotype) reverted to the range of the unchallenged control, and no further increase was observed until 7 dpi. at 9 dpi, the agp level increased almost 3-fold in both challenged groups (p < 0.001). these findings appear to reflect viral invasion but do not appear to be associated with pathogenicity. in this study, we confirmed that the pro-inflammatory response could be induced on a diverse scale depending on the ibv genotype. the kiia genotype (kr/adl110002/2011), which is closely related to the nephropathogenic strain, induced clinical symptoms of depression and a stronger pro-inflammatory response. on the other hand, an active infection of the chvi genotype isolate kr/adl120003/2012, which resulted in an increase in serum agp level at 9 dpi (table 3) , evoked only a limited range of pro-inflammatory responses. according to the data collected at 9 dpi, the excessive production of pro-inflammatory cytokines was temporally associated with clinical signs related to renal damage, such as an increased drinking of water and lesions observed in the kidney, including renal tubular necrosis. these data suggest that excessive pro-inflammatory cytokine production contributes to disease severity. in particular, the excessive production of il-6 in ib infections was already suspected to contribute to the exaggerated disease severity in specific genetic lines (asif et al., 2007) . our data have different implications than the results of that study; we have demonstrated that specific ib genotypes induce pro-inflammatory cytokines and that differences in the scale of the induction could contribute to the disease severity induced by ibv of diverse genotypes. in addition to the il-6 profiling, it was meaningful to observe the pattern of change in the levels of 2 cy-tokines, il-1β and litaf, during ib infection because these immune mediators do not act alone in vivo (perrone et al., 2008) . in chickens, litaf has been investigated as a substitute for tnf-α in viral infections (ruby et al., 2006; esnault et al., 2011) , even though its involvement in viral infections was not demonstrated. in mammals, litaf was reported to regulate tnf-α gene expression (myokai et al., 1999) and to modulate pro-inflammatory cytokines (tang et al., 2005b) . it is still debatable whether litaf could be used as an indicator during viral infection, but our findings at 9 dpi demonstrate the upregulation of litaf following ib infection, so a subsequent increase in transcription of tnf-α could be anticipated. in mammals, il-1β and tnf-α are known to have tissue-damaging potential, including inducing apoptosis (dinarello, 1996; eizirik, 2001; hehlgans and männel, 2002) and local damaging effects on the kidney (van de kar et al., 1992; jo et al., 2002; malaponte et al., 2002) . in this study, these features, which were not previously reported in chickens, suggest that these cytokines may contribute to the pathological changes of renal disease in ib infection. at 7 and 9 dpi, temporal associations between peak viral copy number and peak cytokine production were observed. however, the kinetics of viral load were not precisely in accord with those of the pro-inflammatory cytokines. in previous studies of influenza viruses, the differential hyper-induction of cytokines was not explained by differences in viral replication kinetics (chan et al., 2005) , but the accumulation of doublestranded rna within the h5n1-infected cell appears to induce pro-inflammatory cytokines in the absence of productive virus replication (chan et al., 2012) . in case of sars-cov infection, the upregulation of the pro-inflammatory cytokine il-6 was observed during in vivo studies (wong et al., 2004) , but a similar finding was not observed in in vitro studies of productively in-fected cells (tang et al., 2005a) . instead, the induction of il-6 was more significant in macrophages primed by bacterial lipopolysaccharide (tseng et al., 2005) . thiel and weber (2008) speculated that the pro-inflammatory response appears to be a secondary response of immune activation, not a direct response of cells against viral infection (thiel and weber, 2008) . based on these alterations of pro-inflammatory cytokine mrna expression level in the trachea and kidney at 1, 3, 5, 7, and 9 d postinoculation (dpi). the results are expressed as relative expression; the average normalized ratio of the unchallenged control group (group 1) was adjusted to 1 and was compared with those of the challenged groups (groups 2 and 3). column values represent the mean of triplicates, and vertical bars represent the sd value. *statistical significance (p ≤ 0.05) was determined by the mann-whitney u test. group 1: unchallenged control group; group 2: challenge group inoculated with an isolate of the kiia genotype (kr/adl110002/2011); group 3: challenge group inoculated with an isolate of the chvi genotype (kr/adl120003/2012); litaf: lps-induced tnf-α factor. reports, we assume that the pro-inflammatory response is not a direct sequela of viral infection but is instead a secondary response, and the discordance in the kinetics is consistent with that assumption. in conclusion, we confirmed that a different proinflammatory response was associated with isolates of ibv with different genotypes. the induction of excess cytokine production appears to have a detrimental effect on tissues, 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cytokine mrna expression in heterophils isolated from salmonella-resistant and -susceptible chickens comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229e lps induces the interaction of a transcription factor, lps-induced tnf-alpha factor, and stat6(b) with effects on multiple cytokines interferon and cytokine responses to sars-coronavirus infection pathology of fatal human infection associated with avian influenza a h5n1 virus tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome viral diseases of respiratory system rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with salmonella enterica serovar typhimurium plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome key: cord-286332-cdg4im5h authors: van beurden, steven j.; berends, alinda j.; krämer-kühl, annika; spekreijse, dieuwertje; chénard, gilles; philipp, hans-christian; mundt, egbert; rottier, peter j. m.; verheije, m. hélène title: a reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted rna recombination date: 2017-06-12 journal: virol j doi: 10.1186/s12985-017-0775-8 sha: doc_id: 286332 cord_uid: cdg4im5h background: avian coronavirus infectious bronchitis virus (ibv) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. major advances in the study of the molecular biology of ibv have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, ibv strain beaudette. however, most ibv strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. methods: we established a reverse genetics system for the ibv strain h52, based on targeted rna recombination in a two-step process. first, a genomic and a chimeric synthetic, modified ibv rna were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) ibv intermediate (mibv). herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (mhv), allowing for the selection and propagation of recombinant mibv in murine cells. in the second step, mibv was used as the recipient. to this end a recombination with synthetic rna comprising the 3′-end of the ibv genome was performed by introducing the complete ibv spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. results: targeted rna recombination allowed for the modification of the 3′-end of the ibv genome, encoding all structural and accessory genes. a wild-type recombinant ibv was constructed, containing several synonymous marker mutations. the in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental ibv strain h52. conclusions: targeted rna recombination allows for the generation of recombinant ibv strains that are not able to infect and propagate in continuous cell lines. the ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated ibv vaccines and for studies into the biology of ibv in general. electronic supplementary material: the online version of this article (doi:10.1186/s12985-017-0775-8) contains supplementary material, which is available to authorized users. avian coronavirus infectious bronchitis virus (ibv) primarily infects the upper respiratory epithelium of chickens, causing a respiratory disease that is frequently complicated by secondary bacterial pathogens [1] . in addition, some ibv strains affect the renal tubuli, oviduct and parts of the gastrointestinal tract, leading to pathological lesions in these organ systems, with subsequent reduced weight gain and a drop in egg production. the virus has a worldwide presence in both commercial and backyard chickens, appearing in a wide variety of geno-, sero-and protectotypes [2] . ibv is currently regarded as one of the economically most relevant viral pathogens in the poultry industry. infectious bronchitis virus is the prototype gammacoronavirus in the family coronaviridae, order nidovirales [3] . the enveloped virus particles have a positive-sense rna genome of 27.6 kb (fig. 1a ) [4] . the 5′ two-third of the viral genome comprises gene 1, divided into two large open reading frames 1a and 1b, which code for 15 nonstructural proteins primarily involved in rna replication and transcription. the 3′ one-third of the viral genome codes for structural proteins: spike protein (s, encoded by gene 2), envelope protein (e, encoded by gene 3c), and membrane protein (m, encoded by gene 4), each located in the viral envelope. the nucleocapsid protein (n, encoded by gene 6) occurs in the ribonucleoprotein core [5] . interspersed between the structural genes, coronaviruses carry a variable number of genus specific accessory genes [6] . most of their gene products are nonstructural, and their expression is not essential for virus replication in vitro [7] [8] [9] [10] [11] [12] . the ibv genome contains the accessory genes 3 and 5, encoding the proteins 3a and 3b, and proteins 5a and 5b, respectively [4] . in addition, an open reading frame located in the intergenic region was identified between genes 4 and 5 [13] . the typical coronavirus spikes are formed by trimers of the type 1 membrane protein s, which is often proteolytically cleaved into two subunits, s1 and s2 [4, 14] . the glycosylated s1 domain forms the 'head' of the spike and contains the receptor-binding domain [15] . avian gammacoronaviruses typically interact with glycans on the host cell surface. ibv in particular requires α2,3-linked sialic acids for attachment and entry [16] [17] [18] . the s2 domain builds the remaining part of the ectodomain (the 'stalk'), the transmembrane domain and the internally located endodomain. the s protein is the main determinant of the coronaviral host species tropism [19] . many mammalian coronaviruses of the genera alphacoronavirus and betacoronaviruses can be propagated in cultured cells, unlike most avian coronaviruses of the genus gammacoronavirus. ibv can, however, readily be propagated in, and isolated from, embryonated fowl eggs. during passaging in embryonated eggs adaptation occurs, often leading to attenuation. for example, ibv strain h52 represents the 52nd serial passage of a massachusetts-like ibv strain isolated in the netherlands [20] , which causes embryonic death within 48 h post infection (hpi), and still has a residual virulence in young chickens. another 68 passages resulted in the ibv strain h120, which is more attenuated and has a lower pathogenicity in young chicks. similar serial passaging of another ibv strain of the massachusetts serotype isolated in the usa resulted in the generation of the non-pathogenic and cell-culture adapted ibv strain beaudette [21] . in order to study its characteristics, several research groups have independently developed a reverse genetics system (rgs) for ibv which allow the manipulation of its genome [22] [23] [24] [25] [26] . all these systems are based on the nonpathogenic cell-culture adapted ibv strain beaudette, or the highly attenuated ibv vaccine strain h120. a major drawback of the use of the non-virulent ibv strains beaudette and h120 [20, 21] is the inability to provide insights in the infection process in chickens, as these strains no longer cause a clinically relevant phenotype in vivo. yet, the rgs has provided significant insight in the fundamentals of avian gammacoronavirus replication. key findings include that ibv cell tropism is determined by the spike gene [27, 28] , that the low virulence of ibv beaudette is caused by changes in the replicase gene [29] , and that one or more of the ibv accessory gene products interfere with the hosts' interferon response [30] [31] [32] . targeted rna recombination is another reverse genetics approach, so far only developed for mammalian coronaviruses from the genera alphacoronavirus and betacoronavirus [11, 19, 33] . this system is based on the exchange of the spike gene by that of a coronavirus with a different host tropism, which enables subsequent selection on cells susceptible to the heterologous species [34] . as a consequence, manipulation is limited to the last third of the coronavirus genome, covering all genes encoded that are located 3′ of gene 1, starting with the spike gene. targeted rna recombination has been shown to be easy in use and to allow the rescue of highly defective mutants [11, 19, 33] . however, the system is based on the ability to propagate both the donor and the recipient coronavirus in cell culture, and is hence not implementable for pathogenic ibv. this problem was solved by transfecting ibv genomic rna into otherwise non-susceptible cells, exchanging the ibv spike gene by that of the mouse hepatitis virus (mhv) provided as part of a synthetic rna, and by subsequently rescuing recombinant ibv from infected/ transfected cells in embryonated eggs (fig. 1) . this system was successfully established to introduce marker mutations in the last one-third of the genome of ibv. the resulting recombinant viruses demonstrated growth kinetics in ovo and the in vivo phenotypic characteristics in one-day-old chickens similar to ibv wild type. the results presented here demonstrate for the first time a host species switch for an avian gammacoronavirus by exchanging the spike gene with that of the highly divergent betacoronavirus mhv. this rgs enables the manipulation of the structural and accessory protein genes from the genome of virulent ibv. baby hamster kidney (bhk-21) cells (atcc ccl-10) and murine lr7 cells kuo2000 were cultured in dulbecco's modified eagle medium (dmem) (biowhittaker), supplemented with 4 mm l-glutamine (lonza, basel, switzerland), 10% fetal bovine serum (fbs) (biowhittaker) and 0.05 mg/ml gentamicin (gibco invitrogen), at 37°c and 5% co 2 . virus titers in cells were obtained by determining the 50% tissue culture infective dose (tcid 50 ) per ml at 2 days post inoculation (p.i.) according to the spearman-kärber method [35] . fertilized specific pathogen free (spf) white leghorn eggs (animal health service, deventer, the netherlands) were incubated at 37.5°c and 45-65% relative humidity. a b c d fig. 1 coronavirus genome organization and schematic overview of targeted rna recombination. a schematic genome representations of ibv (blue) and mhv (red). the first two-third of the genome is truncated, the structural and accessory genes are drawn to scale. the lengths of the first two-thirds and last one-third of the ibv genome are indicated at the top. the different domains of the spike gene are indicated: ss = signal sequence; ec = ectodomain; tm = transmembrane domain; en = endodomain. pcr amplicons are depicted as black bars drawn to scale above the genomes, with encircled letters referring to the primer sets in table 3 and fig. 3 . b stage 1 in targeted rna recombination: an interspecies chimeric murinized ibv with a mhv spike ectodomain (mibv) is generated by a single recombination event of ibv genomic rna with synthetic rna transcribed from donor plasmid p-mibv in the 3′-end region of the 1b gene (indicated by a black curved line). murinized ibv is selected on murine lr7 cells. plasmid inserts are indicated above p-mibv, with numbers in black circles referring to the plasmid junctions. c stage 2 in targeted rna recombination: a recombinant ibv with the ibv spike gene (ribv) is recreated by a single recombination event of mibv with synthetic rna transcribed from donor plasmid p-ibv. recombinant ibv is selected on embryonated chicken eggs. d nucleotide sequences of the plasmid junctions, marked with corresponding numbers in the schematic donor plasmid drawings. nucleotide sequences are indicated for wild-type ibv and donor plasmids p-ibv and p-mibv, with restriction enzyme sites in italics, mhv spike gene sequences in lower case, and spike domains (i.e. ss, ec, tm and en) separated by vertical dashes. stop codons are highlighted in red. open reading frames (orfs) are underlined, overlapping orfs are double-underlined, and orf translations are indicated as amino acids below the nucleotide sequences if applicable eight-day-embryonated chicken eggs (ece) were inoculated via the allantoic cavity unless stated otherwise, and candled twice daily. upon embryonic death or no later than 7 days p.i., eggs were transferred to 4°c for 16-24 h prior to allantoic fluid (af) and chorio-allantoic membrane (cam) collection. virus titration in ovo was based on the determination of the 50% embryonic infectious dose (eid 50 ) per ml, as determined at day 7 p.i. according to reed and muench [36] . for the production of a virus stock, ten 8-day-old ece were inoculated with 100 eid 50 , incubated for 24 h, and subsequently cooled for 16-24 h before the af was harvested and pooled. ibv strain h52 (boehringer ingelheim (bi), ingelheim, germany) was propagated in embryonated spf eggs and titrated. ibv strain beaudette (animal health service, deventer, the netherlands) was propagated and titrated in bhk-21 cells. mouse hepatitis virus (mhv) strain a59 was propagated and titrated in lr7 cells. monoclonal antibody (mab) ch/ibv 26.1 against the ibv s2 protein was obtained from prionics (thermo fisher scientific, waltham, ma, usa) [37, 38] . the production of rabbit polyclonal antiserum k134 against mhv was described previously [39] . chicken polyclonal antiserum was derived from a spf chicken vaccinated with ibv strain h120 (bi, ingelheim, germany). secondary fluorescently-labeled antibodies alexa fluor 488 goat anti-chicken igy, alexa fluor 568 goat antirabbit igg, and alexa fluor 488 goat anti-mouse igg (invitrogen by thermo fisher scientific) were stored in 50% glycerol at −20°c. cams were collected from eces, washed in pbs, fixed in neutral buffered 10% formalin in pbs for 24 h, stored in 70% ethanol and finally paraffin-embedded. four micrometer sections of cam were mounted on glass slides and subsequently deparaffinized and rehydrated in alcohol series. next, the sections were subjected to endogenous peroxidase inactivation and antigen retrieval as described before [40] . sections were washed in phosphate buffered normal antibody diluent (nad, scytek laboratories, logan, usa) containing 0.1% tween-20, and after primary antibody incubation with pbs 0.1% tween-20. sections were incubated for 60 min at room temperature with mab ch/ibv 26.1 diluted 1:100 in nad. antibody binding was detected by dako envision hrpo labeled polymer anti-mouse (dako, by agilent technologies, santa clara, usa) diluted 1:1 in nad, and visualized by 3-amino-9-ethylcarbazole (aec, dako). slides were counterstained with hematoxylin, mounted with aquatex (merck, darmstadt, germany), and viral antigen presence was assessed by light microscopy (bx60, olympus, tokyo, japan). rna was isolated from harvested af using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to manufacturer's protocol. reverse transcription (rt) was performed using the transcriptor first strand cdna synthesis kit (roche, basel, switzerland) according to manufacturer's protocol, with random hexamers for standard pcr, or with specific primers for sequencing and cloning purposes. pcr was performed with recombinant taq dna polymerase (thermo fisher scientific) for plasmid characterization or with phusion hot start ii high-fidelity dna polymerase (thermo fisher scientific) for sequencing and cloning purposes. one-step rt-qpcr was used to semi-quantitatively assess virus load in af. forward primer ibv.rdrp.f41 (3′-catgcagtttgttggagatcct-5′) and reverse primer ibv.rdrp.r41 (3′-gtgacctggttttaccgt ttga-5′) targeting the conserved region of gene 1b (nucleotide position 13,412 to 13,580 in ibv beaudette genbank accession number m95169.1) coding for the rna-dependent rna polymerase protein. primers were obtained from biolegio (nijmegen, the netherlands) and used at a final concentration of 300 nm each with the itaq universal sybr green one-step kit (bio-rad laboratories, hercules, california, usa). the rt-qpcr reaction was carried out in a bio-rad cfx connect realtime pcr system, starting with 10 min at 50°c and 1 min at 95°c, followed by 40 cycles of 10 s at 95°c and 30 s at 60°c, and ending with a dissociation step for the determination of the melting point of the obtained pcr fragment. the complete genome sequence of ibv h52 bi was determined by sanger sequencing using primers as described by zhou et al. [25] . the 5′-and 3′-utr sequences were identified using the 2nd generation 5′/3′ race kit (roche, basel, switzerland). the ibv h52 bi genome sequence was 27,640 nucleotides (nt) in length, including an annotated 10 nt polya tail. the design of the donor plasmids principally followed the strategy previously described by kuo et al. [19] . the final donor plasmid p-ibv was constructed from the stepwise ligation of fragments derived from five plasmids (fig. 1b and table 1 , and described below in detail): plasmid (p)ibv-5 comprises a t7 rna polymerase promotor, 2 g nucleotides, and the near full-length 5′-untranslated region (utr), with an unintended a to c substitution at position 54. plasmid ibv-1b comprises the last 754 nt of the pol 1b gene, including the 50 nt overlap with the spike gene, and the first 66 nt of the spike gene, including the signal sequence. plasmid ibv-s contains the near full length ectodomain of the spike gene, 3211 nt in length. plasmid ibv-sir comprises the last 212 nt of the spike gene (the transmembrane and the endodomain), the accessory gene 3, the envelope gene, the membrane gene and half of the intergenic region. plasmid ibv-3 t comprises the 3′-terminal region of the ibv genome, including the second half of the ir, the accessory gene 5, the nucleocapsid gene, the 3′-utr and a 100 nt poly-a sequence. all plasmids were generated by gen-script (piscataway, nj, usa) and provided in the plasmid puc57-simple, a standard cloning plasmid with the polylinker removed. in order to allow cloning of the fragments in a stepwise approach, naturally occurring restriction enzyme sites (res) located in the viral cdna were used, except for the bstbi site between p-ibv-5 and p-ibv-1b, which is only partly present in the 5′-utr, and the xhoi site between p-ibv-1b and p-ibv-s, which was introduced without changing the amino acid sequence (silent mutation). restriction enzyme sites were made unique by silently removing these res from other parts of the genome included in the donor plasmid (additional file 1: table s1 ). in addition, semi-unique res were introduced by silent mutations within 200 nt up-and downstream of the accessory genes 3 and 5. finally, unique res mssi and paci were included after the poly-a sequence, allowing linearization of the plasmid by a single restriction enzyme digest. all genome fragments were ligated step-by-step into p-ibv-5 using the restriction enzymes specified in table 1 . each ligation mixture was subsequently transfected into hb101 competent cells and plasmid dna was isolated by performing midiprep dna isolation (qiagen, hilden, germany). the final plasmid consisted of p-ibv-5-1b-s-sir-3 t, now called p-ibv (fig. 1c) . the composition of each plasmid was confirmed after each cloning step by pcr, restriction enzyme digestion and sequencing of each of the inserts (macrogen, amsterdam, the netherlands). the ectodomain of the mhv a59 spike gene was amplified from ptug [41] by pcr using primers with an xhoi overhang (table 2 ) and ligated into pjet1.2 resulting in p-mhv-s. site directed mutagenesis (sdm) with the q5 sdm kit (new england biolabs, ipswich, usa) was used to silently remove an ecori and an xhoi res interfering with subsequent cloning steps ( table 2 ). the ectodomain of mhv spike was ligated into p-ibv-5-1b, followed by subsequent cloning steps using the ibv fragments sir and 3 t. this resulted in the plasmid p-ibv-5-1b-mhvs-sir-3 t, now called p-mibv (fig. 1b) . a plasmid comprising the nucleocapsid gene and 3′-utr sequence of ibv h52 bi was generated by pcr amplifying the respective region using primers ibv-h52.n.atg.fw and ibv-m41#2.ir.rv ( table 2 ). the amplicon was ligated into pjet1.2 downstream of the t7 promotor sequence, resulting in p-ibv-n, and the correctness of the insert was verified by sequencing. capped, run-off donor transcripts were synthesized from p-ibv, p-mibv and p-ibv-n using the mmessage mmachine t7 kit (ambion by thermo fisher scientific). in brief, p-mibv was paci-linearized, and p-ibv and p-ibv-n were mssi-linearized. linearized plasmid dna was ethanol precipitated. transcription reactions were prepared according to the manufacturer's instructions, using 1.5 and 0.5 μg linearized dna per 10 ul reaction for p-(m)ibv and p-ibv-n, respectively. after 1 h of incubation at 37°c, production of rna was verified by analyzing 1 μl of the reaction volume by gel electrophoreses. after an incubation of 2 h the reaction was stopped by transferring the reaction tubes to ice. targeted rna recombination and rescue of mibv the ibv spike gene was replaced by a chimeric mhv-ibv spike gene in the ibv genome by targeted rna recombination between p-mibv generated donor rna and recipient virus (ibv) rna, as described before [19] . ibv h52 viral rna was transfected into bhk-21 cells, a cell line known to support replication of ibv [25] , but not infection with ibv h52 (data not shown). thus, 20 μl of ibv h52 bi rna obtained from allantoic fluid, mixed with 10 μl transcript reaction mixtures of p-mibv and p-ibv-n each were transfected into bhk-21 cells by electroporation using two pulses at 850 v and 25 μf in a gene pulser electroporation apparatus (bio-rad). transfected bhk-21 cells were seeded onto monolayers of lr7 cells having an approximate confluence of 70-80% and incubated at 37°c. two days after seeding, when syncytia in the lr7 monolayer were observed, the cell culture supernatant was harvested and rescued viruses were purified by two rounds of plaque purification on lr7 cells. characterization of the last one-third of the genome of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the obtained cdna-fragments, using the primer sets specified in table 3 . murinized ibv (mibv) strain #1b3-iia was selected based on sequence analysis, and virus stocks were propagated and stored at −80°c. aliquots were titrated on lr7 cells. recombinant ibv (ribv) was generated by substituting the ibv spike ectodomain back into the mibv genome by targeted rna recombination between p-ibv-generated donor rna and recipient virus mibv. lr7 cells were infected with mibv at a multiplicity of infection (moi) of 0.4 for 4 h. capped, run-off donor transcripts from p-ibv were transfected into the mibv-infected lr7 cells by electroporation with two pulses at 850 v and 50 μf. electroporated lr7 cells were resuspended in 2 ml dmem (at 37°c) and tenfold dilutions (up to 10 −3 ) were prepared. two hundred microliters of lr7 cell suspensions were inoculated into the allantoic cavity of 10-day-old eces, using 5 eggs per dilution. the eggs were candled twice daily and scored for embryonic death. upon death, or at 7 days p.i., the eggs were transferred to 4°c. sixteen to twenty-four hrs later the af was collected aseptically for rt-qpcr, and the cams were fixed in 10% formalin for immunohistochemistry (ihc). the af from eggs inoculated with the highest dilution of electroporated lr7 cells, in which virus was detected by rt-qpcr and ihc, was subjected to two additional rounds of end-point dilution in 8-day-old ece. genetic characterization of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the region encoding the structural and accessory genes using the primer sets specified in table 3 . biological characterization of the chimeric nature of mibv was performed by immunofluorescence (if) double staining for ibv and mhv. bhk-21 and lr7 cells were grown on coverslips and inoculated with ibv beaudette, and mhv a59 and mibv #1b3-iia, respectively, at an moi of 1.0. cells were fixed with pbs 4% paraformaldehyde (aurion, wageningen, the netherlands) for 20 min at room temperature after 5¼, 8, and 10 hpi for mhv, ibv, and mibv, respectively. subsequently, cells were permeabilized with pbs containing 0.1% triton x-100, blocked with goat serum (gibco by life technologies), and incubated for 45-60 min with a combination of two primary antibodies in ngs; rabbit anti-mhv polyserum k134 diluted 1:400 and chicken anti-ibv-h120 serum diluted 1:400, or rabbit anti-mhv polyserum k134 diluted 1:400 and mouse mab ch/ibv 26. animals were housed in separate groups and inoculated via eye-drop with 10 3 eid 50 in 0.1 ml of ibv h52 bi (n = 5), ribv-wt (n = 5), or not inoculated (n = 5, negative control). clinical symptoms monitored included ruffled feathers, decreased consciousness, depression, gasping, coughing, tracheal rales, and nasal discharge. seven days p.i. animals were euthanized, and evaluated for their tracheal ciliary activity. to this end, the trachea was sliced into 10 transversal sections: 3 from the upper part, 4 from the middle part, and 3 from the lower part. ciliary activity was examined by low-magnification microscopy within 2 h after sampling. ciliostasis of each tracheal section was scored on a scale from 0 (100% ciliary activity) to 4 (no ciliary activity, i.e. complete ciliostasis), with the maximum score for each trachea being 40. finally, the mean ciliostasis score for each group of animals was calculated. a unpaired t-test was performed to analyse whether there were differences in ciliostasis scores between ibv h52 bi and ribv-wt. generation and antigenic characterization of mibv and ribv-wt viral rna of ibv h52 bi and rnas transcribed from plasmids p-mibv and p-ibv-n were co-transfected into bhk-21 cells and seeded onto monolayers of lr7 cells. at 2 days post transfection, syncytia were observed in the lr7 monolayers, suggesting the successful generation of recombinant mibv. after two rounds of plaque purification on lr7 cells, candidate recombinants were characterized antigenically and genetically. if staining of lr7 cells infected with mibv showed positive staining with both anti-ibv and anti-mhv sera, indicating the chimeric nature of mibv (fig. 2a) . if staining with an anti-ibv-s2 mab was positive for ibv beaudetteinfected bhk-21 cells (taken along as positive control for if), but not for lr7 cells infected with mibv, indicating the absence of ibv s protein in mibv (fig. 2b) . lr7 cells infected with mibv and subsequently transfected with rna transcribed from plasmid p-ibv were inoculated in tenfold dilution series into the allantoic cavity of 10-day-old eces. no embryonic death was observed up to 7 days p.i., but embryos in the lowest dilutions showed signs of stunting and curling typical for embryos infected with ibv. the presence of replicating recombinant ibv was demonstrated by rt-qpcr on viral rna extracted from the af (data not shown), and by ihc on cam tissue (fig. 2c) . in contrast, cams of eggs inoculated with mibv-infected lr7 cells (not transfected) did not show any positive signal for ibv in ihc. during the first and second passage of ribv-wt in eight-day-old eces for endpoint dilution purposes, infected embryos died between 2 and 3 days p.i. using specifically located primers ( fig. 1 and table 3) , the intended genome structure and sequence of mibv and recombinant ibv wild-type (ribv-wt) were verified by rt-pcr (fig. 3) . the most important findings were that (1) murinized ibv contained the correct 5′ s gene sequence (primer set [f] -with a forward primer located in the ibv 1b gene at a position upstream of the 1b sequence present in p-mibv and a reverse primer located in the mhv spike gene -resulted in a detectable pcr product for mibv, but not for ibv or p-mibv); (2) murizined ibv contained the mhv spike gene at the location of the ibv spike gene (as both primer set [f] and [h] -each with a primer in the mhv spike gene and one in ibv gene 1b [f] or in the m gene [h] -resulted in a detectable pcr product for mibv, but not for ibv or mhv); (3) the ibv spike gene was absent from the mibv genome (as primer set [d] targeting the ibv spike gene resulted in a detectable pcr product for ibv, but not for mibv or mhv(4); recombinant ibv was the result of recombination between genomic rna from mibv and rna transcribed from p-ibv (as primer set [c] -with a forward primer in ibv gene 1b located upstream of the 1b sequence present in p-mibv and a reverse primer located in the ibv spike gene -resulted in a detectable pcr product for ribv-wt, but not for mibv or p-ibv); (5) recombinant ibv contained the ibv spike gene at the location of the mhv-derived spike in mibv (as both primer set [c] and primer set [e] -each with one primer in the ibv spike gene and the other in ibv orf 1b [c] or the m gene [e] -resulted in a detectable pcr product for ribv-wt, but not for mibv or mhv); (6) the mhv spike gene was absent from the ribv-wt genome (as primer set [g] targeting the mhv spike gene resulted in a detectable pcr product for mhv and mibv, but not for ribv-wt). sequence analysis of the 3′ 9 kb of the mibv genome (starting 1 kb upstream of the bstbi res in gene 1b, which marks the start of p-mibv) confirmed the expected genetic identity of mibv as observed after rt-pcr analysis (additional file 2: figure s1 ). the 3′ 9 kb of mibv and ribv-wt were exactly as designed, including the deliberate synonymous mutations listed in additional file 1: table s1 . a single spontaneous silent mutation (t to c) was observed in the spike of ribv-wt at position 22,644. in ovo growth kinetics of ibv and ribv-wt were assessed after inoculating eces with 10 2 eid 50 per egg by determination of the relative viral load in the af of five eggs per virus at 6, 12, 24, 36 and 48 hpi by rt-qpcr. at 12 hpi, the parental wild-type ibv showed somewhat higher viral loads as compared to ribv-wt, while from 24 hpi onwards, viral loads were comparable for both viruses (fig. 4a) . the virus titers remained at the same level until embryos started to die between 36 and 48 hpi. no differences in embryonic death between ribv-wt and ibv h52 bi groups was observed ( fig. 4b ; p > 0.05). the pathogenicity of ribv-wt was compared to that of the parental ibv h52 bi strain by inoculating one-dayold spf chickens. during the course of the infection, no clinical symptoms were observed in any of the groups (data not shown). at 7 days p.i. the animals were euthanized and the mean ciliostasis scores were determined as a readout for the ability of the respective viruses to infect and cause lesions in the primary target organ, the trachea. as expected, the negative control animals scored very low (fig. 5) , while ibv h52 bi infection resulted in a score of 26. ribv-wt had a mean score of 19 (p > 0.05), indicating that ribv maintained the ability to a b c fig. 2 antigenic characterization of mibv and ribv-wt. a immunofluorescence analyses of ibv beaudette, mhv a59 and mibv #1b3-iia infected cells. lr7 cells infected with mibv were fixed and double-immunolabeled with a polyclonal against ibv (green) and a polyclonal antibody against mhv (red). ibv beaudette-infected bhk-21 cells and mhv-infected lr7 cells were taken along for comparison. nuclei are visualized with dapi (blue). overlay pictures (merge) are shown on the right. b similar to (a), except that a monoclonal antibody against ibv s2 was used instead of a polyclonal against ibv, indicating the absence of ibv s2 protein in mibv infected cells. c immunohistochemistry of ibv h52 bi and ribv-wt infected cam tissues. ten-day-old embryonated chicken eggs were inoculated with ibv h52 bi (positive control), mibv-infected and p-ibv transcript electroporated lr7 cells (resulting in generation of ribv-wt), mibv infected and p-ibv transcript, but not electroporated, lr7 cells (mibv + p-ibv mock) or pbs (mock). formalin-fixed and paraffin-embedded cam tissues were immunohistochemically stained using a monoclonal antibody against ibv s2. replication of (r)ibv in the epithelial cells of the cam is indicated by red cytoplasmic staining, which is absent in eggs inoculated with mibvinfected non-transfected lr7 cells infect one-day-old chickens and induced lesions to a similar extent as the parental virus strain. here we developed a novel rgs based on targeted rna recombination, which allows manipulation of the genome of virulent ibv. the resulting recombinant virus has the same characteristics as the wild type ibv h52 both in embryonated eggs and in one-day-old chickens. by adapting the classical targeted rna recombination approach [11, 19, 33] to ibv, the inability to culture ibv strains like h52 on continuous cell lines has been overcome. for this, a cell-line known to support the replication, but not the entry of, ibv was used. this observation was used to create, by co-transfection of ibv viral rna and a transcript of chimeric ibv carrying the mhv spike gene, the recombinant mibv virus. upon transfection of synthetic ribv donor rna into mibv-infected murine lr7 cells, subsequent infectious ibv virus particles could be rescued in ece. the feasibility of the approach was demonstrated by the generation of recombinant ribv virus carrying silent marker mutations. the inability to select for individual ibv recombinants by plaque purification was circumvented by a combination of three approaches. first, the mibv-infected and ribv donor rna-transfected lr7 cells were inoculated into eces by end-point dilution. second, early rt-pcr and sequencing based screening of the genetic make-up of recombinants helped to identify and discard erroneous recombinants. third, two subsequent end-point dilution series were executed in eces, each leading to the selection of ribv-wt. finally, the genetic identity of the 5′ 9 kb of ribv-wt, i.e. the part of the ibv genome had been subject to manipulation, was confirmed by sequence analysis. the replication and pathogenicity of ribv-wt in ece was comparable to that of the parental ibv h52 bi. viral loads in the af were similar with respect to maximum virus titers (fig. 4) and embryonic death induced by both viruses did not differ (not shown). the pathogenicity of ribv-wt in one-day-old chickens was also comparable to that of parental ibv h52 bi, as demonstrated by comparable mean ciliostasis scores at 7 days p.i. (fig. 5) . taken together, ribv-wt has the same properties as ibv h52 both in ovo and in vivo and can thus be used to provide insights in the infection process in chickens. previously described rgs based on non-pathogenic ibv [21] [22] [23] [24] [25] can be used for in vivo studies only upon introduction of virulence factors including spikes from other ibv serotypes [26] [27] [28] . our newly developed rgs for ibv-h52 directly allows the elucidation of factors that determine the pathogenicity of ibv, as well as studying its protective immunity in vivo. fig. 3 genetic characterization of wild type viruses, recombinant viruses and donor plasmids. pcr was performed on cdna templates of viral rna extracted from infected lr7 cell culture supernatants (mhv and mibv) and allantoic fluid of inoculated embryonated eggs (ibv and ribv-wt), plasmid dna (p-mibv and p-ibv), or no template control (−).primer set letters a to h correspond with letters depicted in fig. 1 ; detailed information on the primers is given in table 3 here, for the first time host species switching of an avian gammacoronavirus to mammalian cells was demonstrated, by exchanging the spike gene with that of the betacoronavirus mhv. manipulation of coronavirus genomes by targeted rna recombination using an interspecies chimeric coronavirus has been demonstrated for alpha-and betacoronaviruses, thereby switching species tropism between mammalian hosts. our observation confirms the spike gene as the principal determinant of host species tropism of both avian and mammalian coronaviruses. in summary, a novel reverse genetics system (rgs) based on targeted rna recombination that allowed manipulation of the genome of virulent ibv was developed. this system makes use of an interspecies chimeric coronavirus, which is created by replacing the ectodomain of the ibv spike protein by that of mhv. the spike ectodomain exchange results in a host species tropism switch, which enables replication of mibv in cell culture. upon recombination of mibv with synthetic donor rna carrying the ibv spike gene, ribv a b fig. 4 in ovo characteristics of ibv and ribv-wt. a growth kinetics of ibv and ribv-wt were assessed by quantitative rt-qpcr analysis of rna extracted from allantoic fluid of inoculated embryonated eggs collected at 6, 12, 24, 36 and 48 hpi. data points represent means and standard deviations of 5 eggs per condition, with all samples run and analyzed in triplicates, using a tenfold dilution series of ibv h52 bi as reference for quantification. b embryonic death is indicated as a percentage of all remaining animals at each time point could be rescued in eces. in ovo growth kinetics and in vivo characteristics were comparable for ribv-wt and parental ibv h52 bi, suggesting no attenuating effect of the recombination process or of the introduced synonymous marker mutations. this system will allow the introduction of mutations in the 3′ one-third of the ibv genome, allowing the manipulation of the structural and accessory genes. the use of this system for both fundamental and applied research is promising, and potentially enables the development of a new generation of rationally designed live-attenuated ibv vaccines. additional file 1: table s1 . silent mutations introduced in ribv. nucleotide positions and sequences refer to the ibv h52 bi genome (see additional file 2: figure s1 ). modified nucleotides in recombinant ibv wt are depicted in lower case; n.a. = not applicable. purpose of introduction of restriction enzyme site are indicated for each site; in case of enzyme site removal the purpose was to create unique restriction enzyme sites for cloning; n.a. = not applicable in this study. (docx 19 kb) additional file 2: figure s1 . alignment of 3′ 9 kb of mibv and ribv-wt with ibv h52 bi. alignment of the 3′ 9 kb of mibv 1b3iia p6 (excluding the mhv derived spike ectodomain sequence) and recombinant (r)ibv wild-type (wt) p4 with ibv h52 bi. numbers refer to nucleotide positions in the ibv h52 bi genome. restriction enzyme sites are highlighted in yellow, with the corresponding enzyme indicated above the sequences. an additional thymidine residue to keep the mhv spike gene ectodomain sequence in frame with the ibv spike gene signal sequence at position 20,385 is highlighted in green and marked with a # above the sequence. the long view: 40 years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures family coronaviridae in: ninth report of the international committee on taxonomy of viruses coronavirus avian infectious bronchitis virus 28 coronaviridae accessory proteins of sars-cov and other coronaviruses gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis neither the rna nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses the avian coronavirus spike protein mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity novel receptor specificity of avian gammacoronaviruses that cause enteritis retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the pathogenesis of virulent and avirulent avian infectious bronchitis virus generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection reverse genetics system for the avian coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity activation of the chicken type i interferon response by infectious bronchitis coronavirus infectious bronchitis coronavirus inhibits stat1 signaling and requires accessory proteins for resistance to type i interferon activity infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b switching species tropism: an effective way to manipulate the feline coronavirus genome coronavirus reverse genetics by targeted rna recombination the method of "right and wrong cases" (constant stimuli) without gauss's formula a simple method of estimating fifty per cent endpoints antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions detection by immunofluorescent assay of serotype-specific and group-specific antigens of infectious bronchitis virus in tracheas of broilers with respiratory problems viral protein synthesis in mouse hepatitis virus strain a59-infected cells: effect of tunicamycin contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus geert de vrieze, maartje woelders, maloeke de jong, and alexandra negatsch are acknowledged for excellent technical support. this research was financially supported by boehringer ingelheim, ingelheim, germany. data generated or analyzed during this study and described in this manuscript are included in this manuscript and its supplementary information files. authors' contributions sjvb, em, pjmr and mhv designed the studies. sjvb, ajb, akk, gc, ds, and hcp carried out the experiments. sjvb wrote the manuscript. mhv, em, and pr edited the manuscript. all authors read and approved the final manuscript. submit your next manuscript to biomed central and we will help you at every step: key: cord-272693-432ixb7g authors: phillips, j. e.; jackwood, m. w.; mckinley, e. t.; thor, s. w.; hilt, d. a.; acevedol, n. d.; williams, s. m.; kissinger, j. c.; paterson, a. h.; robertson, j. s.; lemke, c. title: changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus date: 2011-09-10 journal: virus genes doi: 10.1007/s11262-011-0668-7 sha: doc_id: 272693 cord_uid: 432ixb7g full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (ibv) strains of the same serotype was conducted to identify genetic differences between the pathotypes. analysis of the consensus full-length genome for three different ibv serotypes (ark, ga98, and mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75–43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in ibv, ranged from 5.8 to 13.4% of all changes. the attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. however, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype. electronic supplementary material: the online version of this article (doi:10.1007/s11262-011-0668-7) contains supplementary material, which is available to authorized users. avian coronavirus infectious bronchitis virus (ibv) causes a highly contagious upper respiratory tract disease in chickens. live attenuated vaccines are used against the virus but the disease is difficult to control because cross-protection does not usually occur between different serotypes. the respiratory disease caused by this virus can be mild to moderate and can vary depending on the breed of chicken infected as well as the strain of the virus [1] . the virus is worldwide in distribution, and in addition to chickens, ibv has been isolated from peafowl (galliformes) and other electronic supplementary material the online version of this article (doi:10.1007/s11262-011-0668-7) contains supplementary material, which is available to authorized users. gamma-coronaviruses have been isolated from teal (anas crecca), geese (anserinae), pigeons (columbiformes), and ducks (anserfiformes) [2] . coronaviruses are enveloped viruses in the order nidovirales and are classified based on genome organization and antigenic characteristics as alpha (previously group 1), beta (previously group 2), and gamma (previously group 3)-coronaviruses with the avian coronaviruses belonging to the gamma-coronaviruses. subgroups within each group have been reported, and recently, comparative full-length genome analysis placed a novel coronavirus from a beluga whale in subgroup 3b and three new coronavirus isolates from passerine birds in subgroup 3c [3] . infectious bronchitis virus and related isolates as well as turkey coronavirus (tcov) are assigned to subgroup 3a. coronaviruses have a single-stranded positive-sense rna genome ranging in size from 27 to 30 kb, with a 5 0 cap and a 3 0 poly-a tail. transcription occurs through a leader-primed rna synthesis mechanism that results for ibv in six 3 0 co-terminal subgenomic mrna molecules. four structural proteins-spike (s), envelope (e), membrane (m), and nucleocapsid (n)-along with the viral rna make up the enveloped virion. the n protein binds to the viral rna forming the ribonucleoprotein (rnp) complex. the e and the m protein are membrane bound proteins that play a role in virus assembly [4] . the s glycoprotein on the surface of the virus mediates attachment to the host cell, is responsible for fusion of the host cell membrane and viral envelope, and in ibv, it contains epitopes that define serotype and induce neutralizing antibodies [5] . the s glycoprotein of ibv is post-translationally cleaved into s1 and s2 subunits, and the s1 subunit is reported to have three hypervariable regions [6] [7] [8] . mutations, insertions, deletions, and recombination in s contribute to the genetic diversity of ibv, which is recognized as different genetic or serologic types of the virus [5] . two polyproteins 1a and 1ab account for approximately two-thirds of the viral genome-coding region and make up the replication transcription complex (rtc). the polyprotein 1ab is translated through a-1 frame-shift translation mechanism that occurs approximately 20-40% of the time [9] . the ibv 1a and 1ab polyproteins are post-translationally cleaved into 15 nonstructural proteins (nsps), nsps 2 through 16 by a papain-like protease (plp) and the main protease (mpro), also referred to as the 3c-like protease [10] . ibv does not have an nsp 1 equivalent found in some other coronaviruses. the plp contained within nsp 3 is divided into pl1 and pl2 papain-like proteases. the pl1 protease, present in other coronaviruses, is truncated and nonfunctional in ibv, thus pl2 cleaves nsps 2, 3, and 4 [11] . the mpro contained within nsp 5 cleaves nsps 5 through 16 . the biological characteristics of many nsps have been previously reported [9, 10, [12] [13] [14] [15] [16] [17] . in addition to nsps 3 and 5, which contain proteases pl2 and mpro, respectively, nsps 2, 4, and 6 contain hydrophobic residues predicted to play a role in anchoring the rtc to the golgi. nonstructural proteins 7, 8, 9, and 10 are reported to have rna-binding activity. nonstructural protein 11/12 is the rna-dependent rna-polymerase, nsp 13 is a rna helicase, nsp 14 is an exoribonuclease, nsp 15 is an endoribonuclease, and nsp 16 is a methyltransferase. adaptation of ibv to different hosts has been associated with changes in the s glycoprotein, suggesting that spike plays a key role in pathogenicity [18, 19] . however, the ectodomain of the s glycoprotein from the beaudette strain of ibv, an attenuated laboratory strain, was replaced with an s from a pathogenic strain (mass 41 strain) of the same serotype. this chimeric virus was shown to induce an immune response but remained nonpathogenic in chickens, indicating that the s glycoprotein is not solely responsible for pathogenicity of ibv [2, 20] . in another study, a chimeric ibv was created with the replicase genes 1a and 1ab from the attenuated beaudette strain, and all of the structural genes from the pathogenic mass 41 strain including the s gene. this chimeric virus was not pathogenic in chickens, indicating that the replicase proteins also appear to be determinants of ibv pathotype [2, 21] . genetic differences reported in 1a and s between virulent and avirulent strains of ibv also led others to suggest that the replicase proteins, in addition to s, are involved in the pathotype of the virus [22] . to examine the sequence changes in individual genes associated with attenuation of ibv for chickens, we sequenced and compared the full-length consensus genomes of pathogenic ibv viruses and egg-passaged attenuated (for chickens) viruses from three different serotypes. we also examined the replication of pathogenic and attenuated viruses in embryonated eggs and in chickens to determine whether there are differences in growth rate between the pathotypes. pathogenic and attenuated (for chickens) ibv strains from three different serotypes were used in this study. the pathogenic arkansas-delmarva poultry industry ark/ark-dpi/81 and the massachusetts strain mass/mass41/41 were obtained from dr. j. gelb, jr. (university of delaware, newark, de). the pathogenic georgia 98 virus, ga98/cwl0470/98 virus, was isolated in our laboratory in 1998 [23] . the pathogenic viruses were propagated in 10-day-old embryonated chicken eggs (ark/ark-dpi/81 pass 6, mass/mass41/41 pass 8, and ga98/cwl0470/98 pass 8) as previously described [24] . the attenuated viruses of the same strain and serotype were obtained from intervet and were designated ark-attenuated (mildvac-ark), mass41-attenuated (mildvac-h), and ga98attenuated (mildvac-ga-98). whole-genome nucleotide and deduced amino acid sequence analysis viral rna extraction, rt-pcr, library construction, and sequencing were conducted as previously described [25] . briefly, the viruses were filtered through a 0.8-lm filter then through a 0.22-lm filter (millipore, billerica, ma) prior to rna extraction. viral rna was purified using the high pure rna isolation kit according to the manufacturer's recommendation (roche diagnostic corporation, foster city, ca) and re-suspended in depc-treated water. reverse transcription (rt) and polymerase chain reaction (pcr) amplification were performed with the takara rna la pcr kit (takara bio inc., otsu, shiga, japan) using a random primer and an amplification primer in a strand displacement amplification reaction following the manufacture's protocol. the sequence of the random reverse transcription primer was 5 0 -agc ggg ggt tgt cga atg ttt gan nnn n-3 0 , and the amplification primer sequence, which is designed to anneal to the complement of the conserved region on the random primer, was 5 0 -agc ggg ggt tgt cga atg ttt ga-3 0 . both primers were obtained from integrated dna technologies, inc. (coralville, ia). for the rt reaction, a master mix was prepared, which included mgcl 2 (5 mm), 109 rna pcr buffer (19) , dntp mixture (1 mm), rnase inhibitor (1 units/ll), reverse transcriptase (0.25 units/ll), 5 0 degenerate primer (2.5 lm), and rna (5.75 ll/reaction) then 10 ll per sample was aliquoted in a thermocycler tube. the reaction conditions for the rt reaction were 10 min at 30°c for the primer annealing then an hour at 50°c for extension followed by a five-minute incubation at 99°c for inactivation of the enzyme and a five-minute period at 5°c. a pcr master mix-which included at the final concentrations mgcl 2 (2.5 mm), 109 la pcr buffer (19) , sterilized distilled water (32.25 ll), takara la taq (1.25u/50 ll), and 5 0 primer (0.2 lm)-was prepared and 10 ll of the rt reaction was added to 40 ll of the mix. the amplification reaction consisted of a 94°c step for 2 min followed by 30 cycles of 94°c for 30 s, 60°c for 30 s, and 72°c for 3 min. ten pcr were combined for each virus and purified using the qiaquick pcr purification kit (qiagen, foster city, ca) and then run on a 1% agarose gel to visualize the amplified product. the pcr products were size selected by cutting out amplicons between 500 and 1500 bp from the gel. the amplicons were purified using the qiaquick (qiagen) gel purification kit. the topo cloning kit (invitrogen, life technologies, carlsbad ca) was used to clone the pcr products into the pcr-xl-topo vector according to the manufacturer's recommendations. then, one shot topo electrocompetent escherichia coli cells (invitrogen) were transformed using 30 ll of competent cells mixed with 2 ll of the ligation reaction and electroporated with settings at 20 kv and 200 x using a biorad (biorad gene pulser, hercules, ca). the electroporated cells were incubated at 37°c in 480 ll of super optimal broth medium for 1 h on a rotary shaker. the cultures were mixed with 70% glycerol and frozen in -80°c until plated on q-trays (genetix, boston, ma) containing liquid broth agar cat#3002-032 (mp biomedicals, llc, solon, oh) with 50 lg/ml of kanamycin. the q-trays were pre-warmed at 37°c before the entire culture (approximately 500 ll) was spread on the plates and incubated overnight at 37°c, then robotically picked with a q-bot (genetix, boston, ma). plasmid dna from the libraries of cloned cdna fragments for each virus was isolated using an alkaline lysis method modified for the 96-well format, and incorporating both hydra and tomtek robots (http://www.intl-pag.org/ 11/abstracts/p2c_p116_xi.html). cycle sequencing reactions were performed using the bigdye tm terminator ò cycle sequencing kit version 3.1 (applied biosystems, foster city, ca) and mj research (watertown, ma) thermocyclers. finished reactions were filtered through sephadex filter plates into perkin-elmer microamp optical 96-well plates. a 1/12-strength sequencing reaction on an abi 3730 was used to sequence each clone from both the 5 0 and 3 0 ends. each viral genome was sequenced to approximately 109 coverage. the accuracy of the sequence was ensured by generating data in both the 5 0 and the 3 0 directions. gaps and areas with less than 29 coverage were identified and specific primers were synthesized (idt) for rt-pcr amplification and sequencing of the ambiguous areas. the rt-pcr was conducted as described above, and the reaction conditions were 42°c for 60 min, 95°c for 5 min, then 10 cycles of 94°c for 30 s, 50°c for 30 s, 68°c for 90 s, followed by 25 cycles of 94°c for 30 s, 50°c for 30 s, 68°c for 90 s ? 5 s/cycle added. the final elongation step was 68°c for 7 min, and then, the reaction was cooled to 4°c. the pcr products were sequenced in both directions using the abi prism bigdye terminator v3.0 (applied biosystems, foster city, ca) and the specific primers that were used for amplification at a concentration of 15 ng. the amount of cdna added to the reaction ranged from 20 to 30 ng, and the sequencing reactions were analyzed on an abi 3730 (applied biosystems). chromatogram files and trace data were read and assembled using seqman pro, and genome annotation was conducted with seqbuilder (dnastar, inc., v.8.0.2, madison, wi). low-quality segments and vector sequence were trimmed from the ends of each sequence and removed from further analysis. full-length genomes were uploaded to the national center for biotechnology information (ncbi) open reading frame (orf) finder (http://www.ncbi. nlm.nih.gov/gorf/) to identify orfs. nucleotide and deduced amino acid alignments were generated using clustalw, and phylogenetic trees with 1,000 bootstrap replicates were constructed in the megalign program (dnastar, inc.). hydrophilicity analysis using hopp-woods and kyte-doolittle were conducted with the protean program (dnastar, inc.). the viruses were titrated in 10 day of incubation embryonated eggs to obtain a 50% embryo infectious dose (eid 50 ) according to previously published procedures (24). two-week-old chickens were given 1 9 10 4 eid 50 of virus in 100 ll of pbs equally divided intraocularly and intranasally. due to isolator availability, different numbers of birds were tested for each virus. six birds were given ark/ ark-dpi/81, 20 birds were given ark attenuated, 10 birds each were given mass/mass41/41, mass attenuated, and ga98 attenuated, and 12 birds were given ga98/ cwl0470/98. each of the negative control groups consisted of 10 birds. clinical signs and lesions were recorded, and tracheal swabs were collected and placed in 1 ml of ice-cold pbs (ph 7.4) at 5 days post-exposure [26] . the presence of virus in the tracheal swab supernatant was determined by quantitative real-time rt-pcr [27] . tracheas were collected in 10% neutral buffered formalin, routinely processed into paraffin, and 5-lm sections were cut for hematoxylin and eosin staining. epithelial hyperplasia, lymphocyte infiltration, and the severity of epithelial deciliation were scored for each trachea with 1 being normal and 4 being severe [28] . as a measure of adaptation, we examined the growth of the ark/ark-dpi/81, ark attenuated, mass/mass41/41 and mass41-attenuated in embryonated eggs and chicks. because of limited isolator availability, we did not include the ga 98 viruses in this experiment. virus growth in embryonated eggs was examined by inoculating 1 9 10 5 eid 50 of each virus into 30 eggs at 10 days of incubation via the chorioallantoic route. for each virus, allantoic fluid was harvested from five eggs at 12, 24, 36, 48, 72, and 96 h after inoculation. the amount of virus present in fresh (not previously frozen) allantoic fluid was determined by quantitative real-time rt-pcr [27] . to examine virus growth in chicks, 1 9 10 5 eid 50 of each virus was inoculated into 30 specific pathogen-free chicks at 1 day of age via the ocular/nasal route. tracheal swabs were collected from each of five birds at 12, 24, 36, 48, 72, and 96 h after inoculation and placed in 1 ml of ice-cold pbs (ph 7.4). once the birds were swabbed, they were removed from the study. the amount of virus present in the fresh (not previously frozen) tracheal swab supernatant was determined by quantitative real-time rt-pcr [27] . sequences generated in this study were submitted to genbank and assigned the following accession numbers: ark/ark-dpi/81 (gq504720); ark-attenuated (gq504721); ga98/cwl0470/98 (gq504722); ga98-attenuated (gq50 4723); mass/mass41/41 (gq504724); and mass41-attenuated (gq504725). the consensus sequence of the full-length genomes of ark/ ark-dpi/81, ark-attenuated, ga98/cwl0470/98, ga98attenuated, mass/mass41/41, and mass41-attenuated were sequenced, and the genome sizes were found to be 27,651 nt, 27,620 nt, 27,638 nt, 27,621 nt, 27,475 nt, and 27,451 nt, respectively. the genome organization consisting of a 5 0 untranslated region (utr), polyproteins 1a and 1ab, spike, 3a, 3b, envelope, membrane, 4b, 5a, 5b, nucleocapsid, and 3 0 utr was the same for all six viruses (table 1) . gene locations for the nsps in orf 1a and 1ab are shown in table 2 . the 4b protein, previously recognized in m41 [21] , is 94 amino acids long and located downstream from the membrane protein in all the viruses sequenced. a blast search was conducted, and we found the protein to have 96% sequence identity with the 4b protein from tcov (tcov, genbank accession number eu022526.1). in addition, a 6b protein downstream of the nucleocapsid protein was similar to the predicted 6b orf reported for tcov (genbank accession number eu022526.1). the 6b orf was identified in the ark and ga98 viruses but not in the mass 41 viruses. alignment and phylogenetic analysis of the full-length genomes show that ark/ark-dpi/81 has 99.1% sequence identity with ark-attenuated, ga98/cwl0470/98 has 97.1% sequence identity with ga98-attenuated, and mass/ mass41/41 has 92.3% sequence similarity with mass41attenuated (fig. 1) . nucleotide and amino acid sequence differences were identified between each of the pathogenic and attenuated viruses (table 3) . when the genome sequences are compared, there are 249 nucleotide (nt) changes resulting in 62 amino acid changes in the coding regions between the ark viruses, 629 nt changes resulting in 268 amino acid changes between the ga98 viruses, and 1,805 nt changes resulting in 462 amino acid changes between the mass 41 viruses (see table 3 and supplemental data tables 5 and 6 ). the size of the 5 0 utr is 528 nt for all the viruses ( table 1 ). the number of nt differences between the ark viruses for the 5 0 utr was 25 with a 95.6% identity. the ga98 viruses have 6 nt differences with 98.9% identity, and the mass viruses have 12 nt differences with 98.3% identity in the 5 0 utr ( table 3 ). the leader junction sequence, nucleotides 57-64 (5 0 -cttaacaa), were found to be identical for the ark and mass viruses, whereas the ga98/cwl0470/98 pathogenic virus leader junction sequence is 5 0 -ctcaacaa and the ga98 attenuated virus sequence is 5 0 -ctttacaa. the transcriptional regulatory sequences (trs) were identical in all of the viruses and were 5 0 -ctgaacaa-3 0 for mrnas 2 and 3, and 5 0 -cttaacaa-3 0 for mrnas 4, 5, and 6. the size of the 3 0 utrs is 273 nt for ark/ark-dpi/81 pathogenic and ark-attenuated, 276 nt for ga98/ cwl0470/98, 244 nt for ga98-attenuated, and 322 nt for mass/mass41/41, and mass41-attenuated ( table 1 ). the number of nt differences within the 3 0 utrs for the ark viruses is 6 with 98.5% identity. the ga98 viruses have 9 nt differences resulting in 97.1% identity, and the mass viruses have 2 nt differences with 99.4% identity within the 3 0 utrs ( table 3) . the 3 0 utrs contain the s2m motif, which is 41 nt long with a sequence identity of 92.7% or higher between the six viruses. analysis of the locations and number of sequence differences between pathogenic and attenuated viruses of the same serotype for individual nsps in polyproteins 1a and 1a/b (table 3) shows that nsp 3 has the highest number of amino acid differences among all the nsps. in addition, nsp 3 has the greatest number of differences when coding regions across the entire genome are compared. a schematic representation of nsp 3 and number of amino acid changes in each domain is presented in fig. 2 . the nsp 3 orf has 43.66% of all amino acid differences observed between ark/ark-dpi/81 and ark-attenuated (including a ten amino acid deletion in the attenuated virus at positions 789-798), 34.75% of all amino acid differences observed between ga98/cwl0470/98 and ga98-attenuated (including an eight amino acid deletion in the pathogenic virus at positions 901-908 and a three amino acid deletion in the pathogenic virus at positions 950-952), and 37.08% of all amino acid differences observed between mass/mass41/41 and mass-attenuated (including a ten amino acid deletion in the attenuated virus at positions 797-806). these changes represent 1.96, 5.18, and 11.06 differences per 100 amino acids within nsp 3 for ark, ga98 and mass 41, respectively. we also found a virus subpopulation within the ark/ark-dpi/81 strain, which had a ten amino acid deletion in nsp 3 at positions 789-798 similar to the ark-attenuated virus. the catalytic triad of the pl2 protease, amino acids cys623, hys786, asp802 [29] was conserved among all of the viruses, and a hydrophobicity plot of nsp 3 predicted fours transmembrane regions between amino acids 1,000 and 1,300 (data not shown). the fewest amino acid changes for the nsps between pathogenic and attenuated viruses within a serotype are found in nsps 7-10, which are the rna-binding proteins. the polyprotein 1ab-1 frame-shift slippery sequence (5 0 -uuuaaac) is conserved among all six viruses but the location was found at nt 12,328 for ark/ark-dpi/81, nt 12,298 for ark-attenuated, nt 12,321 for ga98/cwl0470/ 98, nt 12,360 for ga98-attenuated, nt 12,391 for mass/ mass41/41 and nt 12,327 for mass41-attenuated. the percent amino acid identity for the s glycoprotein is 97.8% for ark viruses, 96.6% for ga98 viruses, and 97.2% for mass 41 viruses (fig. 3) . the number of amino acid differences within the s glycoprotein between pathogenic and attenuated viruses are 7, 33, and 27 for ark, ga98, and mass 41, respectively ( table 3 ). the s glycoprotein for the ark viruses had 9.86% (0.60 differences/100 amino acids) of all amino acid differences, which is the third most variable orf in the entire genome after nsp 3 and 12. for the ga98 viruses, the s glycoprotein has 13.36% (2.82 differences/100 amino acids) of all amino acid differences, which is the third most variable orf in the entire genome after nsp 3 and orf 6b. the s glycoprotein for the mass 41 viruses has 5.77% of all amino acid differences (2.31 differences/100 amino acids), which was the fourth most variable orf in the entire genome after nsp 3, 2, and 4. orf 3b has the fewest number of differences with no differences observed between the ark viruses, whereas the ga98 and mass viruses each have one amino acid difference. for orf 4b, no amino acid differences are observed for the ark viruses, 16 amino acid differences are observed between the ga98 viruses, and 17 amino acid differences are observed between the mass 41 viruses. the ark virus 6b proteins have only one amino acid mutation and are 99.9% similar to each other, whereas the ga98 virus 6b proteins have 43 amino acid mutations, 3 amino acid deletions, and 1 substitution and are only 41.9% similar. because this protein has not been previously recognized in ibv, a nucleotide blast search rather than an amino acid search was conducted and showed that the ga98/cwl0470/98 virus has 98% identity with mass h120 (fj888351) and the ga98-attenuated virus has 98% identity with ark-dpi (eu418976). to determine whether the ga98-attenuated virus 6b sequence was a subpopulation within the ga98/cwl0470/98 virus, two forward primers (ga98a #1 5 0 -tcacgctcaagttcaagacctg-3 0 , and ga98a #3 5 0 -cagctttaggtgagaatgaact-3 0 ) and two reverse primers (ga98a #2 5 0 -tacgataaaacaa actaatgagaa-3 0 , and ga98a #4 5 0 -ttgataggaa agcacagaaatag-3 0 ) specific for the ga98-attenuated 1m a positions are based on 1ab from tcov (accession number yp_001941164) and presented as the residue position with 1 being the methionine at the beginning of orf 1a and 1ab followed by the single letter code for the amino acid at that position 6b sequence were used in combination in an rt-pcr assay, but no amplicons were observed. the data on pathogenicity of the viruses in 2-week-old spf chicks are presented in table 4 . a birds were given 1 9 10 4 50% embryo infectious doses intraocularly/intranasally and examined for clinical signs, virus, and lesions at 5 days post-inoculation b virus was detected in tracheal swabs by real-time rt-pcr as previously described callison et al. [27] c epithelial hyperplasia, lymphocyte infiltration, and the severity of epithelial deciliation were scored for each trachea with one being normal and four being severe d a representative control group from one of the experiments is presented. all of the data from the negative control groups were the same (fig. 4a) . the ark-attenuated virus, which is adapted to embryonated eggs, only killed chicks inoculated with virus at 1 day of age showed statistical differences (p b 0.1) in the amount of virus detected in the trachea between the ark/ark-dpi/81 and ark-attenuated viruses at 24, 48, 72, and 96 h post-inoculation with the pathogenic ark/ark-dpi/81 having the higher amount of virus at each of the sample times (fig. 4b) . although not statistically different, the chicks given the pathogenic ark/ark-dpi/81 virus also had more virus detected in the trachea than the chicks given the ark-attenuated virus at 12 and 36 h post-inoculation. many studies have examined sequence changes in the structural proteins of ibv and found that most of the changes associated with adaptation to a particular host or with a particular virus pathotype occur in the spike glycoprotein [18, 19, 30] . but only a few studies have examined changes across the entire genome associated with biological characteristics of the virus [22, 31] . ammayappan et al. [22] found a total of 17 amino acid changes between the genomes of ark dpi 11, a pathogenic virus and ark dpi 101 an attenuated virus, with four amino changes in nsp 3 and six amino acid changes in the s1 glycoprotein. based on that data, it was suggested that changes in the replicase sequence in addition to structural proteins might play a role in pathogenicity. fang et al. [31] found 53.06% of all amino acid substitutions across the entire genome were located in the spike glycoprotein following adaptation of an attenuated avian coronavirus to primate cells, suggesting that spike plays a role in host adaptation. in this study, we analyzed the consensus full-length genome for the pathogenic and attenuated viruses of three different ibv types and showed that within a virus type, 34.75 to 43.66% of all the amino acid changes between the pathotypes occurred in nsp 3, whereas changes in spike ranged from 5.8 to 13.4% of all changes. it should be noted, however, that spike had the highest number of differences between different serotypes of the virus, which is consistent with previous reports [5] [6] [7] [8] . a high percentage of differences between pathogenic and attenuated viruses within a serotype in nsp 3 suggests this region plays a key role in pathogenicity. the nsp 3 is a complex protein with multiple domains making it an attractive target for antiviral drug design [9, 32] . it is approximately 1,600 amino acid residues in length and consists of an acidic domain, an adp-ribose 1 phosphatase, the pl2 protease (a deubiquitinating protease), y and transmembrane domains. the acidic domain is of unknown function, however; there is some evidence that it possesses nucleic acid binding activity because it is consistently co-purified with singlestranded rna [33] . previous studies with other organisms indicate that electrostatic interactions from this type of domain play a key role in ligand binding [34] . influenza a viruses also contain a polymerase acidic protein (pa) that is required for the transcription and replication activity of the viral polymerase [34] . differences between pathogenic and attenuated ibv strains within a serotype, including deletions in ark and mass41 viruses, were in and around the acidic domain within nsp 3 (fig. 2) . thus, it is likely that the acidic domain plays a role in attenuation in chickens but the exact function(s) of the amino acids in this domain is unclear. it was interesting that we observed an eight and a three amino acid deletion in the pathogenic virus ga98/cwl0470/98 at positions 901-908 and 950-952, respectively, compared to the ga98-attenuated virus. since sequence insertions are not likely to occur during the attenuation process, the ga98-attenuated virus possibly represents a minor undetected subpopulation in the pathogenic virus, which was selected by passage in embryonated eggs. the adp-ribose-1 phosphatase domain within nsp 3 is relatively conserved between the pathogenic and attenuated strains. this domain has been shown in the beaudette laboratory attenuated strain of ibv not to function as an adp-ribose binding protein [35] . however, the triple glycine sequence that forms part of the adp-ribose binding site (gly47-gly48-gly49), which was not conserved in beaudette, is conserved in all of the viruses sequenced herein [35] . this suggests that the adp-ribose-1 protein may be functional in the pathogenic and attenuated ibv viruses and is consistent with the results of the mass 41-x domain as reported by xu et al. [14] . the adp-ribose-1 phosphatase may be important in pathogenicity of ibv because it has been shown to play a role in adp ribosylation, a post-translational protein modification involved in dna damage repair and transcription regulation [14] . in addition, it was reported that the adp-ribose-1 is dispensable for viral replication in tissue culture, suggesting that this domain is involved in regulation of viral replication rather than the actual replication process [36] . the pl2 domain is a papain-like protease that is responsible for the cleavage of the nsp 2/3 and 3/4 sites. most coronaviruses have two papain-like proteases; however, in ibv the pl1 protease is truncated and is nonfunctional [16] . the structure of the pl2 protease domain was determined to be a ''thumb-palm-finger'' motif [37] . this domain has also been shown to be a potent ifn antagonist by inhibiting the phosphorylation and nuclear translocation of interferon regulatory factor 3 (irf-3) causing a disruption in the activation of the type i ifn response through toll-like receptor 3 (tlr 3) or retinoic acid-inducible gene i (rig-i) [38] . although the catalytic triad of the pl2 protease is conserved, amino acid changes between the pathogenic and attenuated viruses are observed in the pl2 protease, which could affect the efficiency of this ifn antagonist leading to altered viral replication in the cell. the disruption of ifn signaling has been shown in many viral infections, including sars-cov, dengue virus, and paramyxoviruses [39] [40] [41] . the ibv pl2 viral protease was also shown to have characteristics similar to ubiquitin-specific proteases [42] . deubuquitinating proteases, which remove ubiquitin from proteins that have been marked by cellular mechanisms for atp-dependent degradation, could be a potential mechanism by which the virus can alter the cellular environment favoring replication. the y domain, containing transmembrane domains at its n-terminus, was originally described by gorbalenya et al. [43] and has been predicted to consist of three domains y1, y2, and y3, which may act together to form an enzymatic function [32] . the transmembrane domain is inserted into the endoplasmic reticulum (er) membrane co-translationally and plays an important scaffolding role for the replication transcription complex [9] . recently, it was shown that three transmembrane domains were predicted for the sars-cov nsp 3 but only two were found to span the er membrane orienting the protease domain of nsp 3 on the cytoplasmic side where viral replication occurs [13, 15] . in murine hepatitis virus (mhv), five transmembrane domains were predicted but only two domains were found to span the membrane, also locating the protease domain on the cytoplasm side [13, 15] . our sequence data for ibv predicts four transmembrane domains within nsp 3. assuming the protease domain is located on the cytoplasm side of the membrane, we predict that either two or all four transmembrane domains would be used. a chimera ibv containing the replicase genes 1a and 1a/b from the attenuated beaudette strain and the structural genes from the pathogenic mass 41 strain was not pathogenic in chickens, indicating that the replicase proteins appear to be determinants of pathotype in ibv [2, 21] . our data strongly support these studies and further indicate that changes in nsp 3 play a key role in ibv pathotype. it should also be emphasized that pathogenicity in avian coronaviruses is likely polygenic, since we and others [22] observed amino acid substitutions in other viral proteins including spike. the 6b orf detected in tcov (genbank accession numbers acb87503 and acb87504) is identified in ark and ga98 viruses herein. only one amino acid difference was observed between the ark viruses, but 43 differences as well as 3 amino acid deletions and 1 insertion are observed between ga98 viruses. an attempt to identify a subpopulation in the ga98/cwl0470/98 pathogenic virus with the ga98attenuated gene 6b was unsuccessful. it is not clear why gene 6b is so variable between the ga98 viruses but it appears recombination rather than mutations over time may have played a role. a nucleotide blast analysis indicated that the ga98/cwl040/98 virus was 98% similar to mass h120 a vaccine virus and the ga98-attenuated virus was 98% similar to ark-dpi a pathogenic virus, suggesting an origin for those genes. nonetheless, assuming the 6b orf is expressed, it apparently does not play a role in defining pathotype. interestingly, we find differences between pathogenic and attenuated viruses in the 5 0 and 3 0 utrs. the 5 0 and 3 0 utrs play key roles in transcription and replication of coronaviruses [44] . however, the differences between the ark and mass viruses, which are 25 nt and 12 nt, respectively, for the 5 0 utr, and 6 nt and 2 nt, respectively, for the 3 0 utr did not appear to affect replication as determined in embryonated eggs. the trs sequences for generation of the subgenomic mrnas were identical in all of the viruses; however, the leader junction sequences were different for ga98 viruses. different leader junction sequences could be important for attenuation since efficiency of subgenomic mrna production would affect growth of the virus [45] . differences are observed in the amount of virus detected in chickens given viruses with different pathotypes. when the same amount of virus was administered, birds given the attenuated virus compared to birds given the homologous pathogenic virus had less virus detected in the trachea at all sampling times and the difference was statistically significant for most of the time points. thus, it appears that the amount of ibv replication in the trachea correlates with the ability of the virus to cause disease in chickens. attachment and entry, and replication of the attenuated virus (for chickens) were not impaired because it grew to the same titer (with the exception of one time point) as the pathogenic virus in 10-day-old embryonated eggs. inefficient attachment and entry into chicken host cells in vivo could be due to changes in spike. and decreased replication of the attenuated viruses could be due to the inability of the virus to overcome some as yet unidentified innate defense mechanism(s) in chicken cells that is not present in embryonic cells. domains within nsp 3 associated with the deubiquitinating protease or ifn antagonists are likely candidates for further research. in summary, we find that most changes associated with attenuation of ibv for chickens are located within nsp 3 and that the attenuated viruses have reduced replication in chickens but not in 10-day-old embryonated eggs. changes in spike suggest that attachment and entry may have been affected and changes in nsp 3 suggest that the attenuated virus lost the ability to overcome some innate host cell defense mechanism in the mature chicken cell. the exact mechanism(s) surrounding the interaction of virus and host processes affecting virus replication have yet to be determined for ibv, but identifying the sequence changes in the virus responsible for reduced replication and attenuation is an important step in elucidating those mechanisms. finally, changes observed in nsp 3 and spike as well as in other viral genes support the polygenic nature of pathogenicity in avian coronaviruses. infectioius bronchitis epitopes of neutralizing antibodies are located within three regions of the s1 spike protein of infectious bronchitis virus infectious bronchitis, in a laboratory manual for the isolation, identification, and characterization of avian pathogens code of federal regulations, standard requirements for ibv vaccines. animal and plant health inspection service, us national archives and records administration proc. natl. acad. sci. usa 103 acknowledgments this work was supported by usda, csrees award number 2007-35600-17786. the authors appreciate the assistance that was provided by lauren byrd, carey stewart, and joshua jackwood in conducting these studies. key: cord-272666-3uidpr79 authors: doyle, nicole; neuman, benjamin w.; simpson, jennifer; hawes, philippa c.; mantell, judith; verkade, paul; alrashedi, hasan; maier, helena j. title: infectious bronchitis virus nonstructural protein 4 alone induces membrane pairing date: 2018-09-06 journal: viruses doi: 10.3390/v10090477 sha: doc_id: 272666 cord_uid: 3uidpr79 positive-strand rna viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral rna synthesis. infectious bronchitis virus (ibv), a pathogenic avian gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (dmvs), zippered endoplasmic reticulum (zer) and tethered vesicles, known as spherules. these membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. in this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of ibv were compared. three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. in contrast to previously studied coronaviruses, ibv nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate dmvs. this indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble ibv replicative structures. this study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family. viruses rely on their host cell to provide most of what they need to replicate and in order to do this, they hijack many cellular processes. a well-studied example is the ability of positive-sense single-stranded rna viruses (+rna) to induce cellular membrane rearrangements upon expression of viral proteins [1, 2] . this reorganization of cellular membranes is a critical step in the viral replication cycle since these areas of restructured membranes act as a site for assembly of all components required for viral rna synthesis as well as offer protection from detection by the host antiviral defenses [3, 4] . although the structures of these membranes are relatively well-understood, the mechanisms behind their formation, and particularly the viral and host proteins involved, are often not. the precise structure of virally induced membrane rearrangements varies between viruses [5, 6] , but viruses generally cause proliferation of membranes, forming structures, such as convoluted membranes (cm), as well as distinct types of vesicles. most common are double membrane vesicles (dmvs), which are discrete from the cytoplasm and are produced by viruses, such as poliovirus [7, 8] , hepatitis c virus [9, 10] , human norovirus [11] , and recently the equine torovirus, berne virus [12] . spherules, which are invaginated vesicles with a channel connecting them to the cytoplasm, have been found in semliki forest virus [13] , some flaviviruses [14] [15] [16] [17] , as well as brome mosaic virus (bmv), which is able to induce their formation with the expression of just one viral protein [18] . an important +rna virus family, the coronaviruses, include pathogens of both animal and human importance, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), mouse hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and infectious bronchitis virus (ibv). within this subfamily of viruses, we see variations in membrane rearrangements formed. dmvs and cm are found in cells infected with the alphaand betacoronaviruses, such as sars-cov, mers-cov, and mhv [19] [20] [21] [22] [23] [24] . in the case of the gammacoronavirus ibv, although dmvs are found, the virus induces little cm and instead induces membrane zippering to form zippered endoplasmic reticulum (zer) as well as double membrane spherules, which are found tethered to the zer [25] , producing a much more defined structure when compared to cm. subsequent to this discovery, mers-cov infection has also been shown to produce small circular structures similar in appearance to the spherules seen in ibv infection but less distinct [26] . the coronaviral proteins involved in the production of membrane rearrangements have been recently investigated with the three transmembrane non-structural proteins (nsps) nsp3, 4, and 6, which are the focus of these studies. nsps 3, 4, and 6 from different coronaviruses are accepted as functional homologues, although amino acid sequence conservation is low (ranging from 13.4 to 25.9% amino acid homology for nsps 3, 4, and 6 between ibv strain beaur and mhv strain a59). these proteins do, however, have conserved secondary structure and conserved domains, including enzymatic domains in nsp3, transmembrane domains in all three proteins, and cytoplasmic endo-domains in nsps 4 and 6. for a detailed review of the domain organization and known functions of nsps 3, 4, and 6, see [27] . nsp4 of mhv has been shown to be important for the normal function and stability of dmvs, where mutations in nsp4 resulted in attenuated virus and impairment of dmv formation [28] [29] [30] . in addition, nsp3 has been shown to localize to dmvs and cm in sars-cov-infected cells [31] . in a related group of viruses, the arteriviruses, expression of two nsps (nsps2 and 3) was able to produce dmvs [32] [33] [34] . these nsps of the arterivirus are considered functional homologs to coronavirus nsp3 and 4 [35] . upon co-expression of nsp3 and 4 from mhv, both proteins located to areas of curved membranes from where they were shown to be able to recruit nsp2 and 6; however, nsp3 and 4 alone were not able to induce the formation of dmvs [36, 37] . following on from this, it was shown that co-expression of sars-cov nsp3 and 4 induced membrane pairing and with the addition of nsp6 the formation of dmv-like structures [38] . in a subsequent study by others, it was shown that expression of only nsp3 and 4 from either mers-cov or sars-cov was able to induce dmv formation, and furthermore, addition of nsp6 made no difference to their shape or size, and did not induce the spherule-like structures seen following infection with whole virus [26] . interestingly, however, a small molecule inhibitor, k22, has been shown to inhibit the replication of several coronaviruses in vitro. in hcov-229e, k22 impaired dmv formation, while k22 resistance was associated with mutations in nsp6, emphasizing a role for nsp6 in dmv formation [39] . ibv is a pathogen of poultry, causing significant economic losses to the poultry industry worldwide as well as animal welfare problems. various strains of ibv cause disease that varies in severity from mild respiratory problems to virulent strains that can cause nephropathology and reproductive organ pathology. in this study, we compared the membrane rearrangements induced by viral proteins from two different strains of ibv, the pathogenic m41 and the apathogenic beaur. these strains were chosen because beaur and other strains of ibv induce dmv, zer, and spherule formation; however, m41 produces a low spherule phenotype when compared with other strains of the virus [40] . as the role in membrane rearrangements for nsp3 and 4 is well-established for several nidoviruses and considering that nsp6 may also play some role, here we investigated the role that these three nsps play in the formation of ibv membrane rearrangements. avian df1 cells were maintained in dmem (sigma aldrich, gillingham, uk) supplemented with 10% fcs (sigma aldrich, gillingham, uk). ibv strains beaur and m41-ck (here referred to as m41) have been described previously [41, 42] . plasmids expressing tagged nsps derived from either the apathogenic strain beaur or the pathogenic strain m41 were generated to produce pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag, and pcdna3.1(-)-m41 nsp6-3xflag. rna was extracted from virus-infected cells using an rnaeasy kit (qiagen, hilden, germany) following the manufacturer's protocol. rna was reverse transcribed using superscript iii (fisher scientific, loughborough, uk) and a random primer following the manufacturer's protocol. pcr was carried out on cdna using primers specific for each gene, including flanking restriction sites. pcr products were digested and ligated into pegfp-n1 (takara bio europe, saint-germain-en-laye, france) or pmcherry-n1 (takara bio) using xhoi and bamhi restriction sites. plasmid pcdna3.1(-) was modified by insertion of a 3xflag motif between the kpni and hindiii sites to generate pcdna3.1(-)-3xflag. the pcr products were then ligated into this backbone using the xhoi and bamhi restriction sites. plasmid sequences were verified using sanger sequencing. the er marker plasmid pyfp-er was kindly provided by dalan bailey. df1 cells seeded into six-well plates were transfected with pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag or pcdna3.1(-)-m41 nsp6-3xflag, pegfp-c2, pmchery-n1, or pcdna3.1(-)-beaur nsp7-3xflag using lipofectamine 2000 (fisher scientific). cells were transfected with a total of 1000 ng plasmid with a dna:lipofectamine 2000 ratio of 1:3 following the manufacturer's instructions. after 24 h, cells were lysed in cell lysis buffer (25 mm tris-hcl (ph 7.4), 150 mm nacl, 1 mm edta, 1% v/v triton-x100, 5% v/v glycerol, 1× halt protease inhibitor complex (fisher scientific). cell lysates were heated with 4× sample buffer (bio-rad laboratories, watford, uk) and separated on 4-20% tgx gels (bio-rad). proteins were transferred to a nitrocellulose membrane and blocked in 5% milk in pbs-t. membranes were incubated with primary antibodies to detect gfp (biolegend, london, uk), mcherry (abcam, cambridge, uk), or flag (m2; sigma aldrich, gillingham, uk). after 1 h, membranes were washed with pbs-t and incubated with irdye conjugated secondary antibodies (li-cor, cambridge, uk). membranes were imaged using an odyssey clx infrared imaging system (li-cor). df1 cells seeded onto glass coverslips were transfected with pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag, and pcdna3.1(-)-m41 nsp6-3xflag alone or in combination using lipofectamine 2000. cells were transfected with a total of 500 ng plasmid with a dna:lipofectamine 2000 ratio of 1:2 following the manufacturer's instructions. after 24 h, cells were fixed for 20 min in 4% paraformaldehyde in pbs at room temperature. cells were then permeabilized in 0.1% triton x-100 in pbs for 10 min and blocked in 0.5% bsa in pbs for 1 h. primary anti-flag m2 antibody (sigma aldrich) and anti-pdi antibody (enzo life sciences, exeter, uk) were diluted in blocking buffer and cells incubated for 1 h. after three washes in pbs, alexa fluor conjugated secondary antibodies (fisher scientific) were diluted 1/500 and cells incubated for 1 h. after a further three washes in pbs, nuclei were strained using topro3 (fisher scientific) or dapi (sigma aldrich) and coverslips mounted with vectashield (vector laboratories, peterborough, uk). cells were visualized using a leica sp5 confocal microscope (leica microsystems, milton keynes, uk). quantitation of transfected cells was performed manually on three randomly selected fields of view. df1 cells in six-well plates were either infected with beaur and incubated for 1 h at 37 • c when fresh 1× bes medium (mem, 0.3% tryptose phosphate broth, 0.2% bovine serum albumin, 20 mm n,n-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (bes), 0.21% sodium bicarbonate, 2 mm l-glutamine, 250 u/ml nystatin, 100 u/ml penicillin, and 100 u/ml streptomycin) was added, or were transfected with plasmids as described above. at 24 hpi, cells were washed once in 0.9% saline and scraped into the saline buffer. cells were pelleted at 500× g for 5 min at 4 • c and 500 µl 2% glutaraldehyde in 0.1 m sodium cacodylate was added to the pellet. df1 cells were then rinsed three times in 0.1 m sodium cacodylate and incubated in 1% osmium tetroxide for 2 h. after three washes in water, cells were incubated in 2% uranium acetate aqueous for 2 h at 4 • c. cells were dehydrated in increasing concentrations of acetone and then embedded in agar 100 resin (agar scientific, stansted, uk). sections approximately 50 to 60 nm in thickness were cut and stained with 2% uranyl acetate to enhance contrast. data was recorded at 80 kv on a phillips cm20 (amsterdam, netherlands) with a charge-coupled device (ccd) camera. cell sections used here each contained a single visible nucleus, with intact nuclear and plasma membranes. alternatively, df1 cells were seeded onto thermanox coverslips (fisher scientific) and either infected with beaur and incubated for 1 h at 37 • c, after which time fresh 1× bes medium was added, or cells were transfected with plasmids as described above. after 24 h, cells were fixed in 2% glutaraldehyde for 1 h, incubated in 1% aqueous osmium tetroxide solution for 1 h, then dehydrated in increasing concentrations of ethanol. cells were embedded into agar 100 resin and sections of 80 nm were cut, collected on hexagonal 200 thin bar copper grids, and stained with 2% uranyl acetate and lead citrate. data was recorded on a fei tecnai 12 tem (fei, cambridge, uk) used at 100 kv with a tvips f214 digital camera. df1 cells seeded onto thermanox coverslips were transfected and processed as before. sections 250 or 300 nm thick were cut from the resin-embedded blocks and collected on 50 mesh copper hexagonal grids coated in formvar or pioloform-coated copper slot grids. ten or 15 nm gold particles were applied to the grids to serve as fiducial markers for subsequent alignments. data was recorded on a jeol 2100 f tem (jeol, welwyn garden city, uk) used at 200 kv with a tvips f416 digital camera, or on a tecnai 20 tem (fei) used at 200 kv with a fei 4 k × 4 k eagle ccd camera. samples were mounted in a jeol high angle tilt holder or a fischione double tilt tomography holder, respectively. a single axis tilt series was collected using serial em or fei software. each single axis tilt series was collected over 100 • to 130 • in increments of between 1 • and 2.5 • and subsequently aligned and reconstructed in imod [43] . our previous studies have shown that ibv is able to induce diverse membrane rearrangements in vero cells, primary chicken kidney cells (ckcs) and tracheal organ cultures (tocs). these membrane rearrangements include dmvs, zer, and spherules [25, 40] . in order to further characterize membrane rearrangements induced by ibv, we analyzed the membrane rearrangements induced by beaur in df1s. unlike primary ckcs, df1s are a continuous avian cell line that are more easily transfected and are therefore used throughout this study. although the spike protein of beaur has increased tropism to allow for virus entry into additional cell lines, including df1 cells, m41 is not adapted to infect these cells [44, 45] . df1 cells were infected with beaur, fixed after 24 h, processed for em, and imaged. consistent with previous work, dmvs, zer, and spherules were all seen in ibv-infected df1 cells ( figure 1 ). beaur. df1 cells were infected with beaur for 24 h, fixed, and processed for electron microscopy (em). viral particles are indicated with arrowheads, double membrane vesicles (dmvs) with asterisks, and zippered endoplasmic reticulum (zer) and associated spherules with arrows. scale bar represents 500 nm. other viruses in the nidovirales order have been shown to require expression of only two or three nsps to induce membrane rearrangements similar to those seen under virus infection conditions [26, 34, 38] . to begin to understand the roles of ibv nsps in rearranging cellular membranes, nsps4 and 6 from apathogenic beaur and nsps3, 4, and 6 from pathogenic m41 were tagged with fluorescent or epitope tags. it was not possible to generate a plasmid expressing nsp3 from beaur due to presumed toxic sequences, as has been found for this region in other coronaviruses [46] [47] [48] . df1 cells were transfected with these plasmids and after 24 h cells were lysed and proteins separated by sds-page and detected by western blot. all fusions proteins were found to be intact with bands detectable at the predicted molecular weights (figure 2a) , although an additional 49 kda band was present in nsp3-gfp expressing cells, presumably due to a cleavage event within nsp3. it was also noted that nsp6-3xflag from m41 migrated at a higher molecular weight than nsp6-3xflag from beaur, most likely due to differences in post-translational modification. subsequently, df1 cells were transfected with these plasmids and after 24 h cells were fixed, labelled with an anti-flag antibody, and visualized by confocal microscopy. all three nsps showed reticular cytoplasmic labelling consistent with localization to the er (figure 2b ), as has been observed previously [26, 37, [49] [50] [51] [52] [53] . in addition to er localization, nsp4 was found in both small and large puncta in cells where the level of nsp4 expression was higher (comparison shown in figure 2b ). nsp6 was also found in small cytoplasmic puncta when expressed alone (figure 2b ). to confirm er localization, df1 cells were transfected with either the plasmid expressing nsp3-gfp alone or plasmids expressing nsp4 or 6 together with pyfp-er, as indicated. after 24 h, cells were fixed and labelled with anti-flag-and nsp3-expressing cells with anti-protein disulphide isomerase (pdi), a resident er protein. colocalization between yfp-er or pdi and nsp3, 4, and 6 was observed, confirming that these proteins localize to the er (figure 2c ). cells were transfected with plasmids expressing tagged nsps, as indicated, or empty vectors or nsp7-3xflag as controls. cell lysates were separated by sds-page and proteins detected by western blot. from left to right, nsp3-gfp detected using anti-gfp, nsp4-mcherry detected using anti-mcherry, and nsp6-3xflag detected using anti-flag, as labelled. molecular weight markers are shown on the left and asterisks indicate the nsp bands on each blot. (b) df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsp3-egfp, nsp4-mcherry, and nsp6-3xflag from m41. after 24 h, cells were fixed with 4% paraformaldehyde and imaged. nsp3 (green), nsp4 (red), and nsp6 (blue) were imaged as labelled. nuclei were stained with topro3 (grey) and scale bars indicate 10 µm. (c) df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsp3-egfp, nsp4-mcherry, and nsp6-3xflag from m41 together with yfp-er. after 24 h, cells were fixed with 4% paraformaldehyde and imaged. nsp3 (green) and nsp4 and nsp6 (red) were imaged along with markers for the er; pdi (red) or yfp-er (yellow) as indicated. nuclei were stained with dapi (grey) and scale bar represents 10 µm. next, to understand whether co-expression of these proteins results in changes in their localization, df1 cells were transfected with combinations of the plasmids. after 24 h, cells were fixed and labelled with an anti-flag antibody. upon co-expression of some combinations of these viral proteins, this staining pattern changed. expression of nsp3 with nsp4 resulted in both proteins localizing to cytoplasmic puncta, although some signals for both proteins also remained in the er (figure 3 ). co-expression of nsp3 with nsp6, or nsp4 with nsp6, did not result in relocalization of either protein, with nsp3 remaining er-associated, nsp4 remaining both er-associated and localized in cytoplasmic puncta, and nsp6 remaining both er-localized and in cytoplasmic puncta (figure 3) . interestingly, co-expression of nsps3, 4, and 6 resulted in relocalization of all three proteins to cytoplasmic puncta, some containing nsp3 and 4, some nsp6 only, and some puncta containing nsp3, 4, and 6 ( figure 3 ). nsps4 and 6 derived from either beaur or m41 exhibited the same pattern of localization. this demonstrates that co-expression of ibv nsps in the absence of any other viral components can result in their relocalization within the cell, presumably as a result of protein-protein interactions and potentially associated with rearrangement of cellular membranes. figure 3 . co-expression of ibv non-structural proteins results in their relocalization from the er to cytoplasmic foci. df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsps3-egfp, nsp4-mcherry, and nsp6-3xflag from m41 in pairs or in a combination of three, as indicated. solid arrows indicate areas of nsp3 and 4 colocalization, open arrows indicate areas of nsp3, 4, and 6 colocalization. nuclei were strained with topro3 (grey) and scale bar represents 10 µm. to further understand the ability of ibv nsps3, 4, and 6 to rearrange cellular membranes, proteins were expressed in cells and analyzed by electron microscopy (em). initially, to assist with subsequent analysis by em, the percentage of total cells in figures 2b and 3 that were expressing the nsps of interest, as well as the percentage of cells expressing other combinations of nsps, was quantified (table s1 ). df1 cells were transfected with tagged nsp3, 4, and 6 derived from beaur or m41 alone and in combination. after 24 h, cells were chemically fixed, embedded in resin, and visualized using an electron microscope. a phenotype common to all transfected cells was small, tight whorl-like structures which stained more strongly than other structures (figure 4a ). these were considered an artefact of transfection. transfection of cells with empty pegfp-n1, pmcherry-n1, or pcdna3.1(-)-3xflag did not result in changes to cellular membranes (figure 4a ). different types of membrane structures were observed in the transfected cell samples that were absent from mock treated cells, including paired membranes, disordered or piled membranes, and dmv-like structures. nsp4 in other coronaviruses has been shown to be important in membrane modifications, particularly in the formation of conventional dmvs [29, 30] . initially, the effect of expression of nsp4 in df1 cells was investigated. interestingly, it was observed that expression of beaur nsp4 alone was capable of forming paired membranes. this the first time this has been observed for any coronavirus nsp4. these paired membranes were observed both as very large areas of extensive accumulations or as small regions of shorter sections of paired membranes. the paired membranes were tightly apposed, often connected to the er, were largely free of ribosomes, and strongly resembled ibv-induced zer (figure 4b) , although the electron density often surrounding ibv-induced zer was missing here and no spherules were present. transfection of m41 nsp4 also induced membrane pairing (figure 4b) with an appearance comparable to that of beaur nsp4-induced paired membranes. for cells transfected with the beaur nsp4 expression vector, 31 out of 235 cell sections (13%, percentage of total cells not transfected cells) contained piled membranes, and 3 out of 108 (3%) sections for m41, significant to p < 0.00001 by a fisher's exact test. it has previously been shown for other coronaviruses that membrane pairing requires co-expression of nsps3 and 4 or that co-expression of these proteins results in dmv accumulation [26] . therefore, the effect of co-expression of nsp3 with nsp4 was investigated. firstly, the effect of expression of nsp3 alone on cellular membranes was determined. although over 200 cells were examined from multiple experiments, expression of nsp3 was found to have no striking phenotype with cellular membranes appearing unchanged in the presence of nsp3 derived from m41 when compared with untransfected cells. furthermore, surprisingly, expression of nsp3 with nsp4 had no effect on the membrane pairing ability of nsp4 (figure 4c) , with membrane rearrangements appearing comparable to cells expressing nsp4 alone, i.e., paired membranes connected to the er and lacking ribosomes, found covering both large and smaller areas of the cytoplasm. specifically, the numerous dmv-like structures observed in cells expressing nsp3 and 4 from either mers-cov or sars-cov were not observed here [26, 38] . overall, this data confirms that ibv nsp4 alone is the main driving factor in membrane pairing and co-expression of nsp3 does not alter this function. coronavirus nsp6 has previously been linked to autophagy induction when expressed alone [49] . nsp6 derived from sars-cov has also been shown to induce single membrane vesicle accumulation and microtubule organizing center vesiculation [38] . therefore, the cellular membrane rearrangements induced by expression of ibv nsp6 were analyzed. in cells expressing nsp6 alone from either beaur or m41, large areas of tangled single membranes, which appear to be derived from the er, were observed ( figure 5 ). these piled, disordered membranes strongly resemble the disordered membrane bodies seen previously upon expression of sars-cov nsp3 [38] . to determine the effect of expression of nsp6 with other nsps on the formation of disordered membranes or any other structures, samples transfected with plasmids expressing nsp6 and either nsp4 or nsp3 were analyzed. in cells co-expressing nsp3 and 6, it was observed that cells expressing beaur nsp6 formed disordered membranes while those expressing m41 nsp6 did not. co-expression of nsp4 and nsp6 produced the paired membranes associated with nsp4 expression (for both beaur and m41 nsp4). disordered membranes were only found in cells co-expressing beaur nsp6 but none when co-expressing m41 nsp6. this indicates that while nsp6 from either beaur or m41 can induce the formation of disordered membranes when expressed singly, co-expression of nsp6 with either nsp3 or 4 disrupts this mechanism and to a greater extent in m41. finally, the membrane rearrangements induced by co-expression of ibv nsps3, 4, and 6 were investigated by electron microscopy to determine whether co-expression of all three transmembrane nsps could result in the formation of structures comparable to replication organelles in ibv-infected cells. the major phenotype observed following co-expression of all three nsps was the paired membranes induced by expression of nsp4 alone ( figure 6 ). when nsp4 and nsp6 derived from beaur were expressed with m41 nsp3, a very limited number of dmv-like structures was observed (3 in 329 cell sections). in cells co-expressing nsp3, 4, and 6 derived from m41, no dmv-like vesicles were found in 489 cell sections with only nsp4-associated paired membranes being detected. in neither combination were the spherules usually found during virus infection observed. therefore, although co-expression of ibv nsps 3, 4, and 6 may be sufficient for formation of dmvs, this does not seem to be a very efficient process compared with dmv formation by nsp3 and 4 from the betacoronaviruses studied previously [26, 38] and nsp6 is unlikely to be the additional nsp required for ibv dmv formation. in order to further understand the paired membranes induced by expression of ibv nsp4, electron tomography (et) was used to visualize membrane rearrangements in three dimensions. in addition, et was used to confirm that, unlike for other coronaviruses [26, 38] , co-expression of ibv nsp3 and 4 does not result in the formation of dmvs. df1 cells were transfected with plasmids expressing either beaur nsp4 or beaur nsp4 with m41 nsp3. after 24 h, cells were fixed and processed for et. the paired membranes produced by nsp4 expression (indicated by arrows) were found to form sheet-like structures with sections of paired membranes dilating in several places (arrowheads) (figure 7a , video s1). a comparison with cells expressing nsp3 and 4 showed there is no noticeable difference between the areas of paired membranes induced upon expression of these nsps (figure 7b, video s2) . therefore, expression of ibv nsp4 alone results in the formation of paired er membranes. addition of nsp3 does not alter the membrane structures induced with no formation of either dmvs, as seen for other covs or spherules. induction of host cell membrane rearrangements is a tool used by many +rna viruses, such as coronaviruses [1, 2] . these membrane rearrangements vary between the different members of the family, with the alpha and betacoronaviruses inducing convoluted membranes and dmvs and the gammacoronavirus ibv inducing zippered er, spherules, and dmvs [19] [20] [21] [22] [23] 25, 26] . the formation of these membrane rearrangements is, however, a well-conserved mechanism used by these viruses in order to provide a site for viral rna synthesis. although the pool of knowledge about these structures has been growing, the mechanisms behind their formation remain largely unclear. some light has been shed in recent years on the specific viral proteins involved in the formation of these structures; however, these studies were lacking in ibv. in this study, we looked at the involvement of nsps3, 4, and 6, which have all been implicated in the formation of membrane rearrangements. as transmembrane proteins, these are likely candidates in reordering the host cell membranes to the advantage of the virus. we showed firstly that df1 cells are a suitable cell type to use for studying ibv membrane rearrangements in addition to those already tested [40] . in order to assess the involvement of nsps 3, 4, and 6 in virus-induced membrane rearrangements, plasmids expressing gfp, mcherry, or 3xflag fusion proteins were generated. to confirm expression of full-length fusion proteins, western blots were performed using antibodies against the tags. for all the constructs, full-length nsp fusion proteins were detected. however, in cells expressing nsp3-gfp, an additional 49 kda band was seen indicating that as well as full-length protein, a cleavage product corresponding to the c-terminus of nsp3 plus gfp was also being produced. next, we expressed nsps alone or in combination in df1 cells to assess their ability to rearrange cellular membranes. when expressed alone, all three nsps had a reticular, cytoplasmic localization consistent with previous observations that these nsps localize to the er [26, 37, [49] [50] [51] [52] [53] , although nsp4 and nsp6 in addition had a punctate localization with nsp4 in particular forming large foci in some cells. er localization was subsequently confirmed by colocalization of the three nsps with er markers. when nsps3 and 4 were co-expressed, both proteins localized to large and small cytoplasmic puncta with some protein also remaining in the er. this suggests that nsp3 and 4 are able to interact with one another, again consistent with previous findings for other coronaviruses [26, 37] , resulting in nsp3 moving into the nsp4-containing puncta. co-expression of nsp3 and 6 or nsp4 and 6 did not result in alteration of their cellular localization. however, when nsp3, 4, and 6 were co-expressed, nsp3 and 4 colocalized as seen before but some puncta now also contained nsp6, although some puncta contained only nsp3 and 4 or nsp6 alone. this suggests that, as seen in other coronaviruses, nsp3 and 4 together, but not alone, are able to direct nsp6 into the nsp3/4 puncta [36, 37] . subsequently, em was used to identify changes to the structure of cellular membranes upon expression of these three proteins. surprisingly, expression of nsp3 did not induce any notable phenotype. expression of nsp3 from either sars-cov or mers-cov results in the production of disordered membrane bodies likely derived from the er [26, 38] . it is not clear why nsp3 derived from ibv behaves so markedly differently from nsp3s expressed by other coronaviruses. however, the previously studied nsp3s have all been derived from betacoronaviruses so nsp3 from gammacoronaviruses, including ibv, may function somewhat differently. indeed, an amino acid sequence comparison between nsp3 sequences from beaur and the betacoronavirus mhv a59 shows only 13.4% homology and 25.9% similarity. therefore, although these are accepted as functional homologs, there is scope for these proteins to behave differently from one another. furthermore, given that we have previously demonstrated that ibv-induced membrane rearrangements are distinct from those induced by alphaand betacoronaviruses [25] , differences in the mechanism of their formation might reasonably be expected. interestingly, expression of nsp6 alone induced membrane proliferation and the formation of disordered membranes similar to the disordered membrane bodies (dmbs) induced by sars-cov and mers-cov nsp3 [26, 38] . expression of nsp6 alone did not appear to induce microtubule organizing center vesiculation as seen upon expression of sars-cov nsp6 [38] and the presence of autophagosomes was also not apparent [49, 54] , although this is likely due to differences in experimental approaches, namely the use of em in this study compared to immunofluorescence of whole cells used previously [54] . therefore, ibv nsp6 also appears to function somewhat differently to nsp6 from sars-cov in its ability to rearrange membranes. the most striking phenotype came upon expression of nsp4; expression of nsp4 alone was sufficient to induce areas of paired membranes. furthermore, et demonstrated that these are sheet-like areas of paired er membranes, highly similar to zer in ibv-infected cells. it was noted that the paired membranes, although resembling zer in infected cells, lacked the electron density often surrounding the membranes [25] . this reflects the lack of the other viral proteins making up the replication complex, which, presumably, accumulate on the cytoplasmic surface of the zer. nsp4-induced paired membranes were observed as both small regions throughout the cytoplasm and also in extensive areas of paired membranes. these two phenotypes potentially reflect the different localizations observed by confocal microscopy with some cells containing nsp4 localized only to the er and some cells containing large cytoplasmic puncta corresponding to the large areas of paired membranes. use of correlative light electron microscopy (clem) in the future would confirm this. attempts were made to confirm the nsp4 homotypic interaction by co-immunoprecipitation; however, this was not successful. it has previously been shown for mhv that nsp4 can self-associate [37] , although earlier attempts to demonstrate the interaction in sars-cov failed [55, 56] , highlighting that detection of this interaction can be challenging. however, it is likely that self-interaction between nsp4 proteins located in both membranes of the er zippers the two er membranes together to generate the paired membranes seen, although it cannot be ruled out that instead an interaction with one or more cellular proteins is required. significantly, this is the first time for any coronavirus that, regardless of mechanism, a membrane pairing function for nsp4 alone has been described. surprisingly, addition of nsp3 did not alter the membrane rearrangements induced by nsp4 alone. previous work by others has shown that for other related coronaviruses and arteriviruses, membrane pairing requires the expression of nsp3 and 4 (or their homologs) [36] [37] [38] 57] . in addition to this, however, co-expression of nsp3 and 4 for other coronaviruses resulted in the formation of numerous dmv-like structures [26, 38] . despite extensive searching and the use of electron tomography to gain three-dimensional information, we were not able to detect any dmvs in cells expressing nsp3 and 4. the reason for this difference is not clear. here, we used separate plasmids to express nsp3 and 4 but this strategy was also used in previous work and when compared with a cleavable nsp3-4 precursor did not yield different results [26] . therefore, the protein expression strategy is unlikely to be the reason that dmvs were not formed. it is possible that the presence of the shorter nsp3 fragment detected by western blot prevented the formation of dmvs. however, full-length nsp3 was also present and therefore should have been capable of inducing dmvs in combination with nsp4. in addition, dmvs were not detectable in cells expressing either nsp3 from m41 and nsp4 from beaur or cells expressing nsp3 and 4 from m41, indicating that the use of proteins from different virus strains was not the reason for the lack of dmvs. indeed, nsp3 relocalized to both beaur and m41 nsp4-containing foci suggesting that m41 nsp3 is capable of interacting with both nsp4 proteins. again, attempts were made to confirm interaction between nsp3 and nsp4 by co-immunoprecipitation, but this was not successful. interactions between full-length or the c-terminus of nsp3 and nsp4 from other coronaviruses have been shown previously [37, 55] . interestingly, sakai et al. showed that just two amino acid residues in nsp4 are necessary for the interaction with nsp3; however, these residues are only conserved in betacoronaviruses, not in alphaor gammacoronaviruses [58] , so it is likely that the mechanism of any nsp3/nsp4 interaction is different in ibv. overall, the data indicates that dmv formation by ibv requires the presence of additional viral protein(s), either to direct an interaction between nsp3 and nsp4 if it cannot occur directly or because dmv formation is via another mechanism. co-expression of nsps 3, 4, and 6 did appear to result in the formation of a very small number of dmv-like structures. however, these were significantly less numerous and less easily identifiable than those observed by oudshoorn et al. [26] . therefore, nsp6 does not appear to be the ibv protein required, in addition to nsp3 and 4, to induce dmvs and other viral proteins must play a role. throughout this study, we were unable to detect spherules associated with ibv infection, although we did identify membranes highly similar to zer. in our previous work, we demonstrated that m41 virus has a low spherule phenotype and the region of the genome from the 5 end to nsp13 was responsible for this [40] . unfortunately, we were unable to clone nsp3 from beaur due to toxicity problems in escherichia coli. it was also not possible to clone nsp3 from two further strains of ibv. as the nsp3 used in this study was derived from m41, it is possible that this is the reason that spherules were not detected under any conditions. nsp3 from beaur and m41 are highly related with 90.5% amino acid homology and 95.2% similarity with the majority of the differences occurring within the non-functional papain-like protease 1 domain. despite that fact, it cannot be ruled out that these differences are sufficient to prevent spherule formation. in future, cloning the c-terminal part of nsp3 from beaur, as other groups have done for mhv [36] , may provide further insight into the role of nsp3 in membrane modifications. an alternative explanation for the lack of spherules could be that the precise molar ratio of nsps to one another, as well as the presence of cleavage intermediates, generated as a result of expression via a polyprotein during virus infection is critical for spherule formation. in that case, the expression approach taken here of transfecting multiple plasmids into cells would not result in the correct ratio of proteins or presence of cleavage intermediates, thereby preventing spherule formation. however, oudshoorn et al. were also unable to identify cms and spherule-like structures when combinations of nsps were expressed either from separate plasmids or as a polyprotein [26] . instead, it is more likely that additional viral proteins are required for spherule formation. this is not necessarily surprising. for alphaviruses, spherules are only formed in the presence of all nsps and although they are able to form in the absence of rna, the length of rna present directly affects the size of the spherule produced [59, 60] . furthermore, in the case of flock house virus, spherules only form when rna synthesis is actively occurring [61] . therefore, spherule formation by ibv may require expression of additional nsps, including those required for rna synthesis, as well as an rna template. alternatively, it may require expression of additional nsps that direct interaction with cellular proteins that facilitate changes to the membrane. the mechanisms behind the formation of virus-induced membrane rearrangements required for replication organelle formation are doubtlessly complex. although we have identified a clear role for ibv nsp4 in membrane pairing and the formation of zippered er, numerous questions remain and further differences between ibv and members of the betacoronavirus sub-family have been highlighted. the identity of the ibv proteins required for both spherule and dmv formation remain unknown and further study is required to complete our understanding of the critical stage of the virus replication cycle. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/10/9/477/s1, table s1 : cells expressing each nsp compared to the total number of cells counted, video s1: electron tomographic reconstruction of paired membranes in an nsp4-expressing cell, video s2: electron tomographic reconstruction of paired membranes in an nsp3 and nsp4-expressing cell. modification of intracellular membrane structures for virus replication virus factories, double membrane vesicles and viroplasm generated in animal cells organelle-like membrane compartmentalization of positive-strand rna virus replication factories the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites ultrastructure of the replication sites of positive-strand rna viruses architecture and biogenesis of plus-strand rna virus replication factories structural and functional characterization of the poliovirus replication complex complex dynamic development of poliovirus membranous replication complexes identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection membrane alterations induced by nonstructural proteins of human norovirus ultrastructural characterization of membranous torovirus replication factories biogenesis of the semliki forest virus rna replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures ultrastructural characterization of zika virus replication factories a positive-strand rna virus replication complex parallels form and function of retrovirus capsids ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex rna replication of mouse hepatitis virus takes place at double-membrane vesicles qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructural characterization of membrane rearrangements induced by porcine epidemic diarrhea virus infection infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex evolving the largest rna virus genome membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. virology mobility and interactions of coronavirus nonstructural protein 4 severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity reverse genetics system for the avian coronavirus infectious bronchitis virus taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures computer visualization of three-dimensional image data using imod selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate detection of nonstructural protein 6 in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches membrane topology of murine coronavirus replicase nonstructural protein 3 topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome biogenesis and architecture of arterivirus replication organelles two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication template rna length determines the size of replication complex spherules for semliki forest virus partially uncleaved alphavirus replicase forms spherule structures in the presence and absence of rna template nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, rna templates, and polymerase activity the authors would like to thank paul britton and pip beard for helpful discussions. the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord-298078-uqrwq5qk authors: kwak, hoyun; park, min woo; jeong, sunjoo title: annexin a2 binds rna and reduces the frameshifting efficiency of infectious bronchitis virus date: 2011-08-30 journal: plos one doi: 10.1371/journal.pone.0024067 sha: doc_id: 298078 cord_uid: uqrwq5qk annexin a2 (anxa2) is a protein implicated in diverse cellular functions, including exocytosis, dna synthesis and cell proliferation. it was recently proposed to be involved in rna metabolism because it was shown to associate with some cellular mrna. here, we identified anxa2 as a rna binding protein (rbp) that binds ibv (infectious bronchitis virus) pseudoknot rna. we first confirmed the binding of anxa2 to ibv pseudoknot rna by ultraviolet crosslinking and showed its binding to rna pseudoknot with anxa2 protein in vitro and in the cells. since the rna pseudoknot located in the frameshifting region of ibv was used as bait for cellular rbps, we tested whether anxa2 could regulate the frameshfting of ibv pseudoknot rna by dual luciferase assay. overexpression of anxa2 significantly reduced the frameshifting efficiency from ibv pseudoknot rna and knockdown of the protein strikingly increased the frameshifting efficiency. the results suggest that anxa2 is a cellular rbp that can modulate the frameshifting efficiency of viral rna, enabling it to act as an anti-viral cellular protein, and hinting at roles in rna metabolism for other cellular mrnas. ribosomal frameshifing is a recoding process of translation where a specific messenger rna (mrna)-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame. programmed-1 ribosomal frameshifting is the most widely used translational recoding mechanism of many viruses [1] . many viruses employ frameshifting during replication and generate the frameshifted protein critical for efficient viral replication [2, 3, 4] . in the case of retroviruses such as hiv-1, ribosomal frameshifting is required for the expression of protease, reverse transcriptase and integrase. for most other viruses, including coronavirus avian infectious bronchitis virus (ibv), frameshifting generates rna dependent rna polymerases. since translation is a complex process involving many regulatory factors in addition to the ribosome, binding of rna binding protein (rbp) at a nearby rna signal could affect rna conformation and somehow redirect the translational machinery [5, 6] . therefore, the identification of rbps involved in frameshifting is important in elucidating the regulatory mechanism for rna translation. viral frameshifting signals are present in coding regions of mrna, and are generally composed of a ''slippery'' sequence, a stimulatory downstream rna hairpin or pseudoknot structure and an intervening spacer region [7, 8, 9] . rna pseudoknots are structural elements found in almost all classes of rna, which are now recognized as a widespread motif with diverse biological functions [2] . for example, viral pseudoknots in non-coding regions act in the regulation of translation initiation and in template recognition by viral replicase. in contrast, when pseudoknots are present in coding regions, they regulate translation elongation and termination, leading to ribosomal frameshifting [10] . it is not clear whether ribosomal frameshifting occurs through the interaction of ribosome or any rbps, or due to the intrinsic nature of the pseudoknot rna. annexin a2 (anxa2) is a multi-functional protein that has been implicated in a number of cellular functions, including calcium dependent regulation of exocytosis, dna synthesis and cell proliferation [11] . recently, its potential role in rna metabolism was proposed because it was found to bind some mrna transcripts, such as c-myc, collagen prolyl 4-hydroxylasea (i) and its own mrna [12, 13, 14, 15, 16] . since anxa2 binds 39untranslated region (utr) of these mrnas, it was suggested to be involved in mrna localization and translational regulation [15, 17] . interestingly, anxa2 is reported to be the most abundant proteins in cytoskeleton bound polyribosome fraction and also associated with mrnas [17] . these findings led to the suggestion that anxa2 may bind to some rna elements involved in translational regulation. in fact, aberrant expression of anxa2 is somehow related to the carcinogenesis of human cancers, because it has been shown to be clearly absent in some prostate cancers and highly overexpressed in other human cancers [18, 19, 20] . to identify the proteins that might recognize frameshifting signals, we presently utilized ibv pseudoknot rna as a model transcript. we identified anxa2 as a rbp that can recognize viral pseudoknot rna and reduce viral frameshifting. we confirmed the rna binding activity of anxa2 by various methods and tested its role in the regulation of frameshifting. by overexpression and knockdown approaches, we showed it to be an efficient modulator of viral frameshifting. we speculate that cellular anxa2 protein may act as an anti-viral protein that can reduce detrimental viral frameshifting and successful replication of the virus. the present results offer a clue for the role of anxa2 as a rbp for other cellular rna transcripts. since ibv pseudoknot rna was shown to induce ribosomal frameshifting during rna translation, we utilized ibv frameshifting rna sequences as the model transcript [3] . ibv genomic rna contains the frameshifting rna pseudoknot as well as the slippery site and the spacer. sequences of wild-type and mutant ibv pseudoknot rna (72 nts) shown in figure 1a were generated as in vitro transcribed transcript. rna structure of wild-type ibv rna was predicted to fold into pseudoknot structure, whereas mutant ibv rna was not able to form a pseudoknot structure (pknotsrg: http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/submission.html) [21, 22] . these predicted structure are largely consistent with the structure previously reported for ibv pseudoknot rna by brierley et al. (1992) , albeit with minor differences [9] . to search for cellular proteins that directly interacted with ibv pseudoknot rna, a rna pull down assay was performed in the presence of cell extracts (figure 2a ). wild-type and mutant ibv pseudoknot rnas were end-labeled with biotin to serve as bait for cellular proteins that might precipitate with streptavidin-coated beads. in addition, rna was in vitro transcribed in the presence of 4-thio utp that could serve as crosslinker for directly interacting proteins. ibv pseudoknot rna was incubated with cell extracts from human lung fibroblast imr90, which is a natural host cell for the infection of ibv. the complex was irradiated with 365 nm uv light to directly crosslink interacting proteins to 4-thio utp incorporated ibv pseudoknot rna. rna-protein complexes were precipitated with streptavidin-coated magnetic beads to pulldown any proteins bound to biotin-labeled ibv pseudoknot rna. proteins were eluted from the beads, fractionated by sds-page and analyzed by silver staining. comparison of the bands from wild-type pseudoknot rna bound to those from the mutant demonstrated that most of the bound proteins were common for two rna molecules ( figure 2a ). however, a couple of protein bands seemed to present more in wild-type than mutant pseudoknot rna, so we eluted those bands from the gel. identification of the protein by maldi-tof-tof revealed that one of such proteins was annexin a2 (anxa2). the previous experiment indicated the possible interaction of anxa2 to ibv pseudoknot rna. to confirm whether anxa2 could bind wild-type ibv pseudoknot rna but not mutant ibv rna, we performed uv crosslinking assay to show that radiolabeled wild-type ibv pseudoknot rna bound to anxa2 protein ( figure 2b ). after rnase treatment on rna-protein complex, radiolabeled ibv pseudoknot rna bound protein bands were characterized by western blot analysis with anti-anxa2 antibody. radiolabeled rna was found near the size of the cytosolic extracts were prepared from imr90 cells, and the extracts were incubated with either wild-type or mutant ibv pseudoknot rna and precipitated with streptavidin-coated magnetic beads. bound proteins (pellet) were eluted from the beads, fractionated using 10% sds-page and stained with silver. unbound proteins (sup) were also analyzed as a control. input refers to 10% input of imr90 cell extract. bands isolated from the gel are also shown on the right with higher magnification. maldi-tof-tof analysis identified the wild-type bound protein as anxa2, indicated with asterisk. (b) uv crosslinking assay (left). imr90 cell extracts were incubated with the [a-32 p] labeled wild-type (wt) or mutant ibv pseudoknot rna. after crosslinking with uv, rna-protein complexes were untreated (-) or treated (+) with rnase a and fractionated in polyacrylamide gel. two protein-rna complex bands from imr90 cell extract (shown with an arrow on the right side of the gel, an asterisk indicating non-specific band) were shown to be specific to wild-type (wt) ibv rna. competition assay (middle). cell extracts were incubated with the [a-32 p] labeled wild-type (wt) or mutant (mt) ibv rna and non-labeled competitor (16, 106 and 1006, respectively). western blot analysis of uv-crosslinked rna-protein complexes (right). the same gel as shown on the left was blotted and incubated with anti-anxa2 antibody. all data were generated by the results from at least three independent experiments. doi:10.1371/journal.pone.0024067.g002 anxa2 protein, which suggests an interaction between radiolabeled ibv pseudoknot rna and cellular anxa2 protein. significantly, this band specifically competed with wild-type ibv pseudoknot rna but not with mutant ibv rna, also supporting the binding specificity of anxa2 to wild-type pseudoknot rna ( figure 2b ). identity of the rna bound protein was also confirmed by western blot analysis of the same gel ( figure 2b ). these data clearly demonstrated binding of anxa2 to the frameshifting rna pseudoknot in the ibv genome. next, we tested whether anxa2 could bind directly to ibv pseudoknot rna. to explore this, gst-fused recombinant anxa2 protein was purified and used for gst pull-down assay. more radioactivity was recovered after precipitating with gst-anxa2 in comparison to gst, suggesting that recombinant anxa2 could associate with ibv pseudoknot rna. moreover, wild-type ibv pseudoknot rna tended to bind more than mutant ibv rna did ( figure 3a ). we tested whether such binding was dependent on the presence of calcium, given that anxa2 is a calcium binding protein. however, calcium did not cause any change in the binding of anxa2 to ibv pseudoknot rna (data not shown). we performed a rna-emsa to analyze the anxa2 binding pattern ( figure 3b ). rna-protein complexes were resolved using native polyacrylamide gels. only a single shifted band was generated in the presence of anxa2 protein but not with the same amount of the gst protein ( figure 3b ). we also confirmed whether anxa2 bound to wild-type ibv pseudoknot rna in the cells. through the rna-immunoprecipitation assay, we showed that anxa2 specifically interacted with wild-type ibv pseudoknot rna but not with mutant ibv rna in lncap and hek293t cells ( figure 3c and 3d). these data clearly demonstrated the specific binding of anxa2 to ibv pseudoknot rna. anxa2 regulates ribosomal frameshifting of ibv pseudoknot rna since we found that anxa2 bound ibv pseudoknot rna, we next tested whether it could control the efficiency of ribosomal frameshifting on ibv pseudoknot rna in the cells. we constructed dual luciferase reporters controlled by ibv frameshifting sequences with or without pseudoknot rna ( figure 4a ). frameshifting of the dual luciferase reporter expressed firefly luciferase as well as renilla luciferase ( figure 4b ), so the frameshifting could be assessed by the ratio between these two proteins as well as by control reporters. control reporters with either in frame or no expression of firefly luciferase used as positive or negative controls, respectively. to test how anxa2 regulates the frameshifting efficiency of ibv pseudoknot rna, we first overexpressed anxa2 protein in the presence of the reporters and measured the luciferase activities. transfection of flagtagged anxa2 in hek293t cells reduced the relative frameshifting efficiency of ibv pseudoknot rna up to 40% ( figure 5a ). since hek293t cells possess an endogenous protein, overexpression of flag-anxa2 might generate additional exogenous anxa2 protein. to assess more definitively the inhibitory effect of anxa2 on the frameshifting of ibv pseudoknot rna, we used a prostate cancer cell line, lncap, which does not express endogenous anxa2 protein in the cells. more significant reduction of the relative frameshifting efficiency was shown in lncap cells, which likely resulted from the overexpression of anxa2 without any basal level expression of the protein ( figure 5b ). conversely, we showed that the downregulation of the anxa2 by three independent knockdown greatly increased the relative frameshifting efficiency of ibv pseudoknot rna in hek293t cells ( figure 5d and 5e). targeting sequences of shrna and sirna in anxa2 mrna were diagrammed in figure 5c . by independent experiments either with shrna expression vector or with sirna duplex, we clearly demonstrated that frameshifting efficiency was dramatically increased by anxa2 knockdown. these data clearly indicated that anxa2 controls the frameshifting efficiency in the cells, which could act as the cellular inhibitor against the frameshifting of viral rna translation. translation is the complex rna metabolism regulated by diverse factors, such as ribosome, trna and many ribosome associated proteins. here, we identify anxa2 as the regulator of ribosomal frameshifting. since it was previously reported that anxa2 binds rna and regulates rna localization [13, 14, 15, 16, 17] , our proteomic identification of anxa2 as an ibv pseudoknot rna binding protein leads us to propose that it might regulate the frameshifting efficiency of viral rna translation. as shown in figure 5 , knockdown of anxa2 increased the frameshifting efficiency up to seven times, whereas overexpression of anxa2 significantly down-regulated the frameshifting efficiency of ibv pseudoknot rna. these results clearly show that anxa2 reduces ibv viral frameshifting in some way, probably by binding to pseudoknot rna. since the frameshifting of ibv generates rna dependent rna polymerase, which is critical for successful viral replication, the protein that inhibits frameshifting must be a major antiviral regulator in eukaryotic cells. however, many questions remain to be answered. these include the exact mechanism of anxa2 action on ibv frameshifting, possible involvement of anxa2 on frameshifting of other viral rna and unexplored functions of rna metabolism on many other pseudoknot containing cellular rna. rna pseudoknots are structural elements formed upon basepairing of a single-stranded region to a stretch of complementary nucleotides elsewhere in the rna chain. since the first discovery of pseudoknot structure in 39-utr of turnip yellow mosaic virus (tymv), numerous viruses have been reported to contain pseudoknot rna [7, 8, 9] . many other cellular rnas, such as rrna, mrna, tmrna, catalytic rna, telomerase rna, rna components of ribonucleoprotein complex and many artificially selected rna aptamers also form pseudoknot structures [23, 24, 25, 26, 27] . a pseudoknot is not only a structural element of rna, but also a functional regulator of rna. as mentioned previously, rna pseudoknot located in the coding region of virus is involved in the -1 ribosomal frameshifting of viral rna translation, and pseudoknot in 59-utr in some cases regulates the initiation of viral rna replication [28] . since mutational analysis revealed drastic changes of ribosomal frameshifting efficiency upon disruption of pseudoknot structure, this structure is likely to be kept intact in addition to the slippery site and spacer region for efficient frameshifting [29] . recent advances in rna bioinformatics broaden the possibility of finding pseudoknot structure from rna sequences and predicting threedimensional structures of the rna molecules [30, 31, 32] . the detailed mechanism of pseudoknot function on frameshifting is being intensively studied. recent data suggests that the mechanical strength of the pseudoknot structure is correlated with ribosomal frameshifting, with pausing of ribosome itself is not sufficient for frameshifting [33, 34, 35, 36] . while still contentious, several reports describe pseudoknot binding protein factors. for example, 102-kda rna binding protein and eukaryotic elongation factor 1 (eef1) bind rna pseudoknot of 39-utr in the tobacco mosaic virus genome [37, 38] . recently, it was also reported that replicase gene product of mouse coronavirus interacts with its own rna pseudoknot [29] . also, we present data showing that anxa2 ; lanes 4-7 contain gst-anxa2 protein (0.5, 2, 5 and 10 mm, respectively). (c) rna immunoprecipitation assay. lncap cells were cotransfected with reporters (wild-type or mutant ibv pseudoknot plasmid wild-type or mutant) and anxa2 expression plasmids (flag-vector or flag-anxa2). after formaldehyde fixation, immunoprecipitations were performed with flag-m2 agarose beads. bound rna was extracted from the immune complexes and analyzed by rt-pcr (left panel) and qrt-pcr (right panel). pcr products were resolved by electrophoresis in agarose gel and visualized by staining with ethidium bromide. anxa2 mrna and ibv pk rna were also shown as an expression and input controls. immunoprecipitation of flag-anxa2 was confirmed by immunoblotting (ib) using anti-flag antibody. in the qrt-pcr data, wild-type pk rna (wt) in binds pseudoknot rna and inhibits ribosomal frameshifting of ibv. even though it is necessary to determine the exact mode of action for anxa2 on ribosomal frameshifting, evidence is accumulating concerning the role of anxa2 in translational regulation. for example, anxa2 is associated with active and inactive mrnas, polyribosomes and many ribosomal subunit proteins [12, 19] . in addition, the receptor for activated c-kinase (rack1), which resides in ribosome acting as the signal regulator of translation, also interacts with anxa2 [39] . deregulated expression of anxa2 is implicated in many human tumors and diseases [20, 40] . especially, anxa2 has an important role in angiogenesis and tumor progression, and, thus, might be a potential therapeutic target [41] . furthermore, the gene linked to optiz bbb/g syndrome, mid1, is associated with microtubule-associated ribonucleoprotein complex including ef-1a, rack-1, small ribosomal subunit proteins and anxa2, which again suggests a possible role of anxa2 in rna translation and in pathogenesis of the cell [42] . since viral frameshifting is critical for many pathogenic viruses including coronavirus, anxa2-pseudoknot rna interaction might be a novel target for anti-viral drug discovery [43, 44] . plasmids containing wild-type (pfs-cass5) and mutant (pfs-cass5.15) pseudoknot sequences from ibv genomic rna were generous gifts form dr. ian brierley (university of cambridge) [3] . bovine anxa2 cdna was kindly provided by dr. anni vedeler (university of bergen) [12] . human embryonic kidney cell line hek293t were purchased from the american type culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) containing 10% fetal bovine serum (fbs; brl life technology). human prostate cancer cell line lncap were purchased from the atcc and cultured in rpmi1640 with 10% fbs. human lung fibroblast imr90 and human lung adenocarcinoma sk-lu i cells were purchased from atcc and cultured in mem containing 10% fbs using standard techniques. cells were harvested and washed twice with ice-cold phosphate buffered saline (pbs). cell pellets were resuspended in two volumes of buffer a (20 mm hepes, ph 7.9; 10 mm nacl; 1 mm edta; 1 mm dtt) supplemented with protein inhibitor cocktail (1 mm pmsf, sigma-aldrich). after incubating 15 min on ice, cells were lysed with 0.5% nonidet p-40 and centrifuged at 4,000 rpm at 4uc for 30 sec. clarified cytosolic extract was quantitated for protein concentration and stored in aliquots at 280uc. wild-type and mutant ibv rnas were transcribed in the presence of 4-thio utp (ambion) by in vitro transcription with t7 rna polymerase. phosphate was removed from 59-end by calf intestinal alkaline phosphatase (new england bio lab) and c-s 32 was added by incubating with c-s 32 atp (10 mm) with t4 polynucleotide kinase (new england bio lab). biotin was added at the 59-end of the prepared rna by incubating one and half hours with 10 mm peo-iodoacetyl biotin (pierce) in the dark. cytosolic extracts were pre-cleared with streptavidin-coated magnetic beads (new england bio lab) and beads were preblocked with trna. biotin and 4-thio labeled rna was incubated with pre-cleared cell extract in binding buffer (20 mm hepes, ph 7.5; 50 mm kcl; 1 mm dtt; 0.1 mm edta; 5% glycerol; 40 u rnase inhibitor) for one hour in room temperature. ultraviolet radiation (365 nm) was performed to induce binding between 4-thio utp and interacting proteins. pre-blocked magnetic beads were added and incubated for one hour to pellet rna-protein complexes. two volumes of washing buffer (20 mm hepes, ph 7.5; 500 mm nacl, 1 mm dtt, 0.1 mm edta, 5% glycerol) was added and washed seven times, followed by elution with boiling in 26 sample buffer for 5 min. proteins were separated by 10% sds-page and visualized by silver staining. after pulling-down interacting proteins by a biotin pull-down assay as described below, protein identification was performed in the functional proteomics center, korea institute of science and technology. silver stained bands were excised and an automated in-gel tryptic digestion was performed on a mass prep station (micromass). the gel pieces were destained, reduced with dtt, alkylated with iodoacetamide and digested with sequencing grade modified trypsin (promega). resulting peptides were extracted from the gel and analyzed via maldi-tof and maldi-tof-tof. proteins were identified from the mass spectrometry data using mascot (matrix science) and searching the ncbi data base. ibv pseudoknot rna was transcribed and labeled in vitro with t7 rna polymerase (ambion) with a-[ 32 p]ctp. 32 p-labeled ibv pseudoknot rna (1.1610 5 c.p.m) was incubated with 30 mg of cell extract with 2 mg of trna, 0.2 mg of poly d(i-c) and 40 u of rnase inhibitor. the rna-protein binding reaction was carried out in a 30 ml reaction mixture containing 20 mm hepes ph 7.5, 50 mm kcl, 1 mm dtt, 0.1 mm edta and 5% glycerol. the mixtures were incubated at room temperature for 30 min, after which they were uv-irradiated (120 mj/cm 2 ) on ice for 30 min with an uv cross linker (fisher scientific). rnas were digested with 1 mg rnase a, 25 u rnase s1 and 0.1 u rnase v1 at 37uc for 10 min and analyzed by 10% sds-page. anti-anxa2 antibody (bd biosciences) was used for western blot analysis. proteins in sample buffer were resolved by 10% sds-page, transferred to a polyvinylidene difluoride membrane, immunostained by specific antibodies and visualized using a chemiluminescent substrate. gst-pull down assay was performed for confirmation of direct rna-protein interaction. radiolabeled rna transcript (5610 4 c.p.m) and purified proteins were incubated at room immune complex was normalized with input pk rna level and presented as relative enrichment in comparison to mutant pk rna (mt). (d) rna immunoprecipitation assay in hek293t cells. reporter plasmids (wild-type or mutant ibv pseudoknot plasmid) were transfected into hek293t cells and fixed with formaldehyde. sonicated lysates were then incubated with the antibodies as indicated (anti-igg or anti-anxa2). rna-protein complexes were precipitated with protein-g beads. bound rna was extracted from the immune complexes and analyzed by rt-pcr and qrt-pcr. immunoprecipitated anxa2 protein was shown by immunoblotting (ib). relative enrichment of binding was shown as in (c). doi:10.1371/journal.pone.0024067.g003 temperature for 1 h in a binding buffer containing 25 mm hepes ph 7.5, 100 mm nacl and 1 mm mgcl 2 . rna-protein complexes were pulled down by glutathione sepharose 4b (ge healthcare) pre-blocked with trna for 30 min. following washing using binding buffer, the radioactivity in the recovered pellets was measured by scintillation counting. construction of anxa2 expression clone and rna interference cdna for anxa2 was polymerase chain reaction (pcr)amplified and ligated into pcmv-tag2b vector (stratagene). anxa2 was amplified with primers 59 -caggatccatgtc-taccgttca-39 and 59 -ccgaattctcagtcatcccca-c-39. to knock-down anxa2 mrna, small hairpin rna (shrna) was designed using the ambion small interfering rna (sirna) converter website. the anxa2 target sequence was 59-ugcauaugggucugucaa-39 corresponded to coding region nucleotides 66-83. to make psuper-anxa2 (shanx2-1), specific oligonucleotides were synthesized (bioneer, korea) and ligated to psuper vector as previously reported [45] . also, two different sirnas corresponding to coding region nucleotides 109-129 (sianx2-2: 59-cgggaugcuuugaacauugaa-39) and 772-792 (sianx2-3: 59-aaccugguucagugcauucag-39) were designed. sirna duplex were chemically synthesized and contain dtdt 39 overhangs (bioneer, korea). purification of recombinant anxa2 protein cdna of the anxa2 protein was pcr amplified and inserted into the pgex-4t-1 vector (ge healthcare), followed by the transformation of the vectors into the protease deficient strain bl21. gst-anxa2 and gst protein was purified according to manufacturer's instructions. the basic procedure for emsa was previously described [46] . briefly, rna was in vitro transcribed and labeled in vitro with t7 rna polymerase (ambion) with [a-32 p]atp. 32 p labeled ibv pseudoknot rna (1.1610 5 c.p.m) and purified gst-anxa2 or gst protein were incubated at room temperature for 30 min in a binding buffer containing 25 mm hepes ph 7.5, 100 mm nacl, 1 mm mgcl 2 and 4% glycerol. rna-protein complexes were resolved on 5% native polyacrylamide gels. gels were dried and analyzed by autoradiography. the basic procedure for rna-ip was previously described [46] . briefly, lncap and hek293t cells were transiently cotransfected with wild-type of mutant ibv pseudoknot reporters and pcmv tag2b vector (flag-vector) or flag-anxa2 plasmids, and incubated with 1% formaldehyde for crosslinking. sonicated lysates were immunoprecipitated with flag m2 agarose beads (sigma aldrich) or with antibodies as indicated (anti-normal mouse igg or anti-anxa2). pellets were subsequently incubated at 70uc for 1 h to reverse the crosslinks, and the rna was purified with tri reagent (ambion) and treated with dnase i (ambion) to remove reporter plasmid dna. after reverse transcription, cdna was amplified by using primer pairs for ibv pseudoknot rna (59-gtcgactttaaactgatacggggt-atc-39 and 59-gaaggatcccagctgaaaggc-39). qrt-pcr was performed with stepone real-time pcr system (applied biosystems). reactions were amplified using selective primers as described above with power sybr master mix (applied biosystems) according to the manufacturer's instructions. quantification was carried out with the stepone tm software (ver 2.2). percentages of pk rna binding to anxa2 were expressed as the to make ibv dual luciferase reporters, a frameshifting sequence (72 bp) was amplified from pfs-cass 5 and pfs-cass 5.15. the sequence was inserted between renilla and firefly luciferase genes of a dual reporter, p2luc vector (kindly provided by dr. yang kyun kim, sungkyunkwan university) [47] . wildtype reporter is composed of slippery site, stop codon, spacer linker and pseudoknot region of ibv genomic sequences. the mutant reporter is as same as the wild-type reporter except for a disrupted pseudoknot. the in-frame reporter has wild-type pseudoknot but not frameshifting regulatory elements, where no-frame reporter has wild-type frameshifting regulatory elements except for the slippery site. wild-type and mutant ibv pseudoknot sequences were amplified by pcr with primers 59-aatgtcgactt-taaactgatacggg-39 and 59-taaggatcccagctga-aaggctc-39 from of pfs-cass 5 and pfs-cass 5.15, respectively. in frame sequence was amplified with primers 59-aa-tgtcgacacggggtatcagtc-39 and 59-taaggatcc-cagctgaaaggctc-39. no frame sequence was amplified with primers 59-aatgtcgacctgatacggggtat-39 and 59-taaggatcccagctgaaaggctc-39. cells were cultured and transfected with frameshifting reporters using lipofectamine tm (invitrogen). for the luciferase assay, cells were scraped into 100 ml of passive lysis buffer (promega). luciferase activity in the lysate was determined with a dualluciferase reporter assay system (promega), according to the manufacturer's instruction and measured with a turner luminometer td-20/20. firefly luciferase activity was normalized to the activity of renilla luciferase. frameshifiting efficiency was calculated by the following formula: where fe = frameshifting efficiency, f = wild-type or mutant luc activity, fn = no-frame luc activity and fp = in-frame luc activity. programmed ribosomal frameshifting in hiv-1 and the sars-cov viral rna pseudoknots: versatile motifs in gene expression and replication decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1 a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal trading translation with rna-binding proteins translation matters: protein synthesis defects in inherited disease an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot mutational analysis of the "slipperysequence" component of a coronavirus ribosomal frameshifting signal structure, stability and function of rna pseudoknots involved in stimulating ribosomal frameshifting annexins -unique membrane binding proteins with diverse functions annexin a2 is a novel rna-binding protein annexin a2 binds to the localization signal in the 39 untranslated region of c-myc mrna annexin a2 recognises a specific region in the 39-utr of its cognate messenger rna translational control of collagen prolyl 4-hydroxylase-(i) gene expression under hypoxia the mrnabinding site of annexin a2 resides in helices c-d of its domain iv an au-rich stem-loop structure is a critical feature of the perinuclear localization signal of c-myc mrna medicationrelated problem type and appearance rate in ambulatory hemodialysis patients annexin ii is associated with mrnas which may constitute a distinct subpopulation loss of annexin ii heavy and light chains in prostate cancer and its precursors equal loading of proteins were confirmed by anti-b-actin antibody. (b) luciferase reporter assay after overexpression of anxa2 in lncap cells. relative frameshifting efficiency was measured for the vector and flag-tagged anxa2 transfected cells. expression of anxa2 was confirmed by western blot analysis with anti-anxa2 antibody. anti-b-actin antibody was used as a loading control. (c) diagram of sirna or shrna target sequences (dashed line) in the coding regions anxa2 mrna (filled line). locations of a shrna and two sirnas were indicated (shanx2-1: 66-83 nt, sianx2-2: 109-129 nt, sianx2-3: 772-792 nt). (d) dual luciferase reporter assay following knockdown of anxa2 in hek293t cells. relative frameshifting efficiency was measured with psuper vector (vector) and with psuper-anxa2 (shanx2-1) transfected cells. western blot analysis was performed after shrna transfection. anti-b-actin antibody was used as a loading control. (e) luciferase reporter assay after knockdown of anxa2 in hek293t cells. relative frameshifting efficiency was measured from control sirna (sigfp) or two independent anxa2 sirnas (sianx2-2 and -3) transfected cells. western blot analysis was performed after sirna transfection. anti-b-actin antibody was used as a loading control pknotsrg: rna pseudoknot folding including near-optimal structures and sliding windows design, implementation and evaluation of a piratical pseudoknot folding algorithm based on thermodynamics structure and function of telomerase rna structural basis of a ribozyme's thermostability: p1-l9 interdomain interaction in rnase p rna folding of the sam aptamer is determined by the formation of a k-turn-dependent pseudoknot endogenous expression of a high-affinity pseudoknot rna aptamer suppresses replication of hiv-1 rna aptamers that bind the nucleocapsid protein contain pseudoknots genetic interactions between an essential 39 cis-acting rna pseudoknot, replicase gene products, and the extreme 39 end of the mouse coronavirus genome mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses beyond mfold: recent advances in rna bioinformatics the mc-fold and mc-sym pipeline infers rna structure from sequence data knotseeker: heuristic pseudoknot detection in long rna sequences the 9-ã� solution: how mrna pseudoknots promote efficient programmed -1 ribosomal frameshifting correlation between mechanical strength of messenger rna pseudoknots and ribosomal frameshifting a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency isolation and characterization of the 102-kilodalton rna-binding protein that binds to the 5 and 3 translational enhancers of tobacco mosaic virus rna eukaryotic elongation factor 1a interacts with the upstream pseudoknot domain in the 39 untranslated region of tobacco mosaic virus rna regulation of eukaryotic translation by the rack1 protein: a platform for signalling molecules on the ribosome the annexinopathies: a new category of diseases the role of annexin ii in angiogenesis and tumor progression: a potential therapeutic target the opitz syndrome gene product mid1 assembles a microtubule-associated ribonucleoprotein complex coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus identification of novel ligands for the rna pseudoknot that regulate -1 ribosomal frameshifting specific inhibition of gene expression using a stably integrated, inducible small-interfering-rna vector b-catenin stabilizes cyclooxygenase-2 mrna by interacting with au-rich elements of 39-utr from the cover: specific mutations in a viral rna pseudoknot drastically change ribosomal frameshifting efficiency we appreciate drs. brierly (university of cambridge), vedeler (university of bergen) and yang kyun kim (sungkyunkwan university) for plasmids. we especially thank dr. myeong-hee yu (director of functional proteomics center, korea institute of science and technology) for allowing us to use proteomics facility and providing us excellent analysis. key: cord-286473-sl5zy8nj authors: gomaa, m.h.; barta, j.r.; ojkic, d.; yoo, d. title: complete genomic sequence of turkey coronavirus date: 2008-05-12 journal: virus res doi: 10.1016/j.virusres.2008.03.020 sha: doc_id: 286473 cord_uid: sl5zy8nj turkey coronavirus (tcov), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in ontario, canada, and the full-length genomic sequence was determined. the full-length genome was 27,632 nucleotides plus the 3′ poly(a) tail. two open reading frames, orfs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream orfs were identified. a gene for hemagglutinin-esterase was absent in tcov. the region between the membrane (m) and nucleocapsid (n) protein genes contained three potential small orfs: orf-x, a previously uncharacterized orf with an associated putative trs within the m gene (apparently shared among all group iii coronaviruses), and previously described orfs 5a and 5b. the tcov genome is organized as follows: 5′ utr – replicase (orfs 1a, 1b) – spike (s) protein – orf3 (orfs 3a, 3b) – small envelop (e or 3c) protein – membrane (m) protein – orf5 (orfs x, 5a, 5b) – nucleocapsid (n) protein −3′ utr – poly(a). tcov genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (ibv). this is the first complete genome sequence for a tcov and confirms that tcov belongs to group iii coronaviruses. turkey coronavirus (tcov) is associated with highly contagious gastroenteritis in young poults. first identified in 1951 (peterson and hymass, 1951) , tcov causes high morbidity, some mortality, and poor long-term growth of the affected birds, resulting in significant economic losses in the turkey industry. outbreaks have been reported in different areas in the usa including minnesota, north carolina, and indiana as well as in quebec, canada (dea et al., 1986) . turkey coronavirus was subsequently determined to be the causative agent of blue comb disease during an investigation of an outbreak of acute, highly contagious enteritis in a flock of young turkeys (nagaraja and pomeroy, 1997) . in addition to turkeys, tcov can infect a variety of avian hosts including chickens, pheasants, sea gulls, and quail (deshmukh and pomeroy, 1974) . recently, tcov was incriminated as one of the most important causative agents of poult enteritis and mortality syndrome teixeira et al., 2007) . coronaviruses are divided into three groups (i, ii, and iii) based on the genome structure and organization (holmes and lai, 1996) . the coronavirus genome is a single-strand positive-sense rna with a 5 cap and poly (a) tail at the 3 end. two large open reading frames (orfs) occupy the 5 proximal two thirds of the genome and are involved in polyprotein processing, genome replication, and subgenomic rna synthesis, and the 3 one third of the genome codes for structural proteins. orf1 consists of two overlapping orfs (orf1a and orf1b) that are translated into 1a and 1a/1b polyproteins by a ribosomal frame-shifting mechanism. orf1a encodes 2 proteases; papain-like cysteine protease (plp) and picornavirus 3c-like chymotrypsin protease (3clp). both proteases cleave the polyproteins into at least 16 cleavage products (sawicki et al., 2007) . an intergenic consensus sequence of about seven bases is found immediately upstream of each gene, which plays an important role for subgenomic rna synthesis (sawicki et al., 2007) . most coronaviruses code for four major structural proteins; spike (s) glycoprotein, membrane (m) protein, small envelope (e) protein, and nucleocapsid (n) proteins, in addition to hemagglutinin esterase (he) glycoprotein in some group ii coronaviruses. the genome structure and organization are not known for any tcov, and this lack of genetic information has made it difficult to develop effective detection and control measures for tcov. the goal of the present study was to complete the full genomic sequence of tcov and establish a better understanding of the virus at the molecular level. clinical specimens consisting of intestinal tracts were collected from turkey poults during an outbreak of diarrhea in a turkey farm in ontario, canada. the tissue samples were processed by washing several times with phosphate buffer saline (pbs) and cut into small pieces prior to grinding using a sterile mortar and pestle. coarse particles and tissue debris were removed by centrifugation at 3000 × g for 30 min at 4 • c. the supernatants were filtered through 0.22-m filters (millipore, bedford, ma). virus was isolated from the filtrates by inoculating embryonated turkey eggs. allantoic fluids were collected 3 days post-inoculation then clarified by centrifugation 3000 × g for 30 min at 4 • c and the supernatant were filtered through 0.22-m filters and stored at −80 • c until use. complementary dna (cdna) was synthesized using 10.5 l of the first strand mixture (invitrogen, carlsbad, ca) containing 0.2 g of random hexamers and 2 g of total rna isolated from the intestinal specimens. the mixture was incubated at 70 • c for 10 min and then quick-chilled on ice for 5 min. rt master mix was composed of 4 l 5× rt buffer (invitrogen), 2 l 10 mm dtt, 2 l 10 mm dntps (amersham, piscataway, nj), 1 l superscript ii reverse transcriptase (invitrogen), and 0.5 l rnase inhibitors (invitrogen). this rt master mix was added to 10.5 l of the first strand mixture and then incubated at 42 • c for 2 h. the reaction was terminated by heating at 95 • c for 10 min then chilling on ice for 5 min. fifty microliters pcr reactions included 2 l of cdna and 48 l of the master mix composed of 5 l 10× buffer (100 mm tris-hcl, 500 mm kcl), 1.5 l of 15 mm mgcl 2 , 1.5 l of 10 mm dntps, 2 pmol of upstream and downstream primers, two units of taq high fidelity dna polymerase (invitrogen), two units of amplitaq gold (roche molecular systems, inc., usa) plus 38 l nuclease free water. pcr was performed for 35 cycles as follow; 95 • c for 1 min, 65 • c for 1 min, 55 • c for 30 s, 72 • c for 3 min, followed by final extension at 72 • c for 10 min. the pcr products were analyzed by 1% agarose gel electrophoreses and visualized by staining with ethidium bromide and uv illumination. thirteen overlapped pcr fragments spanning the entire viral genome were amplified using specific primer sets (table 1) . the pcr products were purified using the qia quick pcr purification kit (qiagen, valencia, ca) and cloned in pgem-t easy (promega, madison, wi). transformants were screened by restriction enzyme digestion and sequencing using primers specific for t7 and sp6 promoter. the sequences were analyzed using the sequencher 4.5 sequence analyses program, and a single contiguous sequence comprising the entire tcov genome was constructed. prediction for orfs was conducted using vector nti advanced 10 (invitrogen), and the sequences were analyzed using lasergene dna star (version 7, lasergene corp, madison, wi). the pairwise nucleotide identity was determined using vector nti advanced 10 and multiple sequence alignments were generated using clustal-w (thompson et al., 1997) . comparative analyses of tcov with other coronaviruses were conducted using the coronavirus database (covdb, huang et al., 2007) . cdna clone representing the 5 end of the tcov-mg10 genome had been synthesized according to the 5 race system for rapid amplification of cdna ends (invitrogen). the antisense primer had been designed based on the available tcov-mg10 sequence (5 -cgccaggtgttattttgtca) then cdna was synthesized as mentioned before. the cdna was purified using qiagen column purification kits. tailing of the cdna was done using dctp and dtd. pcr had been done to amplify the dc-tailed cdna with the abridged anchor primer together with the designed primer (5 -gttgtcactgtctattgtatg) according to the instructions of the kits. the 3 end of tcov genome had been done using the 3 race system for rapid amplification of cdna ends (invitrogen) according to the instruction of the kite. cdna was synthesized using the adaptor primer then the cdna was amplified with pcr using the tcov-mg10 specific primer (5 ctatcgccagggaaatgtct 3 ) and the universal amplification primer according to the instruction of the kits. the obtained pcr products had been cloned and sequenced in both directions as mentioned earlier. the obtained sequence aligned with the rest of the genomic sequence and run through the poly(a) tail. phylogenetic relationships among coronaviruses were investigated using the following complete genomes representing groups i, ii, and iii: bcov-ent (nc 003045), hcov-nl63 (dq445912.1), hcov-oc43 (nc 005147.1), ibv-m41 (ay851295), ibv-p65 (dq001339.1), ibv-cal99 (ay514485), ibv nc (nc 001451), sarscov-bj202 (ay864806.1), hcov-229e (nc 002645.1), mhv-jhm (nc-006852.1) and fipv wsu-79/1146 (nc 007025.1). whole genome sequences were aligned using clustalw (thompson et al., 1997) and subsequently optimized by eye using the geneious software (version 3.0.6, biomatters ltd, auckland, new zealand) (kumar et al., 2004) . aligned nucleotide sequences were analyzed using paup (version 4.0), and maximum likelihood (hky85 model with transition/transversion ratio of 2) and maximum parsimony (transversion/transition ratio of 2) search criteria were used with branch and bound search strategies (swofford, 1991) . bootstrap supports for the resulting trees were determined using 100 replicate heuristic tree searches in both parsimony and likelihood analyses using the same search criteria. for more detailed ingroup analysis of relationships among the group iii coronaviruses, amino acid sequences for the spike glycoprotein (s), envelope protein (e, also known as 3c) and the nucleocapsid (n) protein were aligned using clustalw and subsequently analyzed using maximum parsimony (branch and bound search algorithm) followed by bootstrap analysis using a heuristic search method. the following additional group iii coronavirus sequences used for the comparative sequence analyses: tcov-gh s gene (ay342356), tcov-gi s gene (ay342357), quail coronavirus italy/elvia/2005 s1 gene (ef446155.1), tcov-nc95 n gene (af111997), tcov-minnesota n gene (af111996), tcov-indiana n gene (af111995). all ingroup translation products (s, e and n proteins) from the whole genomes of the same group i and ii coronaviruses used above were included in these analyses. the group i and ii coronaviruses were considered functional outgroups for determining relationships among the group iii coronaviruses. the tcov full genomic sequence described in this report was deposited in the genbank database with accession number eu095850. a tcov was isolated from an outbreak in an ontario turkey farm and designated tcov-mg10. subsequently, the full-length genomic sequence of tcov-mg10 was determined by sequencing of overlapping pcr fragments in both directions. the sequences were assembled into one contiguous sequence to represent the entire viral genome. the sequence of 27,632 nucleotides was obtained, plus the polyadenylation tail at the 3 end. the entire genome has a gc content of 38.3%. the tcov genome contained two large slightly overlapping orfs in the 5 two thirds of the genome and multiple additional orfs in the 3 one third of the genome (fig. 1) . both termini were flanked with untranslated regions (utrs). the tcov genome was similar overall in its coding capacity and genomic organization to those of other coronaviruses. eleven orfs were identified in the genome (table 2) . gene 1 was 19,806 nucleotides comprising orf1a and orf1b, located between nucleotides 529 and 20,333. this gene contained motifs common in all coronaviruses including ribosomal frameshifting and slippery sequences, as orf1b is translated in the −1 frame. the typical coronavirus structural genes encoding the spike (s), small envelope (e, also known as 3c), membrane (m), and nucleocapsid (n) proteins were identified following gene 1 (table 2, fig. 1 ). the tcov genome had polycistronic genes 3 and 5 interspersed between the s and e genes, and between m and n genes, respectively. in ibv, gene 3 is believed to be tricistronic consisting of 3a, 3b and 3c, where 3c has been shown to encode the small envelope (e) protein. gene 5 contained a coding potential for two products, 5a and 5b, of unknown function. a third orf of 282 nucleotides was located between the m gene and gene 5a. this gene, designated orf-x, contained a coding potential for a protein of 94 amino acids that had no structural or sequence homology with any known protein. blast searches failed to identify any protein homologs, but identified highly similar nucleotide sequences from other tcov isolates as well as from numerous ibv isolates. orf-x had a relatively distant but highly conserved putative transcription regulatory sequence (trs). in summary, the genome organization for tcov was determined as follows: 5 utr-gene 1 (orf1a, 1b)fig. 1 . turkey coronavirus (tcov) mg10 genome organization. the full-length tcov genome is 27,632 nucleotides excluding the polyadenylation tail at the 3 end. center: diagrammatic representation of the genome organization shows the predicted genes and their relative sizes and positions along the tcov genome. s, spike glycoprotein gene; 3a, 3b, and 3c (e), tricistronic gene 3; m, membrane protein gene; orf-x, unique orf conserved among group iii coronaviruses; 5a and 5b, bicistronic gene 5; n, nucleocapsid protein gene; utr, untranslated region. scales indicate relative positions of the various genes within the genome in kilobases. top: expanded representation of the two orfs (orf1a and orf1b) comprising the polycistronic gene 1 and the likely cleavage products and cleavage sites after proteolytic processing of the 1a/1b polyprotein. bottom: expanded representation of the s gene indicating the signal peptide (sigp), putative cleavage site (s1/s2), endodomain (clear bars), endodomain (hatched bar) and a short transmembrane region (solid bar). s-gene 3-(orfs 3a, 3b, e)-m-gene 5 (orfs x, 5b, 5c)-n-utr-3 ( fig. 1) . the 5 terminus of the tcov genome was characterized by the presence of a 528 nucleotides long utr with relatively higher gc content of 50.4% compared with the genome as a whole. the tcov 5 utr showed a high degree of sequence similarity with that of most ibv isolates such as ibv-nc (98% identity). following 5 utr, gene 1 of 19,806 nucleotides was located. gene 1 included two slightly overlapping orfs, 1a and 1b. orf1a was 11,853 nucleotides in size enabling it to code for a protein of 3951 amino acids, and orf1b was 7992 nucleotides in length for a coding potential of 2664 amino acids. those two orfs overlapped by 40 nucleotides. orf1b did not contain a typical aug translational initiation codon but instead started with gaa at position 12,342 ( fig. 2a) . the heptanucleotide slippery sequence (uuuaaac) was present which is conserved among all coronaviruses (fig. 2b) , and therefore the ribosomal frame-shifting mechanism seemed to be applicable to tcov and orf1b was believed to be translated by the ribosomal frame-shifting mechanism as a fusion with orf1a to make the polyprotein of 1a/1b. for most coronaviruses, the polyprotein is processed into 16 cleavage products, while the ibv polyprotein is likely cleaved into 15 products (ziebuhr et al., 2000) . as with other coronaviruses, the tcov polyprotein was also believed to undergo proteolytic processing by viral proteinases. the tcov replicase protein was similar to that of ibv in its processing patterns. since the potential cleavage sites were conserved for both viruses (table 2) , the tcov polyprotein was assumed to be processed in the similar manner. two main proteases used by coronaviruses have been identified; plp (papain-like proteinase) which produces 2 or 3 n-terminal products of the polyprotein and 3clp (3-c like protease) which produces the central and the c-terminal region of the polyprotein by cleaving 11 sites (ziebuhr et al., 2000) . similar to sars-cov, ibv encodes only one plp, whereas other coronaviruses code for two plps. in the case of ibv, plp equivalent to plp2 of other coronaviruses cleaves the polyprotein at the qs dipeptide to produce the n-terminal 100 kd proteins, and tcov plp seemed to function in a similar fashion. tcov 3clp was mapped in nsp5. based on the sequence comparisons between tcov and ibv, the replicase cleavage products and their putative functions were predicted (fig. 1, table 2 ). it has been suggested that nsp1 does not exist in ibv (ziebuhr et al., 2007) . due to the high degree of similarity between tcov and ibv in the n-terminal region of the polyprotein 1a/1b, tcov also seemed not to contain nsp1. thus, for tcov, nsp2 through nsp11 would be produced from orf1a, while nsp12 through nsp16 would be produced from orf1b. the n-terminal most cleave product nsp2 was predicted to locate between nucleotide positions 529 and 2549. the sequence analysis for nsp1/2 reveals 44%, 55%, and 90% identity to bcov, hcov-229e, and ibv, respectively. plp1 was suggested to have been lost during the ibv virus evolution (ziebuhr et al., 2001) , and our sequence data also supports that tcov contained only plp2. therefore, plp2 would be responsible for cleavage of the n-terminal part of the polyprotein at two sites between nsp2/nsp3 (ag/gk) and nsp3/nsp4 (ag/gi). plp2 was identified in nsp3. nsp3 was the largest subunit of the replicase cleavage products and was highly conserved among tcov-mg10 and ibv-baudette's strain, as both viruses share 89% identity on the nucleotide level. in contrast, 3clp would cleave 1a/1b polyprotein at 11 sites and generate nsp5 through nsp16 (table 2 ). the motif for 3clp was found in nsp5. the 1a/1b polyprotein contained the motifs found in other nidoviruses (fig. 1, table 2 ). an earlier study identified a 87 kd (p87) protein encoded in this region (lim and liu, 1998) , and the p87 homolog was also found in tcov-mg10 as a 673 amino acids protein in the same region. 3clp was located in nsp5 of 307 amino acids. nsp5 was similar by 52%, 44%, and 92% at the nucleotide sequence level and by 39%, 44%, and 93% at the amino acids level to hcov-229e, bcov, and ibv, respectively. this region was believed to play a critical role for orf1b processing since the deletion of nsp5 in ibv resulted in the unprocessing of 1b protein (liu et al., 1997) . nsp3, nsp4 and nsp6 were predicted to carry a hydrophobic transmembrane domain which may play an important role in the transcription/replication process as recently discussed for other coronaviruses (sawicki et al., 2007) . nsp10 was rich in cysteine and histidine and was predicted to contain a metal binding domain as well as ntp binding helicase domain as with other coronaviruses (gorbalenya et al., 1989) . nsp11 was predicted to be a small peptide of 23 amino acids in length, which is likely the c-terminal most cleavage product of 1a. nsp12 contained the rnadependant rna polymerase (rdrp) activity, which would likely be involved in the genome replication and transcription (liu et al., 1994) . the rdrp motif was highly conserved among all coronaviruses, and tcov-mg10 also showed a high degree of sequence identity for rdrp by 64%, 62%, 94% at the amino acid level to hcov-229e, bcov, ibv, respectively. nsp13 has previously been suggested to play a role for genome replication by unwinding double-strand rna (gorbalenya et al., 1989) . nsp14 was assumed to possess the exonn domain. this domain may be associated with rna metabolism such as proofreading ability and recombination. both coronaviruses and toroviruses contain one exonn motif while roniviruses contain two copies of the exonn motif (snijder et al., 2003) . nsp15 contained the motif for nendou activity. in sars-cov and hcov-229e, nendou cleaved the double-strand rna at the uridylate-containing sequence, and this activity was essential for rna synthesis and progeny virus production (ivanov et al., 2004; posthuma et al., 2006) . nsp16 contained a motif for 2 omethytransferase (mt) in other coronaviruses , and tcov also contained nsp16 as the most c-terminal cleavage product of 1ab. tcov orf1a showed a 46% sequence identity to both bcov and hcov-oc43, which are group ii coronaviruses, and a 90% identity to ibv-nc, ibv-m41, and ibv-p65 which are group iii coronaviruses (table 3) . tcov orf1b was more conserved than orf1a, as tcov orf1b showed a 59% identity to groups i and ii coronaviruses, and 93% identity to ibv which is a group iii coronavirus. as with other coronaviruses, tcov was characterized by the presence of 4 major tructural genes, located in the 3 one-third of the genome: spike (s) glycoprotein, small envelope (e) protein, membrane (m) protein, and nuclocapsid (n) protein genes (fig. 1) . the s protein gene was located immediately downstream from orf1b for a predicted protein of 1,226 amino acids. the s gene was fecov 46 43 59 40 37 43 38 hcov-229e 60 45 59 40 40 43 40 hcov-nl63 50 45 60 41 42 44 39 bcov-nc 46 46 59 45 41 50 44 hcov-oc43 48 46 59 44 42 51 44 mhv-jhm 46 43 58 43 43 51 42 sars-cov 45 41 59 44 31 45 41 ibv-beaudette 94 90 93 57 90 94 92 ibv-p65 87 90 93 57 88 92 92 ibv-m41 87 90 93 57 88 92 93 tcov-mg10 100 100 100 100 100 100 100 tcov-g1 n/a b n/a n/a 96 n/a n/a n/a tcov-gh n/a n/a n/a 96 n/a n/a n/a tcov-nc95 n/a n/a n/a n/a n/a n/a 94 tcov-min n/a n/a n/a n/a n/a n/a 94 tcov-ind n/a n/a n/a n/a n/a n/a 93 a fecov, feline coronavirus; hcov, human coronavirus; bcov, bovine coronavirus; mhv, mouse hepatitis virus; sars-cov, severe acute respiratory syndrome coronavirus; ibv, infectious bronchitits coronavirus; tcov, turkey coronavirus. b n/a: data not available. the most variable gene in the tcov genome as compared to that of other coronaviruses. the s gene showed only 40% and 44% identities to that of group i and group ii coronaviruses, respectively, while the similarity to different strains of ibv was 57% (table 3 ). the tcov s gene showed the highest sequence identity to that from other tcov isolates, tcov-gh and tcov-gi, by 96% for both. the sequence variability was mainly due to the hypervariable and the receptor binding regions in the s protein. the high degree of sequence identity for s among tcov isolates suggests that tcov is less heterogenic as with ibv than other coronaviruses (cavanagh, 2005) . tcov s was slightly larger than ibv s as tcov s was 3678 nucleotides, while ibv s was 3453 nucleotides in size. the coronavirus s protein is responsible for receptor binding and virus-host cell membrane fusion. for groups ii and iii coronaviruses, the s protein is cleaved into two subunits; the n-terminal s1 product and the c-terminal s2 product. tcov s was also presumed to be cleaved into s1 (542 amino acids) and s2 (684 amino acids) with the putative cleavage site characterized by presence of basic amino acids (arg-arg-ser) between 542 and 543 ( fig. 1 ) as for ibv s (cavanagh, 2007) . the receptor-binding domain in s1 is not well-identified for ibv and thus was not possible to predict for tcov. tcov s was likely highly glycosylated as it contained 24 potential n-linked glycosylation sites. tcov s contained three hydrophobic transmembrane domains; two ectodomains and one endo-domain. tcov gene 3 was thought to be tricistronic (orf 3a, 3b, and 3c). the small membrane (e) protein of 99 amino acids was potentially encoded by orf3c. both orf 3b and e genes overlapped by fig. 3 . illustration of conservation of the orf-x amino acid sequence and the putative trs found upstream of the orf-x gene among a number of group iii coronaviruses. tcov and ibv share a highly conserved 94 amino acids hypothetical protein, designated as orf-x. a highly conserved trs motif (gtcaacaa) for orf-x is found 288 nt upstream, within the m gene, in all group iii coronaviruses. the beaudette strain of ibv and strains derived from it such as ibv-p65 have a 49 nt deletion in orf-x but the remaining sequence aligns unequivocally with all other group iii coronaviruses when this deletion is taken into account. 1 n/a, no sequence available for this region; 2 single nucleotide (a) from position 446 (af072911.1) was deleted to maintain reading frame; 3 17 amino acid deletion (51 nucleotides for 17 codons) in these sequences. 16 nucleotides. the tcov e gene showed a high degree of sequence identity to ibv e by 90%. the e protein was reported to be a viroporin which played a role in the membrane permeability in sars-cov and porcine reproductive and respiratory syndrome virus, another member of nidoviruses (wilson et al., 2004; lee and yoo, 2006) . the membrane (m) protein gene was 699 nucleotides in size and was able to make a protein of 223 amino acids. the m gene seemed to be highly conserved within group iii coronaviruses since tcov m showed a 94% nucleotide identity to ibv m. the m protein contained a single putative n-linked glycosylation site at amino acid position 4 as well as 3 potential sites for o-linked glycosylation. it remains to be determined whether these sites are functional for tcov. the nucleocapsid (n) protein gene was 1,227 nucleotides with a coding capacity of 409 amino acids. the tcov n gene showed a 93% sequence identity to that of various ibv strains. the n protein was shown to be a serine phosphoprotein in other coronaviruses and arteriviruses (alexander et al., 2005; wootton et al., 2002) . the tcov n protein contained 20 serine residues but no tyrosine residue. gene 3 (orf3) is possibly tricistronic as with other coronaviruses. orf3 was able to code for two non-structural proteins and the small envelope (e) protein. orf3a and 3b were 171 and 192 nucleotides in length capable of coding for 57 and 64 amino acid proteins, respectively. orf5 is potentially bicistronic to code for 5a and 5b proteins. orf5a was 195 nucleotides in length for 65 amino acids while orf5b had a potential for 82 amino acids. orfs 5a and 5b overlapped by three nucleotides, while orf5b and the downstream n gene overlapped by 57 nucleotides. the presence of gene 3 and gene 5 were highly suggestive that tcov was related to group iii coronaviruses. a recent study for the role of gene 3 and gene 5 for ibv replication showed that deletion mutant viruses succeeded in replication in a similar manner to the wild-type virus, suggesting that those genes were non-essential for ibv replication (casais et al., 2005) . in contrast, orf5a and orf5b were not found in mammalian coronaviruses, and thus the presence of gene 5 may be considered a characteristic feature of avian coronaviruses including ibv and tcov. tcov was characterized by the presence of an additional orf, designated orf-x. orf-x was 282 nucleotides in length with 33.3% gc contents. this orf was located upstream of gene 5 and started immediately following the m gene. this gene was able to encode a hypothetical protein of 94 amino acids. a blast search for this orf using the amino acid sequence found no homology to described proteins; however, at the nucleotide level, this orf was strongly conserved among group iii coronaviruses including all ibv strains and all available sequences for turkey coronaviruses in the genbank database. for several ibv strains, this region contained a wellidentified orfs including initiation (aug) and stop codons (fig. 3) . interestingly, a sequence, that might be the putative trs (gucaa-caa) for this particular orf was found 288 nucleotides upstream of the initiation codon for orf-x within the m gene (table 4) ; this putative trs was conserved at the same relative location in virtually all ibv sequences in the genbank database spanning the putative trs region (fig. 3) . further studies are warranted determine the significance of this apparently expressed protein found only in group iii coronaviruses as far as is known (fig. 3) . a 3 utr of 352 nucleotides was present immediately downstream the n gene of the genome. it has been previously shown that the 3 utr of both tcov indiana and minnesota strains were 502 nucleotides long, while tcov-nc95 strain has a 3 utr of 349 nucleotides which lack the first 153 nucleotides at the 5 end (breslin et al., 1999) . also, tcov-mg10, like ibv and some other coronaviruses, contained a conserved stem-loop structure in the 3 utr as illustrated in fig. 5 . the tcov genome contained putative trss located upstream of the start codon of each gene (table 4 ). although the distance between the putative trs and the downstream initiation codon varied among the various genes in the tcov genome, the particular distance for each gene was similar in both ibv and tcov. in all cases, the putative trs contained a conserved aacaa motif. the leader trs (cuuaacaa) was found in the 5 utr at genomic positions 57-64 which is 465 nucleotides upstream of the orf1a initiation codon. the trs for s gene, cugaacaa, differed from the leader trs by replacement of u at the 3rd position with g, and was located 53 nucleotides upstream from the s gene start. the m gene trs was identical to the leader trs and was located 77 nucleotides upstream of the m gene start. in the same manner, the n gene trs fig. 4 . phylogenetic trees for tcov and other coronaviruses. aligned nucleotide sequences of complete coronavirus genomes representing coronavirus groups i, ii, and iii were utilized to construct maximum likelihood (ml) and maximum parsimony (mp) trees using the paup software package followed by bootstrap analysis using a heuristic search method. for each tree, the bootstrap support for each branch is indicated for each branch and the horizontal lengths of branches are proportional to the amount of hypothesized evolutionary change. all trees are rooted using the group i coronaviruses as a functional outgroup. the ln likelihood for the ml tree and the consistency index (ci) and retention index (ri) are provided for the mp trees. for ingroup analyses of relationships among the group iii coronaviruses, aligned amino acid sequences for the spike glycoprotein (s), envelope (e) and nucleocapsid (n) proteins were analyzed using maximum parsimony (branch and bound search algorithm). hcov, human coronavirus; fipv, feline infectious peritonitis virus; bcov, bovine coronavirus; mhv, murine hepatitis virus; ibv, infectious bronchitis virus; tcov, turkey coronavirus. alignment of highly conserved stem-loop (s2m) sequences present in the 3 utr of a variety of coronaviruses. this highly conserved region, flanked by highly divergent sequence (not shown), is found in tcov, ibv and other avian coronaviruses. the s2m sequence is also found in the atypical group iii coronavirus sars-cov and the recently sequenced coronavirus isolated from an asian leopard cat. was identical to the leader trs and located 93 nuclotides from the n gene start. phylogenetic reconstruction of the whole genomes of 12 coronaviruses using maximum likelihood and maximum parsimony produced well-supported trees (fig. 4) that placed tcov-mg10 as a sister taxon to the four ibv isolates included in the analysis. bootstrap support for both analyses was 100% at each node indicating significant support for the branching order. group i coronaviruses (fipv, hcov-nl63, hcov-229e) formed a monophyletic clade as did the group ii coronaviruses (sars-cov, mhv, bcov, hcov-oc43). sars-cov was basal to the other group ii coronaviruses in the whole genome analyses. the solid support for the monophyly of tcov-mg10 with four ibv isolates supports the conclusion that this tcov should be classified as a member of the group iii coronaviruses. analyses of aligned amino acids sequences for the e, n, and s proteins of various coronaviruses produced phylogenetic trees that largely reflected the nucleic acid-based whole genome trees (fig. 4) . the n protein produced a well-supported tree that had a largely unresolved polychotomy consisting of five tcov isolates (three usa isolates, one uk isolate, and isolate mg10 from canada) and the four ibv isolates used in the whole genome analyses. tcov-mg10 was most closely related to tcov-nc95 and these two isolates formed a well-supported monophyletic group based on the n protein; however, this protein did not contain sufficient information to infer relationships reliably among group iii coronavirus. similar results were obtained with a large number of ibv sequences and five tcov sequences with the m protein (data not shown). the e protein analysis placed tcov-mg10 as the sister taxon to the three ibv isolates used in the analysis; however, there was only a single tcov available for that analysis so no conclusions was drawn regarding the utility of this protein for inferring evolutionary relationships among these group iii coronaviruses. unlike the analyses based on aligned s or n protein amino acid sequences, group ii coronaviruses did not form a monophyletic group in the analysis based on aligned e protein sequences; sars-cov did not group with the other three group ii coronaviruses included in this analysis. using available sequences for the s protein, three tcov isolates (gh, g1 and mg10) formed a monophyletic group closely related to a quail coronavirus (qcov) isolate from italy. together, these isolates formed the sister group to a number of ibv isolates. the phylogenetic reconstruction based on the s protein, unlike other analyses based on the amino acid sequences of the n, e or m proteins (data not shown), produced a well-supported clade containing only tcov strains and suggests that the s protein may be a more useful molecule for inferring relationships among the group iii coronaviruses. the family coronaviridae is included in the order nidovirales along with the arteriviridae and roniviridae families. coronaviruses are divided into three groups (i, ii, and iii) based on the genome structure and organization (holmes and lai, 1996; lai and cavanagh, 1997) . group i coronaviruses include porcine epidemic diarrhea virus (pedv), tgev, canine coronavirus (ccov), feline infectious peritonitis virus (fipv), hcov-229e, and a newly identified hcov-nl63, whereas group ii includes murine hepatitis virus (mhv), bcov, hcov-oc43, rat sialodacryoadenyleitis virus (sadv), canine respiratory coronavirus (crcov), and equine coronavirus (ecov). group iii coronaviruses are ibv as well as the newly discovered pheasant coronavirus. group iii coronaviruses are characterized by modification of gene 3 to a tricistronic structure that codes for genes 3a and 3b as well as the e gene, and the insertion of an additional unique sequence designated gene 5. the e gene is common in all coronaviruses but is incorporated into the tricistronic gene 3 located between s and m genes only in the group iii coronaviruses as far as is known. in contrast, gene 5 located between the m and n genes had only been reported previously from ibv. in tcov-mg10, gene 5 has two small orfs (orf5a and orf5b) that code for products of 65 and 82 amino acids, respectively; this is identical in length to the gene 5 products of ibv. the presence of gene 5 has been suggested as a genetic maker for group iii coronaviruses . in vitro work with ibv has demonstrated that gene 5 is not essential for virus replication in cell culture (casais et al., 2005) . whether or not gene 5 is unnecessary for in vitro replication of tcov is unknown. since first identified in 1951 (peterson and hymass, 1951) and despite the economic importance to the turkey industry, tcov has remained one of the least characterized among all known coronaviruses. early studies suggested that this virus could be a group ii coronavirus along with bcov and hcov-oc43 based on serology and partial sequences for n and m genes. (verbeek and tijssen, 1991) reported that the tcov n gene was 100% identical to bcov n gene. this suggestion was supported by a study using the minnesota strain of tcov that demonstrated hemagglutination activity of tcov for rabbit erythrocytes (dea et al., 1990) . a he gene is found in many group ii coronaviruses. despite these early confounding observations, , using antibodies to discriminate among coronaviruses, suggested that tcov was more closely related to ibv than bcov (dea and tijssen, 1989; dea et al., 1990) , which is in agreement with our genomic data. our completion of the full genome sequence of a field isolate of a tcov shows clearly that the tcov genome structure and sequence are much closer to ibv than any other coronavirus. like ibv, the genome structure of tcov-mg10 is: 5 utr -replicase (orf1a and orf1b) -spike (s) protein -orf3 (orfs 3a and 3b) -small envelop (e or 3c) protein -membrane (m) protein -orf-x -orf5 (orfs 5a, and 5b) -nucleocapsid (n) protein -3 utr-poly(a), in order (fig. 1) . the non-structural protein gene immediately downstream of orf1b and the further downstream he gene that are both commonly found in group ii coronaviruses were entirely absent in tcov. our data did not support the presence of any gene similar to he and we concluded that tcov does not contain the he gene. as is obvious from table 3 , tcov-mg10 and other tcov isolates demonstrate much higher sequence similarity to strains of ibv that they do for any other coronavirus. for example, the n gene of tcov was only 44% identical to bcov and hcov-oc43 but had 92% identity to the n gene of ibv. this apparent discrepancy with previous sequencing results (e.g. verbeek and tijssen, 1991) may be explained by several reasons. one possibility is genome recombination between tcov and bcov. it is also possible that a cell line commonly used for coronavirus cultivation, hrt-18, may harbor a latent infection with one of the human coronaviruses which was then activated upon infection with another coronavirus. laboratory contamination of bcov is also a possibility. our data shows a high degree of sequence identity between ibv and tcov in the replicase, e, m, and n genes with greater than 90% sequence identity for each (table 3 ). the s gene is most variable among ibv strains and between ibv and tcov, perhaps reflecting the role that the s protein has in determining receptor binding in coronaviruses (cavanagh, 2005) . despite the relatively large genetic variation in the s gene of various ibv strains, tcov also showed a relatively higher sequence identity to ibv (up to 57%) while the sequence identity to the s gene of bcov was only 45%. in contrast to ibv, our limited study indicates that tcov s is relatively conserved among isolates, suggesting that the tcov s genes might be less varied than the ibv s genes. ibv and tcov have distinct clinical presentations in infected hosts. ibv causes respiratory disease in chickens whereas tcov causes enteric disease in young turkeys. perhaps the tropisms exhibited by ibv and tcov reflect the sequence variation of the s glycoprotein and the resulting differences in receptor affinities. the tcov s gene is larger than that of most ibv isolates. a study is required to understand the functional difference in s for ibv and tcov, which may have a great impact on the antigenic properties and tissue tropism of both ibv and tcov as well as the development of control measures against them. the presence of gene 3 and gene 5 is a unique characteristic for group iii coronaviruses as those genes do not exist in mammalian coronaviruses. as those genes are highly conserved in avian coronaviruses, they might serve as a cis acting elements essential for virus replication. based on the overall structure of the genome and sequence similarities to ibv, we conclude that tcov should be classified as a group iii coronavirus. our sequence data revealed the presence of a novel orf in tcov located upstream of gene 5 and downstream of the m gene. this orf is unique to tcov and ibv, the only group iii coronaviruses for which sequence data in this region of the genome are available. orf-x was strongly conserved among group iii coronaviruses including most ibv strains and all available sequences for turkey coronaviruses in genbank as well as the tcov-mg10 isolate. eight (e.g. beaudette and p65 strains) of 40 or more ibv sequences for orf-x have a 49 base deletion in comparison with other ibv strains; all of the strains with this deletion were laboratory-adapted, cell cultured viruses originating with the beaudette strain of ibv, suggesting that this deletion may be an artifact of cultivation outside of the natural host. the maintenance of this long sequence within a coronavirus genome seems highly unlikely if this was not a functional gene. finding a highly conserved putative trs, 288 bp upstream of the initiation codon for orf-x within the m gene, reinforces the notion that this gene is functional in both ibv and tcov despite the unusually long distance between the putative trs and the initiation codon. this region may be a good marker for group iii coronaviruses. further studies are being carried out to further characterize this particular orf and determine its role, if any, in virus replication. among coronaviruses, only sars-cov contains an orf immediately upstream of the n gene, which is referred to as gene 8. gene 8 plays an important role in the sars-cov replication and induction of apoptosis of its host cells (chen et al., 2007) . the function of the small orf found in tcov remains unknown and the lack of any homology with any known protein makes inferring its function difficult. the 3 utr is believed to be involved in genome replication of coronaviruses (williams et al., 1993) , despite its apparent ability to possess quite variable sequence and sequence lengths. this variation within available 3 utr sequences of tcovs is the same as for ibv strains. the 3 utr of ibv strains (beaudette, kb8523 and cu-t2) are 503-505 nucleotides in length in contrast to 320 nucleotides for ibv strain m41 (boursnell et al., 1985) . the 3 utr of tcov-mg10 was highly conserved with a 94-98% nucleotide identity to most published ibv and tcov 3 utr sequences. in contrast, the identity to bcov was only 45%. some viruses such as ibv, human astrovirus, and turkey astrovirus were reported to contain a stem loop-like motif (s2m) in the 3 utr (jonassen et al., 1998) . this motif appeared to be conserved among those different viruses, suggesting that it might have resulted by rna recombination between different viruses (monceyron et al., 1997) . tcov was found to contain the same motif in the 3 utr (fig. 5) . the presence of the s2m motif in the 3 utr may also suggest that ibv and tcov share a common ancestor. interestingly, sars-cov, but not other group ii coronaviruses, shares the presence of this conserved s2m motif (see fig. 5 ). sequence analyses of the 3 utr from 19 different ibv strains revealed the presence of two distinct regions: region i was highly variable and located immediately downstream of the n gene while region ii was highly conserved and located upstream of the poly(a) tail (dalton et al., 2001) . gobel et al. (2007) reported the presence of the octamer motif (5 -ggaagagc-3 ) within the 3 utr hypervariable region that was highly conserved among all coronaviruses. tcov-mg10 had two copies of the octamer motif in its 3 utr; the first copy between nucleotides 25,690 and 25,697, and the second copy between nucleotides 27,553 and 27,560. the role of this motif for coronavirus replication is unknown, but (gobel et al., 2007) suggested that it might play a role in the coronavirus replication cycle. in summary, this is the first completion of the full-length tcov genomic sequence. this study should lead to a better understanding of the molecular biology of tcov and perhaps contribute to our understanding of poult enteritis mortality syndrome (pems) affecting young turkey flocks thought to result from co-infection of tcov with turkey astrovirus. by completing the first genome of a tcov, we have established the genome organization and coding strategy for the virus that unequivocally establishes that tcov is a group iii coronavirus, closely related to ibv and other avian coronaviruses. in addition, we have identified a putatively functional gene (orf-x) shared among all sequenced ibv and tcov strains that may be a shared feature of all group iii coronaviruses. severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice poult enteritis-mortality syndrome (spiking mortality) of turkeys sequence of the nuclocapsid genes from two strains of avian invectious bronchits virus sequence analysis of turkey coronavirus nucleocapsid protein and 3 untranslated region identifies the virus as a close relative of infectious bronchitis virus gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication coronaviruses in poultry and other birds detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens. avian path coronavirus 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open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf1a products proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kilodalton polypeptide and determination of its cleavage sites molecular characterization of the 3 end of the astrovirus genome coronaviral enteritis of turkeys (bluecomb disease) antibiotic in treatment of unfamiliar turkey disease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle a contemporary view of coronavirus transcription unique and conserved features of genome and proteome of sars coronavirus, an early split-off from the coronavirus group 2 lineage paup: phylogenetic analysis using parsimony, version 3.1 computer program distributed by the illinois natural history survey detection of turkey coronavirus in commercial turkey poults in brazil the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools sequence analysis of the turkey enteric coronavirus nuclocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus analysis of a hypervariable region in the 3 non-coding end of the infectious bronchitis virus genome sars coronavirus e protein forms cation-selective ion channels phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein human coronavirus 229e papin like protease have overlapping specificities but distinct functions in viral replication virus-encosed proteinasees and proteolytic processing in the nidovirales the autolytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogus papin-like proteinase that cleave the same peptide bond this study was supported by nserc canada. mg is a scholarship recipient of the arab republic of egypt. we would like to thank janet swinton for technical assistance. key: cord-306380-msk9p1yy authors: lee, c.-w.; jackwood, m. w. title: evidence of genetic diversity generated by recombination among avian coronavirus ibv date: 2000 journal: arch virol doi: 10.1007/s007050070044 sha: doc_id: 306380 cord_uid: msk9p1yy previously, we demonstrated that the de072 strain of ibv is a recombinant which has an ibv strain d1466-like sequence in the s gene. herein, we analyzed the remaining 3.8 kb 3′ end of the genome, which includes gene 3, gene 4, gene 5, gene 6, and the 3′ non-coding region of the de072 and d1466 strains. those two viruses had high nucleotide similarity in gene 4. however, the other individual genes had a much different level of sequence similarity with the same gene of the other ibv strains. the genome of five ibv strains, of which the complete sequence of the 3′ end of the genome has been determined, were divided at an intergenic (ig) consensus sequence (ctgaacaa or cttaacaa) and compared phylogenetically. phylogenetic trees of different topology indicated that the consensus ig sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. phylogenetic analysis of selected regions of the genome of the de072 serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the s gene. presumably this occurs because the consensus ig sequence serves as the template switching site for the viral encoded polymerase. infectious bronchitis virus causes a highly contagious upper-respiratory disease in chickens. the disease is characterized by increased ocular and nasal secretions, excess mucus in the trachea, decreased weight gain and feed efficiency in broilers, and declines in egg production and egg quality in layers. although live attenuated vaccines are available, ibv continues to be a severe economic problem in commercial chickens because many different serotypes of the virus exist and do not cross protect [3] . [4] . members of the nidovirales order have a single stranded positive sense rna genome and produce a 3 nested set of subgenomic mrnas when they replicate [4] . coronaviruses are divided into three antigenic groups based primarily on their structural proteins. infectious bronchitis virus is the type strain of coronaviruses and is the only virus placed in antigenic group three. characteristics of this group are a cleaved spike (s) glycoprotein, an n-glycosylated membrane (m) protein, and no hemagglutinin/esterase protein [19] . the genome of ibv is approximately 27 kilobases in length [1] . it is organized into six regions, each containing one or more open reading frames (orf's), which are separated by intergenic sequences (ig) that contain the signal for transcription of subgenomic mrnas [1, 17] . the viral rna-dependent rna polymerase is encoded in the 5 two thirds of the viral genome by two overlapping open reading frames (orf1a and orf1b) [1] . the structural protein genes are located 3 to the viral polymerase gene and are in order from 5 to 3 , the s glycoprotein gene (gene 2), the small envelope (e) gene (gene3), the m glycoprotein gene (gene4), and the nucleocapsid (n) gene (gene6) [19, 20] . evolution in ibv has been observed through the occurrence of variant viruses and analysis of known serotypes. more than twenty serotypes within ibv have been recognized worldwide and are thought to be generated by insertions, deletions, point mutations and rna recombination [2, 3, 6, 14] . evidence of natural recombination for several ibv strains has been reported [10, 15, 22] . however, because of the limited sequence information, recombination has only been described for a small part of the genome. so far, the complete sequence of the 3 end of the genome (from the 3 end of the polymerase gene to the poly a tail) of only three strains, beaudette, kb8523 and cu-t2 have been determined [1, 11, 20] . the de072 strain was first isolated in 1992 in the delmarva peninsula region of the usa and initial characterization of this virus indicated this virus was serologically distinct from any other ibv serotypes in north america [7] . previously, we demonstrated that the de072 strain is a recombinant which has a d1466-like sequence in the s1 and s2 genes [18] . d1466 is an ibv vaccine strain of the d212 serotype from the netherlands [7, 13, 14] . herein, we describe the sequences of the remaining genes of the de072 and d1466 strains with the exception of gene 1(the polymerase gene). we conducted phylogenetic analysis by dividing the genome in the ig sequence to elucidate possible role of this sequence in the homologous recombination in ibv. further, we conducted sequence analysis of six isolates of the de072 serotype in order to determine if recombination is frequently occurring in this region in field isolates of ibv. viruses used in this study are listed in table 1 . the viruses were propagated in 9-day-old embryonated specific-pathogen-free (spf) chicken eggs (select laboratories, gainesville, ga, usa). the d1466 strain of ibv was obtained as phenol-inactivated allantoic fluid using usda import permit #42290. viral rna from ibv grown in embryonating eggs was extracted using the high pure pcr template preparation kit (boehringer mannheim, indianapolis, in, usa) according to the manufacturers recommendation. rna from the phenol-inactivated allantoic fluid of d1466 was extracted with a modification in first several step of the high pure pcr template preparation kit. briefly, 1.5 ml of the infectious allantoic fluid was placed into a microcentrifuge tube and centrifuged at 13,000 ×g for 5 min. the aqueous top layer, approximately 200 l, was transferred to new tube. binding buffer (200 l) and 40 l of proteinase k (18 mg/ml) was added and incubated for 10 min at 70 • c. then 150 l of chloroform/isoamyl alcohol (49:1) was added, vortexed gently for 5-10 sec and then placed on ice for 15 min. the mixture was centrifuged at 13,000 ×g for 10 min. the upper phase was transferred to a clean 1.5 ml tube and 100 l of chloroform/isoamyl alcohol (49:1) was added. the mixture was vortexed gently for 5-10 sec. this was centrifuged for 2 min at 13,000 ×g, and the upper phase was transferred to a clean 1.5 ml tube. remaining steps were followed sequentially as described by the manufacturer. gene 3, gene 4, gene 5, gene 6, and a 421 bp hypervariable region (hvr) of the s1 gene were amplified separately using the titan one tube rt-pcr system (boehringer mannheim). primer sets used to amplify gene 3, gene 4, and the hvr in s1 are listed in table 2 . the primers utilized for amplification of gene 5 and gene 6 have been reported [8, 23] . the reaction conditions for rt-pcr were previously described [16, 23] . pcr products were cut from 1% agarose gels and purified using the qia quick gel extraction kit (qiagen, santa clarita, ca, usa). purified pcr products were either sequenced directly or cloned into the ta cloning vector (invitrogen, carlsbed, ca, usa), and automated sequencing with the prism dyedeoxy terminator cycle sequencing kit (perkin elmer, foster city, ca, usa) was conducted at the molecular genetics instrumentation facility, university of georgia. sequencing primers to various regions of the gene for de072 and the relative primer positions were calculated using the atg start site of gene 3 as 1 for primers gene 3 and 4, and atg start site of s1 gene as 1 for primers hvr in s1 d1466 were designed using oligo version 4.0 software (national bioscience, plymouth, mn, usa) and are available upon request. assembly of sequencing contigs, translation of nucleotide sequence into protein sequence, and initial multiple sequence alignments were performed with the clustal v method in megalign software versin 1.03 (dnastar inc., madison, wi, usa). phylogenetic trees for each gene were generated using the maximum parsimony method with 100 bootstrap replicates in a heuristic search using the paup 3.1 software program [21] . the nucleotide sequences reported here have been deposited with the genbank. the accession numbers are as follows: de072 (gene 3), af202998; de072 (gene 4), af202999; de072 (gene 5), af203000; de072 (gene 6), af203001; de072 (3 end non-coding region), af203002; d1466 (gene 3), af203003; d1466 (gene 4), af203004; d1466 (gene 5), af203005; d1466 (gene 6), af203006; d1466 (3 end non-coding region), af203007; 98-2831 (hvr in s1), af206254; 99-5831 (hvr in s1), af206255; 99-5425 (hvr in s1), af206256; 99-5658 (hvr in s1), af206257; 97-6370 (hvr in s1), af206258; 97-6386 (hvr in s1), af206259 the complete sequence of the 3 end of the genome of three strains, beaudette, kb8523 and cu-t2 and gene 6 of holl52 strain have been previously reported [1, 11, 20, 23] . a total of 3839 nucleotide and 3861 nucleotide were found, respectively, in a region beginning from the 5 end of gene 3 to the 3 end of de072 and d1466 genome. the intergenic sequence ctgaacaa or cttaacaa was found immediately upstream of the start site for each gene of both strains. the sequences were identical to those found in the corresponding genomic areas of the beaudette, kb8523, and cu-t2 strains (fig. 1). fig. 1 . the nucleotide sequence alignment of gene 3, gene 4, gene 5, and gene 6, and 3 noncoding region. dots indicates nucleotide identical to that of de072 strain. the conserved nucleotide sequences ctgaacaa or cttaacaa, which is located at the starting site of each gene, is in bold character. heavy underlines indicate the putative start codons, asterisks above the sequence indicate the stop codons gene 3 of both strains contained three orfs, 3a, 3b, and 3c. gene 4 consisted of the m protein gene with a single orf and a non-coding region between the 3 end of the m protein gene and gene 5. gene 5 contained two orfs (5a and 5b). gene 6 consisted of the n protein gene with a single orf and a 3 non-coding region. downstream from the stop codon of the n gene, a 15 base insertion was found in the d1466 genome which also occurs in the genome of the holl52 (fig. 1) . the 3 -terminal 3.8 kb of the genome of five strains and gene 6 of the holl52 strain were compared. the nucleotide sequence similarities among coding regions of gene 3, m, gene 5, and the n protein gene of de072 and other strain were between 83.3-97.6%. those of d1466 and other strains were between 78.7-98.2% identical. d1466 showed only 1.8% nucleotide difference with holl52 in gene 6. gene 3c and gene 5b were relatively more conserved than the other genes (table 3) . genes were divided by ig sequences (ctgaacaa/cttaacaa) and phylogenetic analysis was conducted. the de072 strain clustered with the cu-t2 (fig. 2) . kb8523, which is only the nephropathogenic strain, was solely placed in all genes compared. in order to demonstrate the genetic heterogeneity of the same serotype isolates of ibv, we conducted phylogenetic analysis using six de072 serotype field isolates. phylogenetic analysis of the hypervariable region (hvr) in s1, clustered all the de072 serotype isolates in one group with the prototype strain of the de072 serotype of ibv. this group was far from other serotypes of ibv strains in tree length. however, phylogenetic tree of gene 3 and gene 4 showed differences in tree topology among six isolates. in gene 3, only one isolate, 98-2831, clustered with de072. in gene 4, no isolates clustered with de072 and formed groups randomly with other serotypes of ibv (fig. 3) . de072 is a recent isolate made in 1992 [6] . in a previous study of the s gene, we demonstrated that this virus was closely related to d1466 which is an ibv vaccine strain of the d212 serotype from the netherlands [7, 13, 18] . analysis of gene 4 also reveals a high sequence relatedness between de072 and d1466 (table 3) . however, in the other genes analyzed in this study, de072 shares high sequence similarity with the cu-t2 strain which has also been reported to be a recombinant between arkansas and massachusetts strains [10] . considering the fact that both strains were isolated in the northeastern usa, it is possible that they had undergone similar selection pressure. on the other hand, d1466 shows high similarity with beaudette and holl52 strains in genes other than the s gene. the percent similarity in the n gene and a 15 base insertion in the 3 non-coding region suggests that both d1466 and holl52 are closely related (table 3 , fig. 1 ). the holl52 strain has been extensively used as a live vaccine in europe [5] . this finding provides more convincing evidene that vaccine strains are contributing to the emergence of variants in the field. based on these results, we suggest that de072 and d1466 had the same origin, but diverged a long time ago and evolved independently in different geographical locations. since recombination in coronaviruses is thought to occur by a template switching mechanism [8, 19] , we speculate that ig sequences may serve as 'hot spots' for homologous recombination. so far, recombinations suggested in ibv have been used on a small part of the genome [10, 15, 22] . examining only a small part of the genome may result in misleading conclusions because of point mutations or conserved regions of the gene. we conducted phylogenetic analysis by dividing 3.8 kb of the 3 end of the genome among five ibv strains at the ig sequences. phylogenetic trees of this sequence data had very different topology (fig. 2) , which indicates that recombination had occurred. it has been reported that rna recombination in ibv can occur randomly in non-localized sites in vitro [12] . however, considering the selection pressure in vivo recombination in the ig sequences should be advantageous to virus in two aspects. first, since crossovers occur at the site of consensus ig sequences, there would be no shift in the codon reading frame. second, since whole genes are substituted, there would be no drastic change in the conformation of proteins encoded by individual genes. further, cross-overs at each of the five ig sequences would generate tremendous genetic diversity. this amount of diversity may contribute to persistence and to the continuing emergence of new variants of ibv despite vaccination efforts. finally, we conducted sequence analysis of 6 isolates of the de072 serotype to demonstrate how random recombination occurs within the same serotype. phylogenetic analysis of the hvr in s1 shows that these 6 isolates cluster together because they are the same serotype. however, these 6 isolates had a much different level of nucleotide sequence similarity with each other in gene 3 and gene 4, and clustered randomly with other serotypes of ibv (fig. 3) . based on this result, it is clear that isolates of the same serotype can differ substantially in individual genes. thus, every field isolate of ibv could be unique in each gene sequence because of recombination. completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus location 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infectious bronchitis virus molecular epidemiology of infectious bronchitis virus in the netherlands phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction-fragment-lengthpolymorphism analysis coronavirus: how a large rna viral genome is replicated and transcribed spike gene analysis of the de072 strain of infectious bronchitis virus: origin and evolution the coronaviridae cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus paup: phylogenetic analysis using parsimony. version 3. illinois natural history survey evidence of natural recombination within the s1 gene of infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis viruses and other coronaviruses we express appreciation to dr. yoram weisman for providing d1466 virus and deborah hilt for technical assistance. thanks are also extended to drs. bruce seal, maricarmen garcia, and holly sellers for the review of this manuscript. received december 1, 1999 key: cord-310372-qc6941pm authors: ji, jun; xie, jingwei; chen, feng; shu, dingming; zuo, kejing; xue, chunyi; qin, jianping; li, hongmei; bi, yingzuo; ma, jingyun; xie, qingmei title: phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during 2008-2009 date: 2011-04-22 journal: virol j doi: 10.1186/1743-422x-8-184 sha: doc_id: 310372 cord_uid: qc6941pm background: the nephropathogenic avian infectious bronchitis (ib) caused unprecedented economic losses to the commercial chicken industry of china in 2008-2009. to investigate the prevalence of nephropathogenic ib in china, eighty ibv isolates from different provinces during 2008-2009 were identified by dwarf embryo test and rt-pcr. results: the strains were mostly isolated in winter and spring with a wide age range of ib outbreaks, from 4 to 69 days. by the virus recovery trials, 70/80 of the strains resulted in the deaths or distresses of birds from nephritis. to learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike 1 (s1) protein gene of these strains was rt-pcr amplified and sequenced. compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the s1 genes of these strains and the reference strains displayed homologies ranging from 75.1% to 99.8% and from 73.1% to 99.8% respectively. s1 protein of the major pandemic strains contained 540 or 542 amino acids with the cleavage site of hrrrr or rrfrr. phylogenetic analysis revealed that recent field isolates of ibv in china were mostly belonged to a2-branch (qxibv-branch) and hn08-branch, only one isolate was belonged to gray-branch and m41-branch respectively. most of the 80 strains showed evolutionarily distant from vaccine strains. conclusions: the results of this study suggested that nephropathogenic ibvs were mainly a2-like strains in china during 2008-2009. infectious bronchitis (ib) is a serious and highly contagious disease of chickens, accompanied by decreased egg production and poor egg quality in laying flocks. avian infectious bronchitis virus (ibv) was first reported in the usa, replicating in the respiratory tract and some epithelial cells of gut, kidney, and oviduct [1] [2] [3] . ibv commonly predisposed the birds to secondary infection with some bacterium, such as escherichia coli and mycoplasma gallisepticum, resulting in complicated disease process and increased mortality [4, 5] . the clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry all over the world. ibv belongs to the genus coronaviridae, family coronaviridae, order nidovirales, and possesses a single stranded positive-sense rna genome encoding four structure proteins, phosphorylated nucleocapsid (n) protein, small envelope protein (e), integral membrane glycoprotein (m), and spike glycoprotein (s) [6, 7] . the s glycoprotein on the outside of the virus contains epitopes associated with serotype differences, and is cleaved post-translationally by cellular proteases into the s1 and s2 subunits [8, 9] . the globular s1 subunit forms the tip of a spike, extending outward, plays a role in attachment and entry into the host cell, which has relation to induce virus neutralizing antibody and hemagglutination inhibition antibody, whereas the s2 subunit anchors the s1 moiety to the viral membrane [8] [9] [10] [11] . coding for the heavily glycosylated spike glycoprotein, the error-prone nature of rna polymerase made the s1 gene could easily generate nucleotide insertions, deletions, point mutations, and rna recombination under vaccine pressure, to bring about new variation strain and change of tissue tropism [12] [13] [14] [15] [16] . it is documented that only a few amino acid differences amongst s proteins are sufficient to have a detrimental impact on cross-protection [15, [17] [18] [19] [20] . antigenically different serotypes and newly emerged variants of field chicken flocks lead to vaccine breaks [21, 22] . recently, more than 20 serotypes within ibv have been identified worldwide. the complex epidemiology characterize of ib raised the control difficulty. in china, since ibv strains were first isolated and identified in 1982, various live-attenuated and inactivated vaccines derived from massachusetts (mass) serotype strains have been widely and extensively used in chicken farms to reduce the adverse effect of the ibv [23, 24] . however, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in china, and the situation gets worse as time progressed [25] . it was documented that nephropathogenic type ib has become more and more prevalent in china. the unprecedented economic losses caused by the nephropathogenic ib suggested that selecting the appropriate vaccine strain against the ib outbreaks is of great importance [25, 26] . however, the integrated natures of novel circulating ibv strains in mainland china were not well-learned. the previous study by other researchers has been revealed that the variation in s1 sequences was closely confirmed relative to the emergence of novel strains, and s1 gene sequence was a good predictor of challenge of immunity in chickens [17, 18, 27] . this study was conducted to identify the ibv strains that have escaped immune defenses conferred by vaccination in china. the genetic characterization of recent ibv field isolates in china was performed by sequencing the whole s1 genes, sequence alignment and phylogenetic analysis compared with other reference strains. from unhealthy birds suspected of ibv infection in the vaccinated chicken flocks from guangdong, guangxi, fujian, hainan, jiangsu, zhejiang, chongqing, hubei, sichuan and jiangxi province of china, 80 filed ibv strains were isolated during 2008-2009. the isolation rates in the two years were season-dependent to some extent, 30 strains were isolated in october, while only seven strains were isolated in summer (from june to august). the ages of flocks at the time of the outbreak varied between 4 and 69 days. most of the strains were isolated from the chickens between 10 to 30 days of age. the detailed clinical record of each strain was showed in table 1 . after three passage propagation, ibvs of all isolates induced peripheric lesions and growth retardation of embryo at 72 h post-inoculation. since the fourth day post-inoculation, most of the chicks were listless and huddled together, showed ruffled feathers. the results of virus recovery in chicks indicated 87.5% (70/80) isolates caused serious kidney lesions, which were presented with swollen specked kidney and distended ureters filled with uric acid were nephropathogenic type, and the other ten isolates in the study caused respiratory system signs, which were consistent with the clinical record of each strain (table 1) . homologies among s1 nucleotide and deduced amino acid sequences the obtained strains were characterized phylogenetically by nucleotide sequence analysis of the hyper-variable s1 gene of ibv. the nucleotide and amino acid sequence similarities between the eighty ib strains were ranging from 75.4% (strain cq8 and hy) to 100% (strain pt1 and pt3) and 73.9% to 100%, respectively. compared to the 28 reference strains published in the genbank, the identity of the nucleotide and amino acid sequence among the 108 isolates (including the 80 isolates in this study plus the 28 reference strains) were 75.1 to 99.8% and 73.1 to 99.8%, respectively, indicating low homology and high variation among the isolated and reference strains. as shown in the table 2 and table 3 , s1 genes of the newly strains contain mutations, insertions and deletions, resulting in different lengths of nucleotides. s1 genes of these strains were generated and confirmed from three time sequencing results, contained 1641, 1647, 1650, 1653, 1656, 1659 and 1662 nucleotides, amino acids sequences ranging from 547 (lc strain) to 554 (lc strain). the length differences indicated amino acid insertions and deletions exist among the different strains. through the alignment analysis, the deletions, insertions and mutations of each obtained s1 gene was summarized in table 2 . most variations in the deduced amino acid sequences of chinese ibvs were observed among residues 63-69, 211-212 and 354-358 (numbering was with reference to s1 sequence of the mass41 strain). the precursor protein of s glycoprotein is cleaved into amino-terminal s1 and s2 protein by the protease during viral maturation [9] . in this study, the most common cleavage recognition sites of s1 gene were rrf(s/l) rr (49/80) or hrrrr (28/80) in the china field strains ( table 3 ). the exceptional ones included cq8 (rrtgr), hy52 (rrskr), and hy2 (rrskr). the cleavage sites of these two strains containing amino acids k, t, and g, were novel motifs compared to the reference strains, and quite different with the other isolates of the cleavage site. a phylogenetic tree was constructed from the nucleotides sequences of the s1 glycoprotein genes. as shown in the figure 1 , the 80 isolates ibv strains were clustered into five distinct genetic groups or genotypes which were considerably heterogeneous, including a2type (49 newly isolated strains), 4/91-type (9 newly isolated strains), hn08-type (20 newly isolated strains), gray-type and m41-type. the newly isolated strains mainly belonged to a2-type, 4/91-type and hn08-type branch. the phylogenetic relationship of strains at different times and geographical regions displayed complexity and diversity. strains isolated from hubei, zhejiang, jiangsu, guangdong, guangxi and fujian province mainly belonged to the a2 branch, also including other seven published ibv strains from china (qxibv, ck/ch/ljl/07ii, ck/ch/ ljs/07iv, ck/ch/lsd/08-12, ibvsx4, lz05 and lz07). the isolated strains of hainan province and a few isolated strains from guangdong and fujian province belonged to the hn08 branch, included psh050513 and ck/ch/lcq/08ii. group gray-type was correlative with the american strain (gray), included other two classical american strains (ark99 and holte), one japanese strain (jp9758), and the exceptional field strain (cq08). most of the current vaccine strains (h120, h52, ma5, m41, w93, 4/91 and 28/86) were belonged to the m41 branch, which including one field strains (nj). however, the current pandemic strains were mostly 4/ 91-type, a2-type (qxibv-type) and hn08-type, indicating that the field ibvs co-circulating in chicken flocks in china were evolutionarily distant from the known vaccine strains. infectious bronchitis (ib) is one of the most common and difficult-control poultry diseases in china, caused persistent but infrequent outbreaks in commercial chicken farms [24, 25, 28] . commercial vaccines based on h120, h52, 28/86, ma5, w93 and m41 strains, have been widely used to control the disease [2, 29] . natural outbreaks of ibv often are the result of infections with strains that differ serologically from the vaccine strains. come to the rapid and complicated evolutionary of ibv, it is imperative to learn profoundly the circulating ibvs, facilitate selecting the candidate vaccine strain against the infections [2, 24] . in this study, 80 ibv strains were isolated from the vaccinated chicken flocks, with a wide age range of ib outbreak. the chickens infected before the age of 5 days which might be caused by the vertical transmission of ibvs or the maternal antibody could not provide pertinent protection against the prevalent strains [30] . furthermore, there was accumulating evidence indicated that the nephropathogenic ibvs have become prevalent in china in last several years [23, 26, 31] . through clinical records and the virus recovery trials, 70 identified isolates mainly caused typical swollen kidney, different from the respiratory type strains isolated in earlier years, including the major vaccine strains. these findings indicated that all 80 isolated ibv strains from china during 2008-2009 were evolutionarily distant from the vaccine strains used for current, resulting in vaccination failure cases. the s1 protein determined the serotypic evolution, the phenotype change and the genetic diversity of ibvs [32] . in the present study, nucleotide and derived amino acid sequences of s1 protein genes of the 80 field strains were aligned and compared to the representative strains, to determine the relationship of circulating field isolates, vaccine strains and previously described variant strains. newly isolated strains shared between 75.4% to 100% nucleotide sequence similarity with each other, higher similarity than the vaccine strains and other representative ibvs. although the ibvs all over the world shared some common antigenic types, virus strains within a geographic region were unique and distinct, even in different provinces of china. the variants were mostly located in the first 300 amino acids in the n-terminal of the s1 protein of ibv, even though the mutants consisted of insertions, deletions and point mutations were complicated and detailedly different, the hypervariable regions in s1 protein in this study were similar to previous studies [19, 23, 26] . the phylogenetic analysis showed that there were five subgroups of ibvs co-circulating in china, and multiple strains might cause the constant ib outbreaks. the newly isolated strains were mostly derived from a2, 4/91 and hn08. only ck/ch/chongqing/0908 belonged to the branch of gray. the phylogenetic distributions were closely relative to geographical factors. most of the recently isolated ibvs in this study formed the distinct cluster related to the a2 type. however, the routine vaccine strains mainly belong to m41-type branch. a2 strain is closely related to 4/91 serotype, spreading over europe since its first isolation in uk in 1991 [9, 24, [33] [34] [35] [36] [37] . in this study, 61.3% (49/80) field isolates belonged to the a2type branch, which included 85.7% (42/49) nephropathogenic field isolates of this study. the qxibv, first isolated in china and reported associated predominantly with various forms of renal pathology in china, was also representative a2-type strain [25, 31] . the analysis results were according to the prevalence of nephropathogenicity ib. to date, the qx-like ibv strains have been widely isolated in many european countries, and become a dominant genotype [5, 38] . through ib surveys, the european qx-like ibv strains have been reported that caused 86% respiratory signs, 22% litter or enteric problems, only 2% had swollen kidneys [39] . absorbingly, the qx-like ibv strains have undergone divergent evolution paths, brought out different variants in europe and china. similarly, seven exceptional strains located in the a2-type branch caused evident respiratory problems, including three isolates from zhejiang province (qz1, qz2 and qz3) and three isolates from guangdong province (xd2, xd3 and lz2), and gl from guangxi province. the results of our study indicated the strain grouping, such as phenotype and genotype, were not only depended on the geographical factors. the evolutionary pace and the epidemiology characteristics of the ibv were complicated. in conclusion, the data obtained from our study suggest most of present ibv isolates in china are a2-like nephropathogenic strains. to control the prevalence and well prepare for the potential outbreaks of ib, the candidate virus strain for vaccination might be selected timely and specifically in a geographical region, which manifests the importance of continuing surveillance of new ibv strains. this paper is a periodic report on our ongoing surveillance program. we hope the study could contribute to guiding the development of effective vaccines and establishment of control policy for ib. during the period from june 2008 to november 2009, circulating field ibv isolates were selected from suspected broilers and broiler breeders in vaccinated flocks from eastern, southern, southwestern and central china. documented clinical signs of the birds included typical the allantoic fluids containing ibv isolates after 72 h post inoculation were harvested for subsequent experiments, and the remains were preserved in liquid nitrogen. five 1-day-old spf white leghorn chickens were intranasally inoculated with filtration sterilized allantoic fluid of each isolated virus strains, respectively. all of the chicks were examined and recorded daily for clinical signs of infection and mortality for 20 days post-inoculation, the dead birds were necrospied for lesions of respiratory tract or nephritis. finally, all the survivors were sacrificed and necrospied. rt-pcr and s1 gene sequencing a pair of specific primers was designed to amplify the entire s1 protein gene, including the forward primer (s1f): 5'-aagactgaacaaaagaccgact-3', and the reverse primer (s1r): 5'-caaaacctgccataac-taacata-3'. reverse transcription and amplification were performed using the primescripttm one-step rt-pcr kit in 25 î¼l reaction volume containing 20 î¼l of rt-pcr premix (reaction buffer, dntps, 2 î¼l of enzyme mix), 2 î¼l of extracted viral rna and the specific primer pair. reverse transcription and amplification were performed as one cycle of 50â°c for 30 min, 94â°c for 2 min, followed by 30 cycles of denaturation at 94â°c figure 1 the phylogenetic tree of ibvs isolated in mainland china during 2008-2009 based on the viral s1 sequences. the reference strains in this study were marked with "â�²". for 40 s, annealing at 51â°c for 40 s and extension at 72â°c for 2 min, respectively) with a final 10 min extension step at 72â°c. the pcr products were cloned into pmd19-t vector (takara biotechnology, dalian, china) for later sequencing (augct biotechnology, beijing, china). the s1 protein gene sequences obtained in this study were submitted to the genbank database and assigned the accession numbers of gu471864-gu471897, gu471793-gu471805 (table 1) . twenty-eight representative sequences available in genbank were contributed to comparison and phylogenetic analysis in this study, including vaccine strains, h120 (accession numbers: . the multiple-alignment was carried out using dnastar sequence analysis software (dnastar inc., madison, wi, usa). the phylogenetic tree was constructed using the mega 4.1 software with neighbor-joining method and each tree was produced using a consensus of 1000 bootstrap replicates [40] . a nomenclature for avian coronavirus isolates and the question of species status infectious bronchitis protection of chickens against renal damage caused by a nephropathogenic infectious 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infectious bronchitis virus strains in china and analyses of their s1 glycoprotein gene current status of infectious bronchitis virus infections in chickens and prospects for control by vaccines maternal antibody to infectious bronichitis virus: its role in protection against infection and development of active immunity to vaccine a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china genotypic and phenotypic characterization of the california 99 (cal99) variant of infectious bronchitis virus co-circulation of four types of infectious bronchitis virus (793/b, 624/i, b1648 and massachusetts) variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs a survey of the presence of a new infectious bronchitis virus designated 4/91 (793b) a new strain of infectious bronchitis virus infecting domestic fowl in great britain epidemiology of infectious bronchitis virus in belgian broilers: a retrospective study pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes a reverse transcriptase polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from mega4: molecular evolutionary genetics analysis (mega) software version 4.0 phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during authors' contributions jj and jx carried out most of the experiments and wrote the manuscript, and should be considered as first authors. fc and qx critically revised the manuscript and the experiment design. ds, kz, cx, jq, hl, jm and yb helped with the experiment. all of the authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord-303588-bwllypvq authors: ababneh, mustafa; ababneh, ola; al-zghoul, mohammad borhan title: high-resolution melting curve analysis for infectious bronchitis virus strain differentiation date: 2020-03-03 journal: vet world doi: 10.14202/vetworld.2020.400-406 sha: doc_id: 303588 cord_uid: bwllypvq background and aim: belonging to the coronaviridae family, avian infectious bronchitis virus (ibv) causes respiratory, reproductive, and renal diseases in poultry. preventative measures lie mainly in vaccination, while the gold standard for ibv classification and differentiation is based on the sequence analysis of the spike 1 (s1) gene. in this study, we tested a new assay for ibv strain classification that is less expensive and requires reduced time and effort to perform. we carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qrt-pcr/hrm) curve analysis. materials and methods: in this study, qrt-pcr was conducted on a partial fragment s1 gene followed by a high resolution melting curve analysis (qrt-pcr/hrm) on 23 ibv-positive samples in jordan. for this assay, we utilized the most common ibv vaccine strains (mass and 4/91) as a reference in the hrm assay. to evaluate the discrimination power of the qrt-pcr/hrm, we did the sequencing of the partial s1 gene. results: it was shown that hrm was able to classify ibv samples into four clusters based on the degree of similarity between their melting points: the first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the mass-related ibv strain. although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant ii-like (is-1494-06) ibv field strain. finally, the fourth cluster comprised one unique sample that was found to belong to the q1 ibv strain. conclusion: our developed qrt-pcr/hrm curve analysis was able to detect and rapidly identify novel and vaccine-related ibv strains as confirmed by s1 gene nucleotide sequences, making it a rapid and cost-effective tool. avian infectious bronchitis (ib) serves as a major threat to the worldwide poultry industry, and it is a disease endemic to jordan, causing respiratory, renal, and reproductive problems in chickens [1, 2] . ib virus (ibv) is a positive-sense single-stranded rna virus, belonging to the coronavirinae subfamily gamma-coronaviruses [3] . ibv was first recognized as an avian respiratory pathogen in 1930, and its genetic diversity has resulted in the introduction of many ibv vaccines [4] . recently, emerging ibv variants have been reported to cause nephropathogenic and reproductive problems, which are a turn of events that require a dramatic change in vaccination programs [5, 6] . the ibv genome has an approximate size of 27,500 nucleotides and is organized in the following order: utr5'-pol-s-3a-3b-e-m-4b-4c-5a-5b-n-utr3' [7] . the spike glycoprotein gene, encoding spike protein subunit 1 (s1) and 2 (s2), is the most variable gene in the ibv genome [8, 9] . in particular, the s1 protein, which is responsible for viral attachment and entry into host cells, differs significantly with regard to amino acid sequence among ibv serotypes [10] . this variability makes the s1 gene an ideal target in viral typing assays incorporating reverse transcription-polymerase chain reaction (pcr) and dna sequencing techniques. in most cases, high s1 gene homology between two strains can predict the occurrence of cross-protective immunity, where exposure to one strain confers protection to another [10] [11] [12] . ibv strains can be classified into six genotypes that together comprise 32 distinct viral lineages, the majority of which belong to genotype 1 [13] strain identification, sequencing of the full s1 gene or one of its fragments is performed [13, 15] . high-resolution melting (hrm) curve analysis is a newly established pcr-based technique that has been used to differentiate related strains of the same animal or avian virus [16] [17] [18] [19] . this technique was performed on double-stranded dna resulting in rapid genotyping of genetic polymorphisms in diagnostic and routine [20] . this study aimed to optimize an hrm assay for genotyping the ibv vaccine and field strains found in jordan. approval from the jordan university of science and technology animal care and use committee (just-acuc) is not applicable as these samples were collected from recently dead chickens in private farms, and then samples were submitted to our laboratory. ibv vaccines were purchased from local jordanian markets. the most commonly used ibv vaccine strains in jordan are mass-type strains (m41, ma5, and h120) as well as the 4/91 (793/b) vaccine strain (table-1 ). these vaccines were used as reference ibv strains in the qrt-pcr/hrm procedure. twenty-three positive ibv multi-tissue samples collected from different regions of jordan during various outbreaks were also used ( table-2 ). viral rna of these samples and ibv vaccine strains were extracted, then went through cdna synthesis, and then nested pcr amplification of a spike 1 gene fragment. to differentiate between ibv strains, the pcr products from the second round of nested s1 gene pcr were subjected to hrm analysis on the rotor-gene q 5plex hrm platform (qiagen, ca, usa). the ibv vaccine vials were rehydrated with 1 ml of 1× pbs, then mixed by vortexing to dissolve the vaccines. a 150 µl of the rehydrated vaccine was added to 250 µl of tri reagent (zymo research, usa) and suspended for 5 min in a 50°c dry bath. then, 400 µl of absolute ethanol was added, and rna extraction was performed using the direct-zol rna miniprep kit (zymo research, usa). ibv field samples, mostly consisting of tracheal and kidney tissues that were submitted to our laboratory for ibv diagnosis, were homogenized and centrifuged at 3000× g for 5 min. after that, 150 µl of the supernatant was mixed with 250 µl of trizol. a superscript ® iv first-strand synthesis kit (invitrogen, usa) was used according to the manufacturer's instructions to make cdna for both the vaccine and field samples. nested rt-pcr amplification was performed using the synthesized cdna to amplify s1 gene fragments of the ibv genome. the first and second rounds nested pcr was performed in a reaction volume of 25 µl that contained 2.5 µl of 10× pcr buffer without magnesium, 1 µl dntps, 1 µl of each reverse and forward primers, 0.75 µl mgcl 2 , 0.1 µl taq dna polymerase, 1 µl cdna, and 18.15 µl h 2 o. thirty-five cycles of amplification were carried out in a thermocycler, with each cycle consisting of denaturation for 45 s at 94°c, annealing for 1 min at 50°c (for both rounds), and extension for 2 min at 72°c, and a final extension for 7 min at 72°c. electrophoresis of amplified products was carried out using 1.5% agarose gel, which was visualized and photographed under uv light with a 100 bp ladder. nested pcr primer sequences for the s1 gene were as previously reported [14] . for this assay, the nested rt-pcr for the amplification of s1 fragment was repeated. however, the s1 rt-pcr products from the first round of nested rt-pcr were used as a template in the hrm protocol. a type-it hrm pcr kit (qiagen, ca, usa) was employed according to the manufacturer's instructions on the qiagen rotor-gene q 5plex hrm platform (qiagen, ca, usa). the qrt-pcr 25 µl reaction volume contained 12.5 µl of 2× hrm pcr master mix, 1.75 µl of each 10 µm s1 gene internal primers for the second round, and 2 µl of s1 rt-pcr product from two softwares were used for the analysis of the hrm results. the operating hrm software installed on the rotor-gene q 5plex machine and the rotor-gene screenclust hrm software (qiagen, ca, usa) was used to analyze the results of hrm and to assign the different ibv strains into clusters according to their hrm patterns. the hrm analysis using the operating software was done by setting a reference ibv genotype either as 4/91, mass, or variant ii genotypes, after normalizations, the hrm genotype confidence percentages (gcps) were obtained. for the screenclust software, the unsupervised model was used and then data were normalized and clusters were generated to contain all ibv isolates. the typicality and probability of each sample was recorded. the s1 rt-pcr products of the nested rt-pcr amplification were treated with exosap-it pcr product cleanup reagent (cat. no: 78201, thermo fisher scientific, usa) according to the manufacturer's instructions. briefly, 2 µl of exosap enzyme were added to 5 µl of the product then incubated at 37°c for 15 min to degrade remaining primers and nucleotides followed by another incubation at 80°c for 15 min to inactivate the exosap-it reagent. the cleaned rt-pcr products were sent to a sequencing facility (macrogen, south korea) to be sequenced by chain termination technology. s1 sequences were viewed on the bioedit software and edited by the edit sequence interface of the lasergene package. sequence alignment, calculation of the sequence nucleotide similarities, and construction of the phylogenetic tree were done by the megalign interface of the lasergene package. the partial sequences of s1 gene of all ibv isolates included in this study were deposited in genbank under the accession numbers; mk680008-mk6800034. to detect the field and vaccine ibv strains, we used a nested rt-pcr assay to detect the s1 gene. twenty-two out of the 23 samples were positive for the s1 gene after nested rt-pcr. those 22 samples, along with the five ibv vaccine reference strains, were subjected to qrt-pcr targeting the s1 gene followed with hrm curve analysis. table-3 the clusters are produced by the screenclust software into four clusters with the typicality and probability of each sample to be in one of the four clusters ( figure-1) . furthermore, the hrm gcps of selected samples are shown in figure-2 . the sequencing of the partial s1 gene was carried out to confirm the results of the hrm assay (figure-3) . the sequencing of the partial s1 gene for all five ibv vaccine strains matched 100% with their identity. out of the 22 field samples, three samples were found to be ibv 4/91like strain on sequencing and on hrm analysis. out of the 22 field samples, seven samples were confirmed with sequencing and hrm analysis to be of masslike strain. eleven ibv field samples were found to be of ibv variant ii-like (is-1494-06) field strain (on hrm, 8/11 were also of variant ii-like and 3/11 were of mass like) and one field sample was a unique ibv strain (q1 strain). the hrm assay divided the ibv vaccine and field strains into four different clusters. the first cluster consisted of the ibv 4/91 and ibv 4/91-like strains. three vaccines and three field isolates were located in this cluster. the ibv strain 4/91 can be found both as a vaccine and as a field strain and many ibv infections in different countries have been linked to 4/91 field strains. the ibv field samples (d10117, 101216d, and 7p6) belonged to cluster 1, representing the 4/91 and ibv 4/91-like strains, respectively. based on hrm analysis, the melting temperature of the 101216d sample (78.30°c) was close to that of the 4v (1/96) strain (78.09°c), while the melting temperatures of the 7p6 sample (77.49°c) and the 3v (cr88 variant) strain (77.09°c) were similarly close. despite the variation in melting temperatures between the vaccine and field strains in cluster 1, hrm and screenclust analysis were still able to identify the similarities between them by classifying them into the same cluster. the second cluster contained the ibv mass and mass-like strains. two ibv vaccine strains were located in this cluster along with 10 field ibv isolates, three of which were found to be of a variant ii-like (1s/1494/06-like) strains on sequencing (t2, 61216d and 0605p). while cluster 2, which includes ibv mass and mass like strains, has a closer range between the melting temperatures (78.24-78.51) with less variation up to 0.27. this indicates that the viruses of the field samples share close sequences to the mass vaccine strain. available at www.veterinaryworld.org/vol.13/march-2020/1.pdf the third cluster contained the ibv variant ii-like (1s/1494/06-like) strains, and only field isolates were found in this cluster. this cluster has the highest difference in the melting temperature that reaches 0.75. eight samples were of variant ii-like (1s/1494/06 like) ibv strain representing 34% of the total ibv samples. the fourth and final cluster included one ibv field strain (072017), the latter of which was found to be q1-like strain on sequencing. hrm analysis has been used to detect and genotype fowl adenoviruses, avian influenza virus, and avian nephritis virus, among others [17, 21, 22] . the first published study to use hrm analysis to the type of ibv strains was carried out in australia, where an hrm assay that targeted the 3'utr gene was phylogenetic tree analysis of the partial spike 1 gene sequences of ibv vaccine strains and field isolates. the solid circle is the ibv vaccine strains. the solid squares are the mismatched ibv (in hrm analysis, those isolates were clustered in cluster 2 (mass-like type), but in sequencing, they were of is/1494/06 like). the evolutionary history was inferred using the neighbor-joining method. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. there were a total of 315 positions in the final dataset. evolutionary analyses were conducted in mega6. available at www.veterinaryworld.org/vol.13/march-2020/1.pdf performed on local ibv vaccine and field strains [16] . of the 17 ibv-positive cases, only 12 were typed correctly with matching hrm/3'utr and s1 gene sequencing results [16] . as can be seen, our hrm assay can type any mass or 4/91 ibv vaccine and field strain correctly to their corresponding cluster. however, a problem is only encountered when dealing with ibv variant ii field strain. in such cases, we might need to confirm the results of the hrm assay by sequencing of the s1 gene. regarding cluster 3, the variant ii-like (1s/1494/06-like) stain is an endemic ibv strain in jordan that is involved in the respiratory, kidney, and reproductive tract problems [23, 24] . recently, the variant ii-like (1s/1494/06-like) field strain has been found to be subject to multiple recombination events, which might explain the wide range in melting temperatures in cluster 3 [25] . concerning cluster 4, sample 072017 was found to belong to the unique q1 strain, which was first reported in china, in 1996, and then in italy during a 2011 outbreak in broilers [26, 27] . even though the melting point for this sample is similar to those in other clusters, hrm and screenclust analysis were able to differentiate it into a separate cluster. our qrt-pcr with hrm/spike gene assay was able to genotype all vaccine ibv strains and cluster them into separate clusters according to their distinct genotypes (mass and mass-like cluster, 4/91 and 4/91like cluster). most of the ibv field samples belonged to the variant ii-like (1s/1494/06-like) strain. those variant ii-like (1s/1494/06-like) strains were all classified into one cluster expect for three samples that were clustered within the mass and mass-like ibv strains. the current qrt-pcr/hrm curve analysis was able to detect and rapidly identify novel and vaccine-related ibv strains which are confirmed by s1 gene nucleotide sequencing, making it a rapid and cost-effective tool. infectious bronchitis virus in jordanian chickens: seroprevalence and detection global distributions and strain diversity of avian infectious bronchitis virus: a review severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus review of infectious bronchitis virus around the world pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen-free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes phylogenetic analysis of infectious bronchitis coronaviruses newly isolated in china, and pathogenicity and evaluation of protection induced by massachusetts serotype h120 vaccine against qx-like strains first characterization of a middle-east gi-23 lineage (var2-like) of infectious bronchitis virus in europe recombination in avian gamma-coronavirus infectious bronchitis virus polymorphisms in the s1 spike glycoprotein of arkansastype infectious bronchitis virus (ibv) show differential binding to host tissues and altered antigenicity variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo infectious bronchitis virus s1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization s1 genebased phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification presence of infectious bronchitis virus strain ck/ ch/ldl/97i in the middle east genotypes of infectious bronchitis viruses circulating in the middle available at www.veterinaryworld.org application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens high resolution melting analysis: rapid and precise characterization of recombinant influenza a genomes highresolution melting (hrm) for genotyping bovine ephemeral fever virus (befv) a novel hrm assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance high-resolution melting: applications in genetic disorders classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region detection and characterization of coronaviruses in migratory and non-migratory australian wild birds genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in egypt complete genome analysis of iranian is-1494 like avian infectious bronchitis virus successive occurrence of recombinant infectious bronchitis virus strains in the restricted area of middle east diagnostic and clinical observation on the infectious bronchitis virus strain q1 in italy molecular investigation of a full-length genome of a q1-like ibv strain isolated in italy in 2013 the authors would like to thank ms. amneh tarkhan for her excellent technical assistance and valuable comments. the author expresses a deep appreciation and thanks to the deanship of research at jordan university of science and technology for its financial support of this work (grant # 195/2018). ma and oa designed the experiment. oa performed the technical assays. ma and mba supervised the technical assays, ma and oa analyzed the results. ma and oa prepared the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests.veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord-299428-gon6bzat authors: mondal, shankar; chang, yung-fu; balasuriya, udeni title: sequence analysis of infectious bronchitis virus isolates from the 1960s in the united states date: 2012-10-11 journal: arch virol doi: 10.1007/s00705-012-1500-y sha: doc_id: 299428 cord_uid: gon6bzat to better understand the molecular epidemiology of infectious bronchitis virus (ibv) in the united states following the introduction of commercial ibv vaccines, we sequenced the s1 and n structural protein genes of thirteen ibv field isolates collected in the 1960s. analysis of the s1 sequence showed that seven isolates were of the massachusetts (mass) genotype, five were se17, and one was of the connecticut (conn) genotype, suggesting that these three ibvs were circulating in commercial poultry raised in different regions in the united states during the 1960s. the s1 genes of mass-type isolates had high levels of sequence variation, representing 81.3-81.9 % nucleotide (nt) and 77.3-78.7 % amino acid (aa) identity when compared to those of the se17-type isolates. in contrast, the n genes from the same isolates were less variable (>92 % nt and >93 % aa identity) when compared to those of the se17-type isolates. phylogenetic analysis based on the s1 gene indicated that one isolate (l748) was more closely related to the mass type. in contrast, phylogenetic analysis based on the n gene showed that l748 was more closely related to the se17 type, indicating that there had been exchange of s1 genetic materials between massand se17-like viruses. in addition, the mass-type isolates had high levels of sequence identity in the s1 gene compared with widely used modified live vaccines (mass41, ma5 and h120) and modern field strains from the usa and other countries, suggesting a common ancestor. electronic supplementary material: the online version of this article (doi:10.1007/s00705-012-1500-y) contains supplementary material, which is available to authorized users. avian infectious bronchitis virus (ibv) is a ubiquitous, highly contagious respiratory pathogen of chickens that inflicts serious economic losses to the commercial poultry industry worldwide [3, 4] . ibv, along with turkey coronavirus, belongs to the genus gammacoronavirus in the subfamily coronavirinae, family coronaviridae, order nidovirales [2, 5] . ibv has a single-stranded, positivesense rna genome of approximately 27.6 kilobases. the genes (open reading frames [orfs]) encoding ibv structural proteins are located downstream of the viral replicase genes (orfs 1a and 1b) and in order from 5 0 to 3 0 are as follows: s (spike), e (envelope), m (membrane) and n (nucleocapsid). the s glycoprotein is posttranslationally cleaved into a transmembrane domain (s2) and an outer domain (s1), which expresses the serotype-specific epitopes of the virus [4] . the n phosphoprotein, which forms the capsid of the virion, is involved in rna replication and carries group-specific antigenic determinants [10] . both s1 and n proteins play critical roles in the induction of immune responses against ibv [4, 10] . ibv was first isolated in the early 1930s in the united states [19] . the massachusetts (mass)-type viruses were believed to be the only serotype found in the usa until the mid-1950s, when a second ibv serotype connecticut (conn) was reported [12] . immediately thereafter, the use of commercially produced mass-and conn-type modified live virus (mlv) vaccines began [4] . a number of new ibv serotypes, antigenic variants and field strains were isolated in the subsequent years in the usa, and many ibv serotypes are currently known to exist worldwide [4, 21] . mlv vaccines containing strains of ibv from different serotypes are routinely used to protect commercial chickens against infectious bronchitis (ib). mlv vaccine strains are selected to represent the antigenic spectrum of likely challenge viruses by incorporating the serotypes of viruses most commonly circulating in a particular country or region. in the united states, most ibv isolates causing ib belong to the mass, conn, and arkansas (ark) serotypes [4] . disease outbreaks in vaccinated chickens may result when the flock is infected with ibvs that are antigenically unrelated to the vaccines used to immunize them. new variants of ibv can emerge from either wild-type or vaccine viruses by acquiring point mutations and/or genomic recombinations [7] . one of our previous studies indicated that antigenic and genetic diversity existed during the 1940 s [11] , well before the introduction of ibv vaccines. however, many ibv strains from the usa isolated following the introduction of commercial mlv vaccines have never been characterized by sequencing, and it is essential to sequence these isolates to understand the evolution of ibv geographically and to implement effective vaccination strategies to control modern ibv strains that are in circulation. the main objective of this study was to examine a number of archived ibv isolates from the 1960s in order to determine (1) which genotypes of ibv were prevalent in the united states during that time and (2) whether genetic information obtained from these isolates would shed light on the evolution of the currently circulating serotypes. during the 1960s, clinical signs of bronchitis with upper respiratory distress were frequently observed in broiler flocks, even in ibv-vaccinated birds, but rarely in layer flocks [6] . through the cooperation of numerous hatcheries and laboratories, a number of field isolates of ibv were recovered in the mid-1960s from the respiratory tracts of naturally-infected chickens raised in different regions of the united states (winterfield and hitchner, unpublished data). following isolation at cornell university, the viruses were passed a minimum number of times (no more than three) in embryonated chicken eggs, and allantoic fluids from the embryos were lyophilized and stored at 4°c. for the present study, we used thirteen representative ibv field isolates from all regions of the us, generously provided by dr. benjamin lucio-martinez, college of veterinary medicine, cornell university, ithaca, ny ( table 1 ). the lyophilized samples were reconstituted in 1 ml of diethylpyrocarbonate (depc)-treated water and used directly for rna extraction without further passage in chicken embryos. ibv rna was extracted from 200 ll of each sample using trizol ls reagent (gibco brl, grand island, ny) according to the manufacturer's instructions and resuspended in 8 ll of rnase-free water. the reconstituted rna from each isolate was used as the template for rt-pcr amplification of the s1 and n protein genes. briefly, the rt reaction was performed using a superscript iii firststrand synthesis kit (invitrogen, inc., carlsbad, ca) with random hexamer primers according to the manufacturer's instructions. the rna template was removed by digestion with 2 u of e. coli rnase h for 30 min at 37°c. amplification of cdna with gene-specific primers (news1oligo5 0 and degenerate3 0 [15] for s1, and nfor and nrev [23] for n genes) was performed using an advantage 2 polymerase mix kit containing high-fidelity dna polymerase (clontech laboratories, inc., mountain view, ca). the amplicons (1,610 bp [s1] and 1,230 bp [n]) were analyzed by gel electrophoresis using 0.5 % agarose with ethidium bromide (0.5 lg/ml). with the exception of isolate l969, all other isolates resulted in 1,230-bp n gene rt-pcr products, and therefore, only twelve n gene sequences were included in the analysis. rt-pcr products were gel-purified using a qiaquick gel extraction kit (qiagen inc., valencia, ca), and directly sequenced using a bigdye terminator cycle sequencing kit (perkin-elmer, branchburg, nj) and an applied biosystems model abi 377 automated dna sequencing system. a combination of flanking and internal primers was used to sequence both strands of dna in their entirety. assembly of contiguous sequences and translation of nucleotide sequences into amino acid sequences were performed with vector nti advance 11 software (invitrogen inc., carlsbad, ca). the blastn program (http:// www.ncbi.nlm.nih.gov/blast/) was used to search genbank for homologous ibv s1 and n gene sequences. selected sequences from genbank were included in the alignment. after aligning the s1 and n sequences, the geneious program (biomatters ltd, auckland, new zealand) was used to construct phylogenetic trees by the neighbor-joining method. subsequently, bootstrap analysis was performed with 1,000 replicates to determine the bestfitting tree for each gene. pairwise blast searches were also performed when there were no significant hits in the blastn search. all of the s1 and n gene sequences reported herein have been deposited in the ncbi genbank database, and the accession numbers are listed in table 1 . the s1 and n gene sequences of other ibv strains were also obtained from genbank. the 1,611-bp s1 gene sequences (nt 20,368-21,978, numbered according to the beaudette strain, genbank accession number dq001338) of thirteen 1960s isolates were compared with those of 47 other ibv strains (supplementary table 1 ) from the usa and other countries. a phylogenetic tree based on the nt sequences of the complete s1 genes showed that the 1960s ibv isolates were divided into three distinct lineages, mass, se17 and conn genotypes (fig. 1) , which suggests that these three ibvs were circulating in commercial poultry raised in different regions of the usa in the 1960s. seven ibv field isolates (l74, l423, l455, l569, l748, l806, l969) were clustered with mass-type reference strains (beaudette, mass41), vaccine strains (mass/bvial1, mass/cvial1, ma5, h120) and field strains from the usa and other countries. these viruses had 97.6-99.9 % nt (96.3-99.8 % aa) sequence identity when compared to prototype mass41. five ibv isolates (l163, l613, l718, l905, l919) were clustered with se17 virus, and they had 98.7-99.4 % nt (97.9-98.7 % aa) sequence identity when compared to prototype se17. interestingly, only one ibv isolate (l554) clustered with the conn-type reference virus (conn46), the vaccine strain (conn/cvial1), and field strains from the usa and other countries. this virus had 99.8 % nt (99.3 % aa) sequence identity to the prototype conn46 strain. less-frequent isolation of the conn-type virus was probably due to the high level of conn-type immunity from widespread use of the vaccine in young flocks [6] . the s1 sequences of ibvs closely related to the mass-type isolates were 1,611 bp (537 aa) in length. the s1 sequences of all ibvs grouped with se17 were 1,632 bp (544 aa) in length due to an additional 21-nt (7-aa) insertion at positions 358-363 and 427-441. the s1 sequence of the conn-type isolate l554 was 1543 bp long (513 aa), the same as the conn46 virus. the mass-type isolates were genetically distantly related to other 1960s viruses and had only 81.3-81.9 % nt (77.3-78.7 % aa) sequence identity when compared to se17-type isolates and 94.5-95.2 % nt (89-89.9 % aa) sequence identity when compared to conn-type l554 isolate. the s1 gene of the 1960s isolates were also distantly related to those of the gray, jmk, ark99, n-m24 and n-m39 strains, showing 79.8-85.9 % nt (76.5-84.7 % aa) sequence identity. the deduced aa sequence of the s1 proteins had multiple substitutions that were distributed throughout the s1 protein (data not shown). the mass-type isolates had 1-18 aa differences when compared to mass reference (beaudette, mass41) or vaccine (ma5, h120) strains. isolate l748 had a maximum 18 differences, but only two (s24r, l386r) differed from the mass41 sequence. the se17-type isolates had 1-5 differences compared to prototype se17 virus, and the conn-type l554 had three substitutions compared to conn46 virus. the majority of the differences in mass, se17 and conn-type viruses were concentrated between aa positions 51 and 77, and 115 and 151, which correspond to the two hypervariable regions (hvrs) [13] that are known to carry serotype-specific antigenic determinants. the cleavage recognition sites between the s1 and s2 subunits of the 1960s isolates were compared. all mass-type isolates had an rrfrr sequence, as in other mass viruses, and the se17 group had an hrsrr sequence like the gray and ark99 strains (data not shown). the s1 genes of the mass isolates were found to be genetically closely related to those of widely used mlv vaccine strains and modern field isolates from the usa and other countries (fig. 1) , suggesting a possible common ancestry and emergence of virus strains that are essentially similar to the mlv vaccines that are used. previous studies have indicated that some strains of ibv isolated in different countries may have almost identical s1 genes [16, 17] , reflecting some of the common features of ibv evolutionary direction. the spreading of a virus from one region or country to another could be due to its inadvertent introduction by the trading of birds or by the use of mlv vaccines. it is known that the mass strain of ibv is widely used as a mlv vaccine throughout the world [4] . unlike the s1 genes, the n gene sequences of the 1960s isolates were highly conserved, having [92 % identity at fig. 1 phylogenetic tree created from the nucleotide sequences of the s1 glycoprotein genes of infectious bronchitis virus (ibv) isolates. the neighbor-joining tree was constructed from the pairwise nucleotide differences for the s1 genes, which represented the relatedness between 13 isolates from the 1960s and 47 heterologous ibv strains from the united states and other countries. the 1960s isolates (*) clustered with massachusetts, connecticut and se17-type reference (#) or vaccine (^) strains of ibv. the length of each pair of branches represents the distance between the sequence pairs. the scale at the bottom indicates the number of substitution events the nt ([93 % at the aa) level as compared to the n genes of the other 28 ibv strains (supplementary table 2 ). the phylogenetic tree based on the n gene nt sequences showed that the 1960s ibvs were also divided into three clades, representing the mass, se17 (unfortunately, the n gene sequence of prototype se17 was not available in genbank, and we did not have access to the isolate) and conn genotypes (fig. 2) . of them, five (l74, l423, l455, l569, l806) were closely related to the mass strains (mass41, h52), showing 98.2 % nt (98.5 % aa) sequence identity with mass41, and they were even more closely related ([99 %) to isolates ck/ch/lsd/031 from china and ind/ tn/92/03 from india; six (l165, l613, l718, l748, l905, l919) ibv isolates, those clustered with se17 (except for l748 which clustered with mass) based on s1 phylogeny, were closely related to turkey coronavirus (tcov) in the n gene, showing 96.5 % nt (96.8 % aa) sequence identity, and they had a similar (c96 %) genetic relationship to the ark99, conn, and gray strains of ibv. the isolate l554 clustered with conn-type reference (conn46) and field strains, showing c99% sequence identity. the n gene of all the 1960s isolates was 1,230 bp (409 aa) in length and differed from one another by zero to 6.8 % nt (5.4 % aa) (data not shown). our data is consistent with previous reports describing recombination between ibv and turkey coronaviruses [9] . genetic variation was observed in the s1 and n genes of the isolate l748 in this study. phylogenetic analysis of the s1 gene showed that the mass-type isolates, including l748, clustered together (fig. 1) , as would be expected of viruses of the same serotype. interestingly, the l748 isolate had much different levels of sequence similarity to each other in the n gene, and it clustered randomly with other se17-type isolates (fig. 2) . the shift in sequence homology in the s1 and n genes indicated that exchange of genetic material took place subsequent to coinfection with a mass-like virus and an se17-like virus for l748, probably by homologous recombination. a similar recombination event has been observed in some japanese isolates [20] , where the s1 protein was found to be closely related to australian isolates [18] and the n protein was very similar to those of north american viruses [14] . in addition, two us isolates (md27 and pp14) exhibiting 97 % and 95 % identity, respectively, to se17 in the s1 genes are known to be derived from se17 by recombination [1, 22] . se17 was first reported by hopkins [8] as a new serotype of ibv isolated in 1967 from a closed research flock of chickens with classic signs of ib. herein, we report five se17 isolates from samples collected in 1965 (l165, l613) and 1966 (l718, l905, l919) from commercial chickens raised in different regions of the usa. unlike se17, the conn46 and mass (mass41, ma5, h52, h120) strains have been used simultaneously as mlv vaccines in commercial poultry in the usa [4] . the use of se17 or its derivatives as an mlv vaccine is not known. because the chickens from which the 1960s isolates were isolated most likely had been exposed to live mass vaccines [6] , the vaccine might be implicated as a source for the mass-like sequences in the se17 s1 gene. the se17 source of genetic material was probably a strain naturally infecting these affected flocks, since they had not been exposed to se17 vaccine. although this study cannot prove or disprove that recombination has occurred in l748, we can point to the evolutionary trends in the s1 gene as compared to the evolutionary trends in the n gene. this study demonstrates that the s1 gene of ibv tends to evolve more rapidly by mutation and genetic recombination when compared to the more conserved n gene [11] . this suggests that these two genes of ibv are under very different selective pressures and that the exchange of genetic material may have occurred between vaccine strains and field isolates. based on these findings, we conclude that widespread vaccination with mlv vaccines against previously known dominant serotypes (e.g., mass41, ark99), in association with the high recombination and mutation abilities of ibv, have led to the emergence and broad dissemination of the present-day novel serotypes. new strategies to control ibv infection effectively in chickens should be considered with the use of either inactivated or subunit vaccines. complete nucleotide analysis of the structural genome of the infectious bronchitis virus strain md27 reveals its mosaic nature veterinary microbiology coronavirus avian infectious bronchitis virus infectious bronchitis family coronaviridae infectious bronchitis virus types: incidence in the united states the evolution and emergence of rna viruses serologic and immunologic properties of a recent isolate of infectious bronchitis virus recombinational histories of avian infectious bronchitis virus and turkey coronavirus immune responses to structural proteins of avian infectious bronchitis virus genetic and antigenic diversity in avian infectious bronchitis virus isolates of the 1940s immunological differences in strains of infectious bronchitis virus serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s1) gene molecular cloning and sequence comparison of the s1 glycoprotein of the gray and jmk strains of avian infectious bronchitis virus redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de072 strain of infectious bronchitis virus s1 glycoprotein gene analysis of infectious bronchitis viruses isolated in korea phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan sequence analysis of the s1 glycoprotein of infectious bronchitis viruses: identification of a novel genotypic group in australia an apparently new respiratory disease of chicks complete nucleotide sequences of s1 and n genes of infectious bronchitis virus isolated in japan and taiwan infectious bronchitis virus variants: a review of the history, current situation and control measures evidence of natural recombination within the s1 gene of the infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses sequence analysis of 1960s ibv isolates 503 acknowledgements the authors would like to thank dr. benjamin lucio-martinez from cornell university, ithaca, ny, for providing the 1960s ibv isolates used in this study. the authors would also like to thank dr. carol j. cardona from the university of minnesota, st. paul, mn, for reading the manuscript. key: cord-319253-8bssrn9o authors: okino, cintia hiromi; montassier, maria de fátima silva; de oliveira, andressa peres; montassier, helio josé title: rapid detection and differentiation of avian infectious bronchitis virus: an application of mass genotype by melting temperature analysis in rt-qpcr using sybr green i date: 2018-02-27 journal: j vet med sci doi: 10.1292/jvms.17-0566 sha: doc_id: 319253 cord_uid: 8bssrn9o a method based on melting temperature analysis of hypervariable regions (hvr) of s1 gene within a rt-qpcr was developed to detect different genotypes of avian infectious bronchitis virus (ibv) and identify the mass genotype. the method was able to rapidly identify the mass genotype among ibv field isolates, vaccine attenuated strains and reference m41 strain in allantoic liquid and also directly in tissues. the rt-qpcr developed detected the virus in both tracheal and pulmonary samples from m41-infected or h120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. rt-qpcr method tested provided a sensitivity and rapid approach for screening on ibv detection and mass genotyping from ibv isolates. strains [11] . alternatively, a rt-qpcr based on the 3′-utr of ibv (rt-pcr/hrm curve analysis model), could detect and rapidly distinguish novel and vaccine-related ibv strains from australia [15] . finally, a duplex sybr green i based rt-qpcr [1] and a rt-qpcr using specific probes [13, 23] were able to detect and differentiate mass and non-mass strains. finally, considering that hvr i and hvr ii of s1 gene were thought to be closely associated with major neutralization epitopes and consequently a reliable target for genotyping method, we developed a diagnostic method using melting temperature analysis based on these regions aiming to rapid detect and differentiate mass and non-mass ibv strains in samples previously isolated and directly in clinical samples. analytical specificity and sensitivity were also evaluated, and the method was compared to the standard method of virus isolation in embryonated eggs. seven reference ibv strains (m41, h120, h52, ma5, jmk, se17 and connecticut) and six brazilian field isolates (ibvsc01, ibvsc02, ibvpr02, ibvpr03, ibvpr05 and ibvpr07) were provided by empresa brasileira de pesquisa agropecuária-embrapa (concórdia, sc, brazil) and previously molecularly characterized [26] (table 1) . three other non-related viruses (commercial vaccines) were also used to confirm specificity of our method: newcastle disease virus (ndv, la sota vaccine strain), infectious bursal disease (ibdv, lukert vaccine strain) and avian metapneumovirus (ampv, pl-21 vaccine strain). ninety-six one-day-old chicks (cobb-vantress lineage) from a commercial broiler chick hatchery were separated in three positive pressure isolators. at 28 days of age, first group (n=42) was experimentally infected by intra-ocular and intranasal routes with 10 5.0 eid 50 (50% embryo infective dose) of m41 strain. a negative control group (n=42) was mock infected and maintained under the same conditions. remaining group (n=12) was inoculated with h120 live attenuated vaccine. three chickens from the first and second groups were euthanized at 1-10, 12, 15, 17 and 20 days post infection (dpi), while birds from third group were euthanized at 3, 5 e 12 dpi. trachea and lung samples were collected and kept at −70°c until processing by rna extraction or virus isolation. throughout the experimental period, birds received water ad libitum and feed once a day, the room temperature was adapted according with bird's age. all procedures have been approved by the veterinary sciences animal care committee of universidade estadual paulista, according with ethical principles of animal experimentation adopted by brazilian college of experimentation. virus isolation in specific pathogen free (spf) embryonated chicken eggs from tissue samples was performed as previously described [28] . three passages were conducted to confirm negative samples. extraction of rna from virus strains and tissue samples were performed using trizol reagent (invitrogen, carlsbad, ca, u.s.a.). the cdna was synthesized according to instructions provided with superscript iii reverse transcriptase (invitrogen) and random primer (invitrogen). qpcr was carried out in an applied biosystems real-time 7500 instrument (applied biosystems, foster city, ca, u.s.a.) using 10 µl of platinum sybr green qpcr supermix-udg (invitrogen), 10 pmol of each primer (table 2), 2 µl of cdna, in a final volume of 20 µl. the amplification reaction included a preincubation step at 95°c for 8 min to activate the hotstart taq dna polymerase, followed by 40 cycles of amplification including denaturation at 95 for 30 sec, annealing at 49.2°c for 30 sec and extension at 72°c for 30 sec [27] . after amplification by rt-qpcr, a melting analysis curve was performed by raising the incubation temperature from 65 to 95°c in 0.1°c increments with a hold of 1 sec at each increment. the analytical specificity of rt-qpcr was assessed using three non-related rna viruses including newcastle disease virus (ndv, la sota vaccine strain), infectious bursal disease (ibdv, lukert vaccine strain) and avian metapneumovirus (ampv, pl-21 vaccine strain). the analytical sensitivity of rt-qpcr was assessed using seven serial ten-fold dilutions of plasmidial dna containing the orf of s1 gene fragment (1.6 kb) of m41 strain of ibv cloned into the pyes 2.1/v5-his topo vector (invitrogen), using two combinations of oligonucleotides to evaluate the analytical sensitivity. cq (cycle quantification) results were used to calculate the log of ibv copies (log10) using linear equation from a standard curve. the reproducibility was determined by testing in duplicate and in four independent runs, followed by calculation of mean, standard deviation (s.d.) and coefficient of variation (c.v.). tm data of the real-time rt-pcr assay was analyzed statistically by the scott-knott test [30] , and the level of significance was set at p≤0.05. the detection limit of rt-qpcr was 1.32 × 10 2 copies, r 2 =0.997, efficiency=93.06% (fig. 1) . the reproducibility ranged from 0.1575 to 0.7585 cycles, while the c.v. inter-assay ranged from 0.56 to 2.64%. all ibv strains were successfully detected and no positive signal was recorded for the three non-related avian rna-viruses tested. the rt-qpcr developed in this study detected the virus in tracheal samples from birds experimentally infected with m41, or with h120 strains, in the period from 1 to 20 dpi (entire post-infection period evaluated) or from 3 to 12 dpi, respectively, while in pulmonary samples ibv detection varied from 8 to 15 dpi, or only at 3 dpi in m41-infected or h120-infected birds, respectively. conversely, the standard method of virus isolation in spf embryonated eggs detected the virus only from 2 to 9 dpi or at 3 dpi for both types of tissue samples from birds infected with m41 or h120, respectively. no positive signal of amplification and no positive results by virus isolation were found in tissues samples from negative control birds ( table 3) . the absence of amplification signal in other non-related rna viruses and in negative control samples, characterized the specificity of rt-qpcr. f=forward, r=reverse. a) modified, b) annealing site on s1in m41 reference strain of ibv (sequence accession number: ay851295). all tested ibv strains were amplified by rt-qpcr using both primer sets (hvr i and hvr ii). amplicons of expected size for s1 gene of ibv using hvr i or hvr ii primer sets were also visualized by gel electrophoresis analysis. in the first-derivative melting curve analysis, the s1 gene amplicons displayed melting temperature (tm) values ranging from 80.30 to 83.70°c, for hvr i amplicons, and from 76.16 to 78.25°c for hvr ii amplicons. all mass related ibv strains (reference strains and field isolates) tested in this study presented the same tm profile (81.38 ± 0.28°c) for melting temperature analysis of hvr i amplicons, as no significant differences were observed, while all the remaining tested strains presented significant differences in their tm profiles (fig. 2) . interestingly, the ibvpr05 strain, showed two melting peaks, with the highest peak at 83.17°c, and another one at the same tm profile as mass group (fig. 2) . we hypothesized that this sample of brazilian field isolate was composed by two populations or quasispecies, as described previously [17] for a sample of beaudette strain of ibv amplified by rt-qpcr using another pair of primers. however, when the melting temperature analysis was performed using hvr ii region, the mass genotype differentiation was not possible, though the tm profiles for hvr ii amplicons of jmk and se-17 strains and one brazilian variant isolate (ibvpr05) were distinct one each other and from the remaining tested ibv strains. a similar distinction was observed in the tm profile of hvr ii amplicon for m41 strain with regard to h120 strain, in spite of these viruses are classified in the massachusetts genotype. the detection limit of our method is similar to that recorded for a real-time pcr assay based on 5′-utr gene of ibv genome [3] . however our assay is less sensitive than another real-time technique targeting an orf1b of replicase gene of ibv [11] . this difference might be due to the fact that targeting the most conserved and abundant genes of coronavirus, like replicase gene or nucleoprotein gene, can enhance the sensitivity of conventional and rt-qpcr methods [2, 11, 24] . nonetheless, more conserved target genes do not allow differentiation between different genotypes of ibv [24] , as s1 gene rt-pcr does, which remains an important tool for genotyping ibv isolates. there are several reports demonstrating the use of melting temperature analysis to differentiate genotypes of virus pathogens [4, 20, 29, 31, 32] . similarly, the method used here provided differentiation of mass genotype using hvr i region, as all tested strains previously genotyped belonging to mass genotype presented no significant different tm values, while the other strains previously genotyped differently from mass presented significant different tm values (table 1 ). in addition, previous study [16] using primers flanking similar hvrs of s1 gene in rt-qpcr with evagreen succeeded in discriminating ibv strains from taiwan and h120 from m41 strain of ibv. currently, ibv genotype differentiation has been performed using separated molecular assays, which have increased both time and cost. assays for genotyping based on conventional molecular methods such as pcr, rflp, hybridization assay and direct sequencing are time-consuming, more complex and expensive. alternatively, the method described here has allowed simultaneous detection of ibv genome and mass genotype identification in a single reaction. the specificity of mass genotyping by our method was confirmed, comparing with the results obtained previously [21, 26] . in conclusion, the method described here provides a practical and effective screening test for simultaneous detection and mass genotyping of ibv. moreover, this rapid, simple, specific and sensitive test is applicable for both ibv previously isolated and directly in clinical samples. conflict of interest. the authors declare that they have no conflicts of interest. a duplex sybr green i-based real-time rt-pcr assay for the simultaneous detection and differentiation of massachusetts and non-massachusetts serotypes of infectious bronchitis virus rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction development and evaluation of a real-time taqman rt-pcr assay 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china between acknowledgments. the authors thank fapesp (process number 04/12267-2 and 01/14950-3) and cnpq (process number 01/14950-3) for the financial support; embrapa (concórdia, sc, brazil) for supplying ibv strains; merial (campinas, sp, brazil) for provided the embryonated chickens eggs spf and frango sertanejo (ipiguá, sp, brazil) for provided the commercial broilers. we also thank professor dr joao ademir de oliveira for statistical advice. key: cord-301810-vtgdqart authors: aston, emily j.; jordan, brian j.; williams, susan m.; garcía, maricarmen; jackwood, mark w. title: effect of pullet vaccination on development and longevity of immunity date: 2019-02-02 journal: viruses doi: 10.3390/v11020135 sha: doc_id: 301810 cord_uid: vtgdqart avian respiratory disease causes significant economic losses in commercial poultry. because of the need to protect long-lived poultry against respiratory tract pathogens from an early age, vaccination programs for pullets typically involve serial administration of a variety of vaccines, including infectious bronchitis virus (ibv), newcastle disease virus (ndv), and infectious laryngotracheitis virus (iltv). often the interval between vaccinations is only a matter of weeks, yet it is unknown whether the development of immunity and protection against challenge when vaccines are given in short succession occurs in these birds, something known as viral interference. our objective was to determine whether serially administered, live attenuated vaccines against ibv, ndv, and iltv influence the development and longevity of immunity and protection against challenge in long-lived birds. based on a typical pullet vaccination program, specific-pathogen-free white leghorns were administered multiple live attenuated vaccines against ibv, ndv, and iltv until 16 weeks of age (woa), after which certain groups were challenged with ibv, ndv, or iltv at 20, 24, 28, 32, and 36 woa. five days post-challenge, viral load, clinical signs, ciliostasis, tracheal histopathology, and antibody titers in serum and tears were evaluated. we demonstrate that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least 36 weeks, and conclude that the interval between vaccinations used in this study (at least 2 weeks) did not interfere with protection. this information is important because it shows that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent. vaccination against respiratory viral disease is standard practice in commercial poultry operations. both live and killed vaccines are administered to poultry, and live vaccines are commonly used for a variety of pathogens because they are effective when mass applied and are relatively economical [1] . in general, live vaccines induce local and cell-mediated immunity and provide a broader protective response than killed vaccines, whereas killed vaccines primarily induce humoral immunity and tend to be antigen-specific. the duration of immunity achieved following live vaccine administration depends on the age and type of bird, levels of maternal immunity, disease targeted by the vaccine, immunogenicity of the vaccine, method of vaccine application, number of and interval between boosters, virulence and similarity of the field challenge virus, interval between vaccination and challenge, and immunocompetency of the host [1] [2] [3] . avian coronavirus infectious bronchitis virus (ibv) is an upper respiratory tract viral pathogen of poultry and leads to reduced weight gain and feed efficiency, drops in egg production and egg quality, stunted growth, and secondary bacterial infection resulting in airsacculitis [4] . the virus initially replicates in the upper respiratory tract, followed by systemic replication in the reproductive tract and some strains can cause lesions in the kidney [4] . infected birds may exhibit nasal discharge, coughing, sneezing, and tracheal rales [4] . the disease is prevented by vaccination, and live vaccines are commonly used to induce local immunity and protection. live vaccines are generally administered to young birds to achieve early protection, and layers and breeders are also boosted with either live or inactivated vaccines, which vary based on their similarity to the circulating field viruses [2] . newcastle disease (nd) is caused by virulent strains of avian paramyxovirus type 1, which has recently been reclassified as avian avulavirus 1 (aavv-1) [5] . depending on the strain of the virus, clinical signs of nd infection may be absent or may involve depression, inappetence, respiratory signs (nasal discharge, sneezing, coughing), reduced egg production and egg quality, and neurological signs (torticollis, circling, paralysis) [3] . strains of newcastle disease virus (ndv) are characterized as lentogenic, mesogenic, and velogenic, according to their mean death time in embryos [6] . lentogenic strains are of low pathogenicity causing mild respiratory or enteric infections, followed by mesogenic strains, while velogenic isolates are highly pathogenic often causing neurological signs and mortality [3] . vaccination regimes against ndv vary and may utilize a combination of live, inactivated, and virus-vectored vaccines [7] . in the united states, the most widely used traditional vaccine strains comprise lentogenic b1 (or virus clones of the b1 strain) and lasota strains [3] . infectious laryngotracheitis (ilt) is a respiratory disease of poultry caused by gallid alphaherpesvirus i, and is economically important worldwide [8] . clinical manifestations of ilt include increased mortality, reduced egg production, decreased body weight gain, conjunctivitis, tracheitis with expectoration of bloody mucus in severe cases, depression, severe dyspnea and susceptibility to other respiratory pathogens. live vaccines against ilt virus (iltv) may be of chicken embryo origin (ceo) or tissue culture origin (tco), in which they are passaged multiple times in eggs or tissue culture, respectively. although recombinant vaccines for ilt are commercially available, the ceo vaccine is the most widely used vaccine against iltv worldwide. because of the need to protect chickens against different viral pathogens from an early age, vaccination programs typically include multiple vaccines against a variety of pathogens. sample vaccination regimes in different poultry sectors are reviewed in the merck veterinary manual (www. merckvetmanual.com), in which the interval between vaccinations is often only a matter of weeks. however, there is little information showing that the intervals between vaccinations are sufficient for the birds to develop adequate immune protection against challenge for each virus. the literature shows that sequential viral infections may result in viral interference, in which one virus blocks the subsequent infection and/or replication of another virus in the host [9, 10] , but until now it is unknown whether this phenomenon results in reduced protection from serially administered attenuated live vaccines in chickens. interestingly, it has been reported that simultaneous administration of viruses to chickens or turkeys does not result in viral interference [11] . in this study, we investigate how a typical commercial vaccination schedule consisting of a combination of serially administered, live attenuated viral respiratory disease vaccines affects the development and longevity of immunity and protection against homologous challenge. a commercial ibv vaccine mildvac-ga-98 ® (merck animal health, summit, nj, usa) was used in this study. the vaccine was diluted according to the manufacturer's recommendations. the challenge virus used was ibv ga98/cwl0474/98 and was prepared at a median embryo infectious dose (eid 50 ) of 10 3.19 . virus titers were calculated by the reed and muench method [12] . a commercial ndv vaccine b1 (newhatch-c2, merck animal health, summit, nj, usa) was used for both vaccination and challenge and was reconstituted following the manufacturer's instructions. since mesogenic and velogenic strains of ndv require a biosafety level above bsl2, which is not available in our laboratory, our experimental model for protection against ndv involves significantly reduced virus titers or sterile immunity against a second exposure (challenge) with the vaccine virus. an iltv commercial ceo vaccine (trachivax ® merck animal health, summit, nj, usa) and pathogenic iltv georgia broiler strain 63140 [13] were used for vaccination and challenge, respectively. strain 63140 was propagated in chicken kidney cells obtained from 3-to 4-week-old specific pathogen-free (spf) chickens [14] . the ceo vaccine was prepared following the manufacturer's recommendations. after inoculation, the median tissue culture infective dose (tcid 50 ) was confirmed by titration of both viruses in chicken kidney cells as previously described [14] . specific-pathogen-free eggs were obtained at 18 days of incubation and hatched at the poultry diagnostic and research center, athens, ga. chicks were placed on fresh pine shavings in colony houses and pens. chicks were vaccinated with the manufacturers recommended dose in 100 µl via the oculonasal route according to the following schedule; ibv at 1 doa, ndv at 2 weeks of age (woa), ibv at 4 woa, iltv at 8 woa, ndv at 12 woa, and iltv at 16 woa. in addition, a control group was not vaccinated. homologous challenges were conducted at 20, 24, 28, 32, and 36 woa, and necropsies were performed five days post-challenge (dpc). at challenge, birds received one of four treatments: ibv ga98, ndv b1, iltv 63140, or no challenge. the treatment groups for each challenge virus per time point were as follows: non-vaccinated, non-challenged (n = 9-10); vaccinated, non-challenged (n = 9-10); vaccinated, challenged (n = 17-19); non-vaccinated, challenged (n = 9-10). all ibv-challenged birds received an eid 50 of 1 × 10 3.2 per bird in 100 µl intranasally. all ndv-challenged birds received the ndv b1 vaccine in 100 µl intranasally, reconstituted according to the manufacturer's protocol. all iltv-challenged birds received the 63140 pathogenic strain at a dose of 1 × 10 3.5 tcid 50 per bird in 100 µl split equally between eyedrop and intranasal routes. for ibv and ndv challenges, birds were observed at 5 dpc for respiratory signs, as previously described [15] : 0 = absent; 1 = mild; 2 = moderate; 3 = severe. for iltv challenges, birds were observed at 3 and 5 dpc for dyspnea, conjunctivitis, depression, and mortality, as described previously [16] . the choanal cleft (ibv-and ndv-challenged and control birds at 5 dpc) or trachea (iltv-challenged and control birds at 3 and 5 dpc) was swabbed for virus detection, and swabs were stored in pbs at −80 • c. at 28, 32, and 36 woa, 50 µl of tears was collected by adding granulated nacl to the eye. blood was collected by wing or cardiac puncture and added to a microcentrifuge tube to collect serum for antibody detection. birds were humanely euthanized, and the eyelid, harderian gland (hg), thymus, liver, spleen, cecal tonsils, and bursa of fabricius were collected and stored at −80 • c for virus detection and in 10% neutral buffered formalin. the trachea was removed, and one section was placed in 10% neutral buffered formalin, and the remaining portion of the trachea was submerged in tissue culture media for the ciliostasis test described below. the procedures were approved by the university of georgia institutional animal care and use committee (aup #: a2015 05-001-r2). the ciliostasis test was performed on harvested tracheas collected in cell culture media (dulbecco's modified eagle's medium) at 37 • c. for each trachea, five tracheal rings measuring approximately 1 mm thick were cut and represented the proximal, middle, and distal portions [17, 18] . cilia activity was observed using an inverted microscope (olympus, center valley, pa, usa). the scoring system follows: 0 = all cilia beating; 1 = 75% of cilia beating; 2 = 50% of cilia beating; 3 = 25% of cilia beating; 4 = no cilia beating as previously described [17] . the maximum possible score for each trachea is 20. each tracheal ring was scored by three individuals, and the average total score for each trachea was calculated. the ciliostasis protection score for each group was determined by the following formula: 100 − [(total of the individual scores for the group)/(the number of individuals in the group × 20) × 100], and a score ≥50 was considered protected. a section of each trachea was fixed in 10% neutral buffered formalin, processed, embedded in paraffin, and 5-µm sections were cut for hematoxylin and eosin staining. for ibv lesions, epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation were scored for each trachea. scores were determined as follows: 1 = normal, 2 = focal, 3 = multifocal, and 4 = diffuse, as described previously [19] . for ndv lesions, a descriptive analysis was performed. for iltv lesions, microscopic lesions were scored on a scale of 0-5 (normal to very severe), as described previously [20] . for ibv and ndv detection, viral rna extraction from 50 µl of the pbs from each swab was conducted using a 5× magmax-96 viral isolation kit (thermo fisher, waltham, ma, usa) on a magmax™ express-96 deep well magnetic particle processor (thermo scientific, waltham, ma, usa), according to the manufacturer's instructions. the quantitative reverse transcription polymerase chain reaction (qrt-pcr) was performed with the agpath-id tm one-step rt-pcr kit (thermo fisher, waltham, ma, usa), following the manufacturer's protocol. each 25-µl reaction mixture contained 12.5 µl of 2× rt-pcr buffer, 10 µm of each primer, 4 µm of probe, 1 µl of 25× rt-pcr enzyme mix, and 5 µl of viral rna. the qrt-pcr reactions were run on the applied biosystems ® 7500 fast realtime pcr system (life technologies ltd., carlsbad, ca, usa) under the following conditions: one cycle of 50 • c for 30 min and 95 • c for 15 min, followed by 40 cycles of 94 • c for 1 s and 60 • c for 60 s. the primers and probe for the ibv qrt-pcr were previously published [21] , and are comprised of a forward primer ibv5 gu391 (5 -gct ttt gag cct agc gtt-3 ), a reverse primer ibv5 gl533 (5 -gcc atg ttg tca ctg tct att g-3 ), and a taqman ® dual-labeled probe ibv5 g probe (5 -fam-cac cac cag aac ctg tca cct c-bhq1-3 ). primers and probe for the ndv qrt-pcr were previously described [22] and are comprised of a forward primer ndv m+4100 (5'-agt gat gtg ctc gga cct tc-3'), a reverse primer ndv m-4220 (5'-cct gag gag agg cat ttg cta-3'), and a taqman ® dual-labeled probe ndv m+4169 (5'-fam-ttc tct agc agt ggg aca gcc tgc-bhq1-3'). the primers were obtained from integrated dna technologies (coralville, ia, usa), and the taqman probe was synthesized by biosearch technologies (novato, ca, usa). real-time rt-pcr components and thermocycler parameters were previously described [21] . the data are expressed as the average cycle threshold (ct) value for all samples in each group, with positive ct values based on the limit of detection for this test associated with virus detection in eggs [23] . each qrt-pcr reaction plate included a standard curve as an rna extraction control and as a positive control. ga98 ibv isolated from allantoic fluid was used as the template for the standard curve. negative controls were also included in each plate and consisted of pcr reagents with no rna. for iltv, total dna was extracted from the tracheal swabs using the megazorb ® dna extraction miniprep 96-well kit (promega, madison, wi, usa), as described previously [24] . duplex real-time pcr assay that amplifies a fragment of the ul44 viral gene in iltv and a fragment of the chicken alpha 2-collagen gene was performed, as previously described [25] . ibv-specific igg titers were detected using a commercial igg enzyme-linked immunosorbent assay (elisa) ibv antibody test kit (idexx, westbrook, me, usa). briefly, serum samples (stored at −20 • c) were diluted 1:500, and the procedure was performed according to the manufacturer's protocol. tear ibv-specific iga was detected using a commercial igg elisa ibv antibody test kit (idexx, westbrook, me, usa). briefly, tears were serially diluted two-fold in pbs and incubated in duplicate in wells overnight at 4 • c. all wash steps were performed using pbs-tween 20 (0.05% tween 20). plates were incubated at 23 • c for 2 h in monoclonal mouse anti-chicken iga-biot (1:1000, clone a-1, southern biotech, birmingham, al, usa), followed by 1hr in streptavidin-hrp (1:4000, southern biotech, birmingham, al, usa). final antibody detection steps were completed according to the manufacturer's instructions. endpoint titers were determined by reporting the lowest dilution at which the optical density (od), recorded at 650 nm wavelength, was at least three standard deviations above the mean of 12 control wells incubated with no tear samples. data from wells with a pinpoint color change due to residual substrate or air bubbles were excluded from analysis, and results were reported as log 2 of the endpoint titer. the data were analyzed using prism v.6.0 software (graphpad software, inc., la jolla, ca, usa; www.graphpad.com). for viral load data, a one-way analysis of variance (anova) with dunnet's posttest was used to compare treatment groups within each collection period. all other data were analyzed using a kruskal-wallis test with dunn's posttest to compare treatment groups within each collection period. significant differences were determined at p < 0.05. at 5 days following challenge with ibv ga98, vaccinated/challenged birds had significantly lower rna loads compared to positive controls at all collection times and in all tissue samples, with the exception of cecal tonsil at 24 woa (table 1 ). in vaccinated controls, no ibv rna was detected at all collection times except in the cecal tonsils at 20 and 24 woa and in the choanal cleft at 20 woa. ibv loads in negative controls in all tissues were below the limit of detection using the ct value of ≥36.17 as previously reported [23] , at all collection times except the 24 woa hg negative controls. clinical signs of ibv infection measured at 5 days post-challenge were significantly reduced in vaccinated/challenged birds when compared to positive controls in all weeks except 28 woa, but trends in clinical sign scores were numerically lower among vaccinated/challenged birds ( table 2) . clinical signs were absent in non-challenged negative and vaccinated control birds, and signs in vaccinated/challenged birds were not significantly different from signs in non-challenged controls. notes: sem = standard error of the mean. table 2 . clinical signs and microscopic lesions measured 5 days following ibv ga98 challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < 0.05). histopathological examination of tracheas from all groups ranged from within normal limits to focal to multifocal minimal to moderate lymphocytic tracheitis; however, moderate lymphocytic infiltration was more frequently seen in positive controls. in all weeks, the proportion of vaccinated/challenged birds with deciliation or acute tracheal necrosis was significantly reduced compared to positive controls and was not different from negative and vaccinated controls (table 2) . ciliostasis, defined as the cessation of tracheal ciliary movement, was measured at 5 dpc, and the number of birds positive for ciliostasis and ciliostasis protection scores were calculated for each group (figure 1 ). at all collection times, vaccinated/challenged birds were protected from ciliostasis (scores were >50), and positive controls were not protected (scores were <50). the non-challenged negative controls and vaccinated controls were protected (scores were >50) at all collection times. ibv-specific igg titers were measured in serum collected at 5 dpc. at all times except at 32 woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure 2 ). at 32 woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until 36 woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific igg titers were measured in serum collected at 5 dpc. at all times except at 32 woa, vaccinated birds from both non-challenged and challenged groups exhibited significantly higher titers compared to non-vaccinated birds from both non-challenged and challenged groups (figure 2 ). at 32 woa, titers in vaccinated birds, regardless of challenge status, were significantly higher compared to positive controls. titers in vaccinated/challenged birds did not significantly differ from titers in vaccinated controls until 36 woa, when vaccinated/challenged birds had significantly higher titers. compared to negative controls, vaccinated/challenged birds had significantly higher titers at all times. ibv-specific iga titers were measured in tears collected at 5 dpc at 28, 32, and 36 woa. at 28 woa, titers in vaccinated/challenged birds were significantly higher compared to titers in non-challenged negative controls (figure 3 ). at 32 woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly ibv-specific iga titers were measured in tears collected at 5 dpc at 28, 32, and 36 woa. at 28 woa, titers in vaccinated/challenged birds were significantly higher compared to titers in nonchallenged negative controls (figure 3 ). at 32 woa, vaccinated/challenged birds showed significantly lower titers compared to positive controls. in addition, the vaccinated/challenged birds had significantly higher titers than the vaccinated/unchallenged group at 28 woa. no other significant differences were detected. at 5 dpc with ndv b1, vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table 3) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥35.0 as previously reported [22] . at 5 dpc with ndv b1, vaccinated/challenged birds either had undetectable rna loads or significantly lower loads compared to positive control titers at all collection times and in all tissues sampled (table 3) . non-challenged negative controls and vaccinated controls were negative for rna virus using the ct value of ≥35.0 as previously reported [22] . table 3 . average qrt-pcr ct values for ndv rna collected from choanal cleft, harderian gland, and conjunctiva 5 days post-challenge with ndv b1 vaccine. letters (a-c) indicate significant differences among vaccine and challenge groups for each week (p < 0.05). clinical signs measured at 5 dpc were significant at 20 woa in positive controls, after which clinical signs in positive controls were no different from any of the other treatment groups (table 4 ). no significant differences in clinical signs existed between vaccinated/challenged birds and non-challenged controls at any time. in all weeks, histopathological examination of tracheas was within normal limits or revealed focal to multifocal minimal to mild lymphocytic tracheitis, and there were no group-related differences. tracheas were also evaluated for ciliostasis, and no groups exhibited ciliostasis. ndv-specific serum igg titers in vaccinated/challenged birds and vaccinated controls were significantly higher than titers in both positive and negative controls at all collection times ( figure 4 ). there was no significant difference in titers between vaccinated/challenged birds and vaccinated controls at any time. similarly, no significant difference in titers was detected between non-vaccinated/challenged and negative controls, except at 28 woa in which titers from non-vaccinated/challenged controls were significantly higher. table 4 . clinical signs measured 5 days following ndv b1 challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < 0.05). clinical signs at 5 dpc with iltv strain 63140, vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table 5 ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in nonchallenged negative and vaccinated controls, which were negative using the ct value of ≥38.0 as at 5 dpc with iltv strain 63140, vaccinated/challenged birds had significantly lower viral dna loads than positive controls at all collection times and in all tissues ( table 5 ). the dna loads in the trachea and hg were undetectable or low and did not differ significantly from dna loads in non-challenged negative and vaccinated controls, which were negative using the ct value of ≥38.0 as previously reported [26] . in the conjunctiva, dna loads in vaccinated/challenged birds were significantly higher than dna loads from both non-challenged negative and vaccinated controls, except at 28 woa in which no significant difference was detected between vaccinated/challenged birds and vaccinated controls. clinical signs and viral dna were detected in challenged birds at 3 dpc, but signs in positive controls had become more severe by 5 dpc as tracheal viral load increased (table 6 ). clinical sign scores measured at 5 dpc were significantly reduced in vaccinated/challenged birds compared to positive controls, at all collection points except 36 woa when clinical sign scores were numerically reduced. all negative controls and vaccinated controls had no clinical signs, and clinical signs in vaccinated/challenged birds were not significantly different compared to these controls, except at 36 woa when clinical sign scores were significantly higher. table 6 . clinical signs measured 5 days following iltv 63140 challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < 0.05). clinical signs tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, 3/9 and 1/10 in weeks 20 and 28, respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure 5 ). table 6 . clinical signs measured 5 days following iltv 63140 challenge. letters (a-b) indicate significant differences among vaccine and challenge groups for each week (p < 0.05). tracheal histological examination was within normal limits or revealed focal to diffuse minimal to mild lymphocytic tracheitis in tracheas from all groups. for the positive controls, 3/9 and 1/10 in weeks 20 and 28, respectively, had acute focal necrotizing tracheitis with syncytia and intranuclear inclusions ( figure 5 ). no ciliostasis was observed in the tracheas of birds from any of the groups. no ciliostasis was observed in the tracheas of birds from any of the groups. iltv-specific igg titers in serum collected 5 days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure 6 ). iltv-specific igg titers in serum collected 5 days post-challenge were significantly higher in vaccinated birds from both challenged and non-challenged groups, compared to the positive and negative controls ( figure 6 ). in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least 36 weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at 5 days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [9, 10] . although the vaccines in the present study were administered at intervals of 2 or 4 weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to 28 days post-vaccination [27] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and 19 weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [4] . fentie, et al. [28] reported that chickens vaccinated with ndv b1 shed vaccine virus 14 days post-inoculation. in the present study, we demonstrated that pullets serially administered live attenuated vaccines against ibv, ndv, and iltv were protected against homologous challenge with ibv, ndv, or iltv for at least 36 weeks, as determined by challenge virus detection, clinical signs, histopathology and ciliostasis at 5 days after challenge. additionally, our study showed that the age at vaccination and intervals between each vaccination did not interfere with the development of immunity to each virus and consequently protection against homologous challenge. we designed our vaccination protocol to represent a typical vaccination program for ibv, ndv, and iltv in commercial pullets. with the knowledge that live vaccine viruses can persist in flocks, it has been unclear, until now whether the immunity induced by a live vaccines could be compromised because of viral interference, a phenomenon in which one replicating virus blocks the infection and/or replication of another virus [9, 10] . although the vaccines in the present study were administered at intervals of 2 or 4 weeks, it is feasible that virus from a previous immunization was still present at the time of the subsequent vaccination. ibv vaccines have been detected in the respiratory tract up to 28 days post-vaccination [27] , and ibv was isolated from tracheal and cloacal swabs collected at the point of lay and 19 weeks of age in hens that had been virus-negative for several weeks following recovery from inoculation at one day of age [4] . fentie, et al. [28] reported that chickens vaccinated with ndv b1 shed vaccine virus 14 days post-inoculation. in addition, a study by hughes, et al. [29] demonstrated intermittent shedding in the trachea from iltv-immunized chickens between 7 and 14 weeks post-vaccination. few experimental studies of sequential virus infections have been published, and fewer yet have been considered in the context of poultry viral respiratory pathogens. costa-hurtado, et al. [30] demonstrated that chickens and turkeys serially infected with a mesogenic strain of ndv and highly pathogenic avian influenza virus (hpaiv) 3 days apart resulted in an initial decrease followed by a subsequent increase in replication of the second virus. in a subsequent study [11] , the same group found that low pathogenic avian influenza virus given 3 days after a lentogenic strain of ndv did show viral interference whereas viral interference was not observed when the viruses were given simultaneously. we did not measure vaccine virus replication in the present study and, therefore, could not determine whether one vaccine virus compromised the infection and replication of a subsequent vaccine virus. however, our goal was to determine if chickens sequentially vaccinated with all three viruses were protected from viral replication and clinical signs following homologous challenge. thus, regardless of viral interference, our data shows that immunity to individual vaccine viruses was not compromised following sequential administration of multiple live attenuated vaccines targeting different viral respiratory tract pathogens. the detection of ibv rna in the cecal tonsils of vaccinated/non-challenged birds at 20 and 24 woa but not at subsequent times indicates that residual vaccine virus rna remained in the cecal tonsils until at least 24 woa, following the second ibv vaccination. ibv has been isolated from the cecal tonsils at 14 weeks post-infection and is known to persist for several months in various internal organs [4, 31] . ibv rna was also detected in non-challenged negative control hg at 24 woa but was absent from all other tissues collected from negative controls, which displayed no clinical signs of ibv infection. it is not clear why we detected ibv rna in samples from the hg in negative control birds but likely represents cross-contamination during processing of the samples since ibv was not detected in any other tissue. the tracheal histopathological lesions of deciliation in positive controls were consistent with previous reports of ibv-induced histopathology [32, 33] , and were further confirmed by the presence of ciliostasis among positive controls. as expected, ibv-vaccinated/challenged birds were protected from ciliostasis. the european pharmacopoeia states that ciliostasis can be used to evaluate ibv vaccine efficacy, in which a lack of ciliostasis would indicate that the vaccine was efficacious [34] . therefore, these observations further confirm that ibv-vaccinated birds were protected from homologous challenge. the observation of robust ibv-specific serum igg titers in vaccinated birds is consistent with previous studies showing that ibv infection stimulates a humoral response in chickens [35] , but that circulating antibody titers do not correlate with resistance to infection [36] . therefore, the presence of ibv-specific serum igg titers indicates only that the bird has been exposed to vaccine or challenge virus and should not be correlated with other measures of protection. the lack of significant titers in non-vaccinated/challenged birds can be explained by the early time of collection post-challenge. orr-burks, et al. [37] found that significant changes in igg serum titer were not detected until 10 days post-inoculation. orr-burks, et al. [37] also demonstrated a lack of significance in ibv-specific iga titer in tears 5 days after both primary and secondary exposure to ibv, but iga titer was significantly higher between 6 and 16 days after primary exposure to ibv. in this study, ibv-specific iga titers measured in tears at 5 dpc did not reveal consistent trends and may be explained by the early time of collection post-infection. it was beyond the scope of this study to collect samples after 5 dpc, but the production of ibv-specific iga in tears at later time points was demonstrated in a different experiment in which tears were collected between 10 and 14 dpc. in that experiment, naïve chickens challenged with ibv showed a higher trend of specific iga titer compared to non-challenged controls [38] . the decision to use the b1 vaccine for ndv challenge was based on biosecurity regulations and the lack of appropriate biosafety level 3 facilities needed for challenge with mesogenic or velogenic strains of ndv. since we used a lentogenic strain of ndv as challenge virus in our experimental design, protection was primarily based on significantly reduced or no virus detection at 5 days after challenge, which is a measure of local immunity preventing virus infection and/or replication. presumably a bird protected from infection with a lentogenic strain of ndv would also show some level of protection from exposure to mesogenic or velogenic strains. vaccinated birds at each sampling time had significantly lower or no challenge virus rna compared to positive control groups, indicating that the vaccinated birds developed a local immune response and were indeed protected whereas the non-vaccinated positive controls were not. because lentogenic ndv only causes a mild respiratory or enteric infection [3] , it was not surprising that respiratory signs and histological changes were mild or absent despite the presence of viral rna. we observed clinical signs of ndv infection only at 20 woa in positive controls, while only a few birds showed mild clinical signs at 24 and 28 woa, and no clinical signs of disease were observed at later challenge times. this observation is consistent with existing knowledge that nd tends to be more severe in younger birds [39] . the lack of ciliostasis observed in ndv b1-infected positive controls contrasts with previous reports demonstrating that ndv caused ciliostasis in tracheal explants. butler, et al. [40] demonstrated that ndv caused ciliostasis within 2 to 6 days after infection of tracheal explants, and malo, et al. [41] reported that following vaccination of one-day-old chicks with lentogenic ndv, the peak of ciliostasis occurred at 5 and 7 dpv and waned by 13 dpv. the discrepancy between our results and the previous studies may be explained by the use of a different lentogenic ndv strain; b1 in our studies and lasota in previous reports [41] . it is well established that the lasota vaccine strain is more virulent than b1 [42] , which may explain the ciliostasis observed in lasota-vaccinated chicks or lasota-infected tracheal explants, whereas ciliostasis was not observed in our study using b1 vaccine. robust ndv-specific circulating igg antibody responses developed following ndv vaccination and stayed elevated, which was consistent with prior research indicating that antibodies may be detected for up to one year in birds immunized multiple times against ndv [39] . with the exception of 28 woa, the igg titers in non-vaccinated/challenged birds were not significantly increased compared to titers in negative controls. this observation is not surprising given that ndv-specific antibodies are not detected in the serum until 6-10 days after exposure [39] . for the iltv-challenged birds, vaccination prevented challenge virus replication in trachea and hg but did not completely block virus replication in the conjunctiva, especially at 32 and 36 woa. this may suggest a waning of the local immunity against iltv after the second vaccination at 16 woa. in addition, the proximity of the conjunctiva to the inoculation site (eyedrop and intranasal) might explain why the local immune response was not able to completely block viral replication in the conjunctiva but successfully cleared the virus before it could replicate in hg and trachea. very few positive controls (non-vaccinated/challenged) demonstrated histological evidence of ilt infection in the trachea, though all positive controls tested positive for ilt dna by pcr. this finding is not surprising in light of a report by guy, et al. [43] , which illustrated that histological detection of ilt is highly specific (98%) but poorly sensitive (42%). notably, intranuclear inclusion bodies are present only during the initial infection (1-5 days) and disappear following epithelial cell necrosis and desquamation [44] , which may explain the observation that by 5 dpc only 3/9 and 1/10 positive controls at 20 and 28 woa, respectively, had histological evidence of ilt infection despite the presence of ilt dna in the trachea. the absence of ciliostasis observed in both vaccinated and non-vaccinated, iltv-challenged birds at 5 dpc was not surprising given that butler, et al. [40] found that only some strains of iltv caused ciliostasis and did not correlate with virulence. moreover, the authors showed that ciliostasis rarely occurred before 6 days, and sometimes even 9 days, after inoculation. in addition, gerganov and surtmadzhiev [45] also demonstrated ciliostasis in iltv-infected tracheal organ cultures at 7-8 days post-infection. therefore, our results combined with previous studies indicate that measuring ciliostasis may not be a reliable marker of protection from iltv infection and that ciliostasis in iltv infection studies may need to be evaluated at later times post-inoculation. the lack of significant iltv-specific serum igg titers in non-vaccinated positive controls may be explained by the early time of collection post-challenge, as iltv-specific antibodies do not become detectable until 5-7 dpi and peak at 21 dpi [46] . however, it is worth noting that antibody titers are not correlated to protection from iltv infection [44] . igg titers were 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explants alternative methods to compare safety of live-attenuated respiratory newcastle disease vaccines in young chicks evaluation of the effect of live lasota newcastle disease virus vaccine as primary immunization on immune development in broilers rapid diagnosis of infectious laryngotracheitis using a monoclonal antibody-based immunoperoxidase procedure diseases of poultry cultivation of various avian viruses in pheasant trachea organ cultures and chick embryos studies on a serum neutralization test for the diagnosis of laryngotracheitis in chickens the authors declare no conflict of interest. key: cord-271568-qgpi2kcs authors: jackwood, m.w.; rosenbloom, r.; petteruti, m.; hilt, d.a.; mccall, a.w.; williams, s.m. title: avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo date: 2010-01-21 journal: virus res doi: 10.1016/j.virusres.2010.01.006 sha: doc_id: 271568 cord_uid: qgpi2kcs anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated qr448(a), was examined in vitro and in vivo. treatment of avian infectious bronchitis virus (ibv) with qr448(a) reduced the virus titer as measured in two laboratory host systems, vero e6 cells and embryonating eggs. the effect of qr448(a) on ibv in chickens was also investigated. administering qr448(a) to chickens at a 1:20 dilution by spray, 2 h before challenge with ibv was determined to be the most effective treatment. treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral rna in the trachea. treatment with qr448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of ibv over a 14-day period. anti-ibv activity of qr448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. in addition, qr448(a) had activity against both massachusetts and arkansas type ib viruses, indicating that it can be expected to be effective against ibv regardless of serotype. to our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus ibv. avian infectious bronchitis virus (ibv), a coronavirus, causes a highly contagious upper-respiratory tract disease in chickens. it can also be found in other species of birds including peafowl (pavo spp.) and teal (anas crecca) (cavanagh, 2005) . the virus is worldwide in distribution causing huge economic losses to the poultry industry that result from a reduction in feed conversion and weight gain, and condemnations at the processing plant (cavanagh and gelb, 2008) . losses are also due to drops in egg production and poor egg quality in layer type chickens, and for some strains of the virus that are nephropathogenic extensive mortality can occur (cavanagh and gelb, 2008) . infectious bronchitis virus, presents unique challenges regarding its control in commercial chickens. genomic diversity and the abbreviations: cpe, cytopathic effects; eid50, 50% embryo infectious dose; elisa, enzyme linked immunosorbent assay; hma, hexamethylene amiloride; ibv, infectious bronchitis virus; mhv, mouse hepatitis virus; pbs, phosphate buffered saline; rflp, restriction fragment length polymorphism; rt-pcr, reverse transcriptasepolymerase chain reaction; sars-cov, severe acute respiratory syndrome virus; spf, specific pathogen free; tcid50, 50% tissue culture infectious dose. * corresponding author. tel.: +1 706 542 5475; fax: +1 706 542 5630. e-mail address: mjackwoo@uga.edu (m.w. jackwood). ability of ibv to rapidly change have created different serotypes of the virus that do not cross protect (cavanagh and gelb, 2008) . consequently, attenuated live vaccines, used to control the disease, must be selected specifically for the serotype of ibv present in the flock. compounding this situation is the presence of serologically different variant strains for which no vaccines exist. we were interested in exploring alternative methods of controlling ibv that could potentially be effective across all serotypes. antiviral compounds have been used to target proteins involved in coronaviral replication. coronaviruses have a single stranded positive sense rna genome that is approximately 27.5 kb in length for ibv. during coronavirus replication, a 3 nested set of subgenomic viral mrnas are produced by the viral rna-dependent rna-polymerase, and four structural proteins are translated by this enveloped virus: spike, envelope, membrane and nucleocapsid (cavanagh and gelb, 2008) . two large genes, 1a and 1ab from the genomic viral rna encode the viral-replication complex. those genes are translated into polyproteins that are processed by the main protease (m pro ) as well as a papain-like protease 2 (pl2) to form the viral polymerase and associated nonstructural proteins (ziebuhr et al., 2007) . the main protease of coronaviruses appears to be the most attractive target for antiviral compounds (haagmans and osterhaus, 2006; niu et al., 2008; xue et al., 2008; yang et al., 2005 yang et al., , 2006 . inhibitors of m pro include a michael acceptor inhibitor (designated n3), chloromethyl ketones, epoxides, and ag7088 an inhibitor shown to be effective against the rhinovirus m pro homolog (anand et al., 2003; niu et al., 2008; xue et al., 2008) . other antiviral compounds like hexamethylene amiloride (hma) target ion channel conductance, and have been shown to have an antiviral effect against human coronavirus (hcov)-229e and mouse hepatitis virus (mhv) coronavirus in vitro (wilson et al., 2006) . in addition, specific inhibition of ibv has been reported in vitro using lithium chloride (harrison et al., 2007) . however, lithium chloride is a toxin and its usefulness as an antiviral against ibv in vivo remains to be determined. a major problem surrounding antiviral compounds targeting specific viral genes or proteins is the ability of the virus to rapidly mutate during replication to become resistant. some examples include acyclovir resistant human immunodeficiency viruses and herpes simplex viruses (mcmahon et al., 2008) , oseltamivir resistant influenza viruses (collins et al., 2008) , and nucleoside/nucleotide analog resistant hepatitis b viruses (delaney and borroto-esoda, 2008) . alternatively, compounds that have a virucidal effect work like a disinfectant, and do not require replication to inactivate the virus (reichling et al., 2005; schuhmacher et al., 2003) . thus, resistance to virucidal compounds due to mutations generated in the viral genome during replication is unlikely (schnitzler et al., 2007) . virucidal activity of essential oils from botanicals has been reported for a number of viruses including herpes simplex virus, dengue virus, and junin virus (duschatzky et al., 2005; koch et al., 2008; schnitzler et al., 2007) . virucidal activity of essential oils, which are lipophilic by nature, is probably due to disruption of the viral membrane or interference with viral envelope proteins involved in host cell attachment. although selection of resistant mutants is possible, it has been reported that inactivation of virus by lipophilic essential oils is time dependent and that infectious virus remaining after treatment are still sensitive to the essential oils making selection of resistant mutants unlikely (schnitzler et al., 2007) . in this study we examined the effect of a natural based product consisting of a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion designated qr448(a) against ibv in vitro and in vivo. to our knowledge this is the first report on the virucidal activity of botanical extracts against a coronavirus in vivo. the data are important because these types of compounds might also be useful to control other coronaviruses like the severe acute respiratory syndrome coronavirus (sars-cov) as well as other enveloped viruses like avian influenza virus and newcastle disease virus. strains of ibv used in this study are; the vero cell adapted beaudette strain (american type culture collection (atcc, rockville, md) vr-22), the pathogenic massachusetts 41 (mass41) strain (atcc, vr-21), and the arkansas dpi (arkdpi) vaccine strain (intervet, schering-plough animal health, millsboro de). the beaudette strain of ibv was propagated in vero e6 cells (crl-1586), and the mass41 and arkdpi strains of ibv were propagated by injecting the chorioallantoic sac of specific pathogen free (spf) 10day-old embryonating chicken eggs (sunrise farms inc., catskill, ny) using standard methods (gelb and jackwood, 2008) . the vero e6 cells were obtained from the american type culture collection (rockdale, md) and propagated in minimum essential medium (eagle) with 2 mm l-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mm non-essential amino acids, 1.0 mm sodium pyruvate, and 10% fetal bovine serum (invitrogen corp [gibco] , carlsbad, ca) at 37 • c and 5% co 2 . 2.2. rna extraction and real-time rt-pcr for ibv viral rna was extracted with the magmax-96 rna isolation kit (ambion inc., austin, tx) according to the manufacturer's protocol, using a kingfisher magnetic particle processor (thermo scientific, waltham, ma). real-time rt-pcr was conducted using a smart cycler ii (cepheid, sunnyvale, ca) and the agpath-id tm one-step rt-pcr kit (ambion inc.) according to the manufacturer's recommendations. the real-time rt-pcr primers and probe were previously published (callison et al., 2006) and consist of a forward primer ibv5 gu391 (5 -gct ttt gag cct agc gtt-3 ), a reverse primer ibv5 gl533 (5 -gcc atg ttg tca ctg tct att g-3 ) and a taqman ® dual-labled probe ibv5 g probe (5 -fam-cac cac cag aac ctg tca cct c-bhq1-3 ). the rt-pcr primers were synthesized by integrated dna technologies (coralville, ia), and the taqman ® probe was obtained from biosearch technologies (novato, ca). components of the real-time rt-pcr test and the thermocycler parameters were previously described, and a standard curve for the assay, was used to calculate the approximate genome copy number for each sample (callison et al., 2006) . chicks were hatched from spf fertile leghorn chicken eggs (sunrise farms, inc.) at the poultry diagnostic and research center (college of veterinary medicine, university of georgia, athens, ga). meat-type commercial broiler chickens were obtained from harrison poultry (bethlehem, ga). for each experiment, the chicks were randomly divided into different groups, and housed in positivepressure horsfal isolation units or on litter in 10 × 10 colony houses. feed and water were given ad libitum. the experiments described below were designed to determine the effect of qr448(a) obtained from quigley pharma, inc. a subsidiary of the quigley corporation (doylestown, pa), on ibv in vitro and in vivo. qr448(a) is a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion of pharmaceutical grade excipients. the exact composition of qr448(a) is available from quigley pharma, inc. toxicity of qr448(a) on vero e6 cells, and embryonating eggs was examined. then, the minimum effective dose of qr448(a) against ibv propagated in those laboratory systems was determined. the vero e6 cells were grown to 85% confluence in a t75 flask (bd biosciences, franklin lakes, nj) and transferred to two 96-well plates (bd biosciences). when the cells reached 90% confluence the media was removed and 100 l of undiluted and each of the10-fold dilutions of qr448(a) or the diluent (quigley pharma, inc.) prepared in cell culture maintenance medium (containing 1% fetal bovine serum) was added to each of 4 different wells. control wells receiving cell culture maintenance medium only were also included in the experiment. the cells were incubated for 7 days at 37 • c and 5% co 2 and examined twice daily for cytopathic effects (cpe). the experiment was repeated 3 times. specific pathogen free fertile chicken eggs were obtained from sunrise farms (catskill, ny) and incubated at 37 • c for 10 days. the embryonated eggs were inoculated in the chorioallantoic sac with 100 l of undiluted and each of the 10-fold dilutions of qr448(a) or the diluent prepared in pbs (ph 7.4). four eggs were inoculated with each dilution and the experiment was repeated 3 times. negative control eggs that received pbs only were also included. the eggs were incubated at 37 • c and candled daily for 7 days to record mortality. any mortality occurring within the first 24 h was considered to be due to trauma associated with inoculation and disregarded. on the 7th day, all the remaining eggs were chilled to 4 • c and opened to examine the embryos for clinical signs of toxicity. to test the effect of qr448(a) on ibv propagated in vero e6 cells, a constant amount of virus, 1 × 10 3 50% tissue culture infectious dose (tcid 50 ) of the beaudette strain of ibv was added to seven 10-fold serial dilutions beginning with a dilution of 1 × 10 −3 (determined to be nontoxic for the vero e6 cells) of qr448(a) or the diluent prepared in cell culture maintenance medium. the mixtures were incubated at room temperature for 30 min then nine 10-fold serial dilutions (in maintenance medium) of each mixture were prepared for inoculation onto cells. the cell culture media was removed from the cells in a 96-well tissue culture plate and the mixtures were inoculated onto the monolayers. negative control wells receiving cell culture maintenance medium only were also included in the experiment. the cells were incubated for 7 days at 37 • c and 5% co 2 and examined twice daily for cytopathic effects (cpe). the experiment was repeated 3 times. the experimental design to test the effect of qr448(a) on ibv grown in embryonating eggs was the same as above, except that the beginning concentration of qr448(a) and the diluent was undiluted since neither compound was toxic for the embryos. a constant amount of virus, 1 × 10 4 50% embryo infectious dose (eid 50 ) of the ibv beaudette strain, was mixed with undiluted and each of the 10-fold dilutions of qr448(a) or the diluent prepared in pbs. negative control eggs that received pbs only were also included. the mixtures were incubated at room temperature for 30 min then nine 10-fold serial dilutions (in pbs) of each mixture were prepared and 200 l of each dilution was inoculated into the chorioallantoic sac. four eggs were inoculated for each dilution incubated at 37 • c and candled daily for 7 days to record mortality. since mortality occurring within the first 24 h could be due to trauma associated with inoculation, they were disregarded. on the 7th day, all the remaining eggs were chilled to 4 • c and opened to examine the embryos for lesions, and the eid 50 titer was calculated by the reed and muench method (villegas, 2008) . the experiment was repeated 2 times. two-week-old spf leghorn chickens were divided into 11 groups of 10 birds each and housed in horsfal isolation units. the undiluted qr448(a) compound was given intranasally (200 l per bird), by spray (1 ml per bird), or in the drinking water (1 ml mixed with an equal volume of reconstituted non-fat dry milk per bird after 1 h of water starvation to promote drinking). the spray was delivered with a preval ® sprayer (precision valve corp., yonkers, ny) while blocking the fresh air delivery to the isolator. the preval ® spray applicator consists of a 475 ml refillable reservoir and a compressed air power unit. although not directly measured, the average particle size for this type of sprayer is in the range that would be deposited in the naso-oro-pharyngo-laryngeal region of the respiratory tract (phalen and mendez, 2009 ). the spray treatment lasted approximately 10 s and fresh air was resumed 10 min following treatment. for each of the delivery methods, qr448(a) was administered to the birds at either 6 h before challenge, 2 h before challenge, or 2 h after challenge. two non-treated groups of birds served as a challenged control and a non-challenged negative control. all of the treated birds and one of the non-treated groups were challenged with 1 × 10 4 eid 50 of the pathogenic mass41 strain of ibv per bird. the challenge virus was given intranasally and the birds were examined twice daily for clinical signs of the disease. at 5 days post-challenge the birds were killed and necropsied. at necropsy, tracheal swabs were collected and placed in ice cold pbs. the presence of virus was determined by extracting rna directly from the pbs, and quantitative real-time rt-pcr was conducted on the rna. clinical signs and gross lesions were recorded and tracheas were harvested for histopathology. clinical signs were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. the tracheas were collected in 10% neutral buffered formalin, routinely processed into paraffin, and 5-m sections were cut for hematoxylin and eosin staining. epithelial hyperplasia, lymphocyte infiltration, and the severity of epithelial deciliation were scored for each trachea with 1 being normal and 4 being severe. for this experiment, 2-week-old spf leghorn chickens were divided into 20 groups of 10 birds each and housed in horsfal isolation units. half of the birds were treated 2 h before challenge and the other half were treated 2 h after challenge. the birds were treated with either undiluted or a 2-fold serial dilution of qr448(a) starting at a 1:5 dilution to a dilution of 1:1280. dilutions were prepared in sterile distilled deionized water, and 1 ml/bird was delivered via spray with a preval ® sprayer (precision valve corp.) as described in experiment 2. additional control groups of 10 birds each included in the experiment were birds that were not treated and challenged (challenge control group), birds treated by the spray route with undiluted qr448(a) and not challenged, and birds that were not treated and not challenged (negative control group). all of the treated birds unless otherwise indicated above and one of the non-treated groups were challenged with 1 × 10 4 eid 50 of the pathogenic mass41 strain of ibv per bird as described in experiment 2. all of the birds were examined for clinical signs, and necropsied 5 days post-challenge as described for experiment 2. clinical signs were scored as described above. for this experiment 290 1-day-old commercial broiler chickens that were not vaccinated, were divided into different groups and housed in horsfal isolation units. serum was collected from several birds at the beginning of the experiment to determine the level of maternal antibodies to ibv. antibodies were detected using a commercial enzyme linked immunosorbent assay (elisa) test (idexx, inc., westbrook, me). the birds were treated with a 1:20 dilution of qr448(a) in sterile distilled deionized water by spray with a preval ® sprayer (precision valve corp.) then challenged with 1 × 10 3.5 eid 50 of the mass41 strain of ibv at 2 h, 6 h, 1 day, 2 days, 4 days and 7 days after treatment. the birds were challenged intranasally as described in experiment 2. controls included birds that were not treated and challenged at those times, and a negative group that was not treated or challenged. birds challenged at 2, 4, 6, and 12 h were necropsied on the same day, 5 days after challenge, along with one non-treated/nonchallenged control group. birds challenged at 1, 2, 4, and 7 days after treatment were necropsied along with a negative (non-challenged) control group at 5 days after each of those challenge times. all of the birds were scored for clinical signs, and necropsied as described above. in this experiment, 240 commercial broiler chickens that were not vaccinated, were equally divided and housed in two 10 × 10 colony houses on pine shavings on the floor. colony houses were used so an adequate number of birds could be maintained together for the transmission experiment and to mimic as closely as possible commercial chicken house conditions. at 2 weeks of age 20 birds from each house were removed and wing-banded (for future identification) and given a full dose of modified live ibv arkansas type vaccine in the eye according to the manufacturers recommendations (intervet, schering-plough animal health, millsburo, de). the birds were held in isolation for 2 h before reintroducing them back into the colony houses. immediately after reintroducing the birds into the colony houses, all of the birds in one house were treated by spray using a preval ® sprayer (precision valve corp.) with 1 ml of a 1:20 dilution (in sterile deionized-distilled water) of qr448(a) per bird. we monitored the transmission of the virus to contact exposed birds by removing and swabbing the trachea of 25 birds at 3, 7, 10 and 14 days posttreatment. we also removed and swabbed 5 birds given the vaccine at each sample time. birds that were swabbed were removed from the experiment to avoid promoting the spread of the virus by inducing damage to the trachea and to prevent swabbing the same bird twice during the experiment. virus detection in the tracheal swabs was determined by real-time rt-pcr. serum was collected from 10 birds at the beginning of the experiment (2 weeks of age) to determine the level of maternal antibodies to ibv. in addition, we collected sera from all of the birds (n = 12) necropsied on the last day of the experiment and tested them for antibodies to ibv, newcastle disease virus, and infectious bursal disease virus. antibodies were tested using a commercial elisa test (idexx, inc.). virus titers were calculated by the method of reed and muench (villegas, 2008) . means and standard deviations were calculated for ct values and analyzed statistically with student's t-test, and the histopathological scores were analyzed by the kruskal-wallis test coupled with dunn's post-test, and transmission experiment data was analyzed with fisher's exact test using jmp statistical discovery software (sas institute, inc., cary, nc). 3.1. experiment 1: toxicity of qr448(a) in cell culture and in embryonating eggs, and effect on ibv in vitro undiluted and a 1 × 10 −1 dilution of qr448(a) was toxic to vero e6 cells and cells inoculated with a 1 × 10 −2 dilution of qr448(a) had cpe affecting about 50% of the monolayer. cells inoculated with a 1 × 10 −3 dilution or higher of qr448(a) were unaffected at 7 days post-treatment. the vero e6 cells inoculated with undiluted and a 1 × 10 −1 dilution of the diluent had cpe in >90% of the cells, whereas cells inoculated with higher dilutions of diluent did not have any cpe following 7 days of incubation. none of the negative control cells had cpe. disregarding embryo deaths within 24 h of inoculation, none of the embryonating eggs died during the course of the experiment. at 7 days post-treatment, none of the embryos that received qr448(a) or the embryos that received diluent had gross lesions. as expected, none of the negative control embryos had lesions. to determine the decrease in titer of infectious virus, we examined the effect of qr448(a) on ibv (beaudette strain) as determined by titration in vero e6 cells and the data are presented in fig. 1 . treatment with qr448(a) at a 1 × 10 −3 dilution reduced the titer of ibv from an average of 1 × 10 3.0 to 1 × 10 0.8 tcid 50 /ml. treatment at a dilution of 1 × 10 −4 reduced the ibv titer from 1 × 10 3.0 to 5 × 10 2.0 tcid 50 /ml. none of the dilutions of qr448(a) higher than 1 × 10 −4 reduced the titer of ibv. no reduction in virus titer was observed for the diluent at any dilution and, none of the cells in the negative control wells (cell culture maintenance medium alone) had cpe (data not shown). data showing the effect of qr448(a) on ibv (beaudette strain) as determined by titration in embryonating eggs also measured the decrease of infectious virus and the data are presented in fig. 1 . titration of ibv beaudette strain in vero e6 cells following treatment with 10-fold serial dilutions of qr448(a) starting at 1 × 10 −3 to 1 × 10 −9 in cell culture maintenance medium and cell culture maintenance medium plus ibv alone (control) are presented. the 50% tissue culture infectious dose (tcid50) titers reflect the average of three replicates. no reduction in virus titer was observed for ibv treated with the diluent (data not shown). fig. 2 . treatment with undiluted qr448(a) reduced the titer of ibv from 1 × 10 4 to 1 × 10 2 eid 50 /ml. dilutions of qr448(a) at 1 × 10 −1 and 1 × 10 −2 reduced the titer of ibv from 1 × 10 4 to 1 × 10 1.7 and 1 × 10 3 eid 50 /ml respectively. no reduction in ibv titer was observed when dilutions of qr448(a) from 1 × 10 −3 to 1 × 10 −7 were used or when the diluent at any dilution was used. the lesions in the embryos, were typical of those reported for ibv (gelb and jackwood, 2008) . clinical signs were observed in all of the mass41 virus challenged groups of birds regardless of treatment but in the intranasal and spray treated groups, fewer birds had signs and the signs were milder, as reflected by lower average scores (table 1) . virus was detected in the birds treated by the intranasal route 6 h before challenge but no virus was detected in the other intranasal treatment fig. 2 . titration of ibv beaudette strain in embryonating eggs following treatment with 10-fold serial dilutions of qr448(a) starting at 0 (no dilution) to 1 × 10 −7 in pbs, and pbs plus ibv (control) are presented. the 50% embryo infectious dose (eid50) titers reflect the average of two replicates. no reduction in virus titer was observed for ibv treated with the diluent (data not shown). clinical signs and virus detection in 2-week-old spf chickens following treatment with qr448(a) before or after ibv challenge a . water 6 h before challenge 10/10 (3.5) 9/10 23.13 3.50 a water 2 h before challenge 10/10 (3.2) 10/10 23.31 3.40 a water 2 h after challenge 10/10 (3.5) 10/10 24.72 3.39 a challenge control g 10/10 (4.0) 10/10 26.01 3.52 a negative control h 0/10 (1.0) 0/10 neg 1.06 b a the birds were intranasally challenged with 3.1 × 10 4 embryo infectious dose50/bird of pathogenic ibv strain mass41. b clinical signs were recorded 5 days following challenge and were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. c virus was detected directly from tracheal swabs collected 5 days following challenge by real-time rt-pcr. d the average cycle threshold (ct) value for only the positive samples was calculated and indicates the relative amount of virus detected in the trachea (higher numbers = less virus). e the average histopathology score for only the birds with clinical signs was based on epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation with 1 being normal and 4 being severe. numbers with different superscripts are statistically different (kruskal-wallis test, p ≤ 0.05). f negative. g challenge control birds were not treated and were challenged. h negative control birds were not treated or challenged. groups or in the spray treated groups. clinical signs were observed in all of the birds treated with qr448(a) in the water, and the clinical sign scores were higher than the intranasal and spray treated birds. virus was detected in all but one bird (6 h before challenge group, see table 1 ) receiving qr448(a) in the water. severe clinical signs were observed and virus was detected in all of the ibv challenged birds that did not receive treatment. in addition, all of the negative control birds remained negative for clinical signs and virus detection. tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and the birds that did not receive challenge virus had an average lesion score of 1.06. histopathology scores for all of the treated and challenged birds that had clinical signs were statistically different from the non-challenged controls but not from the challenged controls (p ≤ 0.05). birds that did not have clinical signs regardless of the group were not statistically different from the negative control birds (data not shown). a dose response was observed in the birds treated 2 h before mass41 virus challenge (fig. 3) . a less pronounced response was observed in birds treated 2 h after virus challenge. virus was detected in the trachea of all of the challenged birds, but treatment with the lower dilutions of qr448(a) 2 h before challenge, had a more pronounced effect on lowering the amount of virus detected in the trachea. in general, the clinical signs were more severe and involved more birds at the higher dilutions or qr448(a) (see average scores, table 2 ). tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and all of the birds given undiluted qr448(a) were scored as normal (average score = 1.10). statistically lower (p ≤ 0.05) lesion scores (table 2) were observed for qr448(a) at a 1:20 dilution or lower when it was administered by spray treatment 2 h before challenge. spray treatment with qr448(a) did not statistically lower the tracheal table 2 experiment 3: dose titration of qr448(a) spray treatment in 2-week-old specific pathogen free chickens 2 h before or 2 h after challenge with pathogenic ibv. lesion scores when given 2 h after challenge. birds that were not treated and received the challenge virus had an average tracheal lesion score of 3.00. ten birds were bled at 1 day of age and as expected all of the nonvaccinated commercial broilers were positive for maternal antibodies to ibv (average titer = 2542) by elisa (idexx laboratories). the numbers of birds showing clinical signs of ibv infection and the average clinical sign scores are shown in table 3 . the number of birds with clinical signs that were treated with qr448(a) and challenged with mass41 within 4 days of treatment ranged from 0 to 50%. in birds challenged at 7 days after treatment, 90% of the birds had clinical signs. at each of the sample days, we observed 80-100% of the birds (78% at day 1) in the non-treated mass41 challenge control group with clinical signs indicating that maternal antibodies did not prevent the disease. none of the non-challenged birds had clinical signs. in general, clinical signs in the treated challenged birds consisted of watery eyes or mucus in the nares with an occasional bird having both. the signs were not unlike a vaccine reaction, and were considered to be extremely mild as evidenced by the average clinical sign scores. in comparison, the non-treated challenged birds had more severe signs consisting of watery eyes, mucus in the nares, swollen sinuses, and excessive mucus in the trachea resulting in tracheal rales. challenge virus was detected in all of the challenged birds, even in the qr448(a) treated birds (table 4 ), indicating that there was no protection from infection. although the data were not statistically different, qr448(a) treated birds that were challenged at 2 h, 6 h, 1 day, 2 days, 4 days, and 7 days had fewer viral genome copies than non-treated challenge groups at those same times respectively. no virus was detected in the tracheas of birds that were not challenged. tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and the birds that did not receive challenge virus had a normal 1.00 average score ( table 5 ). birds that were not challenged did not have any appreciable lesions in the trachea, and the tracheal lesion scores in the non-challenged birds that were treated with qr448(a) were not statistically different from the nontreated, non-challenged birds (p ≤ 0.01). the tracheal lesion scores for the treated and challenged group were statistically higher than the non-challenged birds but were not as high as the non-treated challenged birds (p ≤ 0.01). the positive control (non-treated challenged) birds all had tracheal lesion scores statistically higher than the other groups (p ≤ 0.01). no virus was detected in the birds vaccinated with arkansas type ibv at 3 and 14 days post-infection (fig. 4) . virus was detected in 1 of 5 vaccinated birds in the treated group at 7 days post-vaccination table 3 experiment 4: clinical signs a in broiler chickens challenged with ibv at various times after treatment with qr448(a) at 1 day of age. clinical signs consisted of watery eyes, tracheal rales, and mucus in the nares and the trachea. b each bird was given 1 × 10 3.5 eid50 of the mass41 strain of ibv intranasally. c number of birds with clinical signs per total (average score). clinical signs were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. average scores with different capital letter superscripts are statistically significant at p ≤ 0.01 (kruskal-wallis test). d clinical sign scores for negative control birds were all normal (1.0). birds were necropsied at 5 days post-challenge, and tracheas were collected in 10% neutral buffered formalin and routinely processed and stained (see section 2), then scored for epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation with 1 being normal and 4 being severe. scores with different capital letter superscripts are statistically significant, kruskal-wallis test (p ≤ 0.01). b birds that received challenge virus were given 1 × 10 3.5 eid50 of the mass41 strain of ibv intranasally. and in 3 of 6 and 2 of 5 vaccinated birds in the treated and non-treated groups respectively at 10 days post-infection. transmission of ibv from vaccinated to contact exposed birds was monitored by detecting the virus in the trachea using real-time rt-pcr and the percent of contact exposed birds positive for ibv is presented in fig. 4 . in the treated group, virus was not detected in the contact exposed birds at 3 and 7 days post-exposure. in the nontreated group 1 of 25 birds was positive at 7 days post-exposure. at 10 days post-exposure, 1 of 24 contact exposed birds was positive in the treated group whereas 3 of 25 birds were positive in the nontreated group. at 14 days post-exposure, 1 of 25 contact exposed and treated birds was positive for virus. this is in contrast to the non-treated group where 5 of 23 contact exposed birds were positive at 14 days post-exposure. the data were statistically different at 14 days (p ≤ 0.03) but not at any other day post-exposure. serum collected from 10 of the nonvaccinated commercial broilers at 1 day of age (these are the same birds bled in experiment 4) had an average maternal antibody titer against ibv of 2,542. antibodies to ibv were not detected by elisa (n = 12) at 2 weeks of age when the birds were vaccinated. serum was collected from all of the birds necropsied at the end of the experiment and none of the birds were positive for antibodies to ibv, none were positive for antibodies to ndv, and two birds in the treated contact group were positive for ibdv (average titer = 1038.5). the virucidal activity of essential oils, which are lipophilic by nature, likely act to disrupt the viral membrane or interfere with viral envelope proteins involved in host cell attachment (schuhmacher et al., 2003) . in this report, we examined the effect of a natural based product consisting of a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion designated qr448(a) on ibv. the minimum effective concentration against ibv was determined in vitro using the beaudette strain of ibv because it is adapted to grow in cell culture. the optimal route and timing of administration, most effective dose, and the duration of effect on ibv in chickens were investigated using the widespread and pathogenic mass41 strain of the virus. because arkansas vaccine type viruses are frequently found circulating in commercial chicken flocks (jackwood et al., 2005) , we also examined the effect on transmission of modified live arkansas type ibv vaccine in 2-week-old broiler chickens. the lowest dilution of qr448(a) that did not cause toxic effects in vero e6 cells was capable of reducing the titer of ibv from 1 × 10 3 to 1 × 10 0.8 tcid 50 /ml. undiluted qr448(a) was not toxic for 10-dayold embryonating eggs and reduced the titer of ibv from 1 × 10 4 to 1 × 10 2 eid 50 /ml. the diluent did not reduce the titer, indicating that the oleoresins and essential oils in qr448(a) were responsible for the effect. next we sought to determine if the effects of qr448(a) are biologically significant in chickens infected with ibv. both positive and negative control groups were included in the in vivo studies however; we did not include a diluent control group because, in the first experiment, the diluent was shown not to have an effect on the virus titer. based on clinical signs, lesions and virus detection, the best routes of administration were determined to be intranasal and spray inoculation 2 h before challenge. in addition, it appears treatment intranasally or by spray 2 h after challenge was somewhat better than intranasal or spray treatment at 6 h before challenge. administration of qr448(a) by water had little effect regardless of treatment timing. based on microscopic lesions, it appears that qr448(a) alone does not adversely affect the epithelium of the trachea. it is interesting that we could not detect virus in some of the groups treated intranasally or by spray in this experiment. it appears that qr448(a) reduced the titer of ibv below an infectious threshold. infection often does not occur when birds are challenged with ≤1 × 10 2.5 eid 50 of the mass41 strain of ibv (jackwood, unpublished data) . it is also possible that inhibitors to the real-time rt-pcr reaction prevented detection; however, we were able to detect challenge virus in the other groups suggesting the presence of inhibitors are inconsistent or that another explanation is likely. since spray is suitable for mass application of commercial chickens, we used that route of administration to examine the minimum effective dose, duration of activity, and effect on transmission of ibv in chickens. birds treated by spray with qr448(a) 2 h before challenge showed a clear dose response between undiluted qr448(a) and a dilution of 1:40 (table 2 and fig. 3) . treatment 2 h after challenge had a less pronounced dose response, indicating that qr448(a) is less effective following virus attachment and entry into the host cell. this data and the timing of administration data above suggests that qr448(a) activity is virucidal rather than antiviral in nature. one-day-old broiler chickens treated with qr448(a) were protected from clinical signs of disease following challenge with pathogenic ibv up to 4 days post-treatment (table 3) . although maternal antibodies in chicks, which generally last from 1 to 2 weeks of age, and can provide some protection against ibv (mondal and naqi, 2001) , we observed clinical signs in most of the nontreated virus challenged control birds indicating that maternal antibodies did not play a role in protection from clinical signs. protection from infection was not observed since challenge virus was detected in all of the qr448(a) treated and challenged birds. however, at all but two challenge times (4 and 12 h) the amount of challenge virus in the trachea was slightly lower in the qr448(a) treated groups but the values were not statistically significant. in a previous study, where different amounts of ibv were given to chickens, it was reported that the amount of virus detected in a clinical sample from trachea correlated with dose at 1 day post-challenge but by 5 days post-challenge all of the birds had similar levels of virus in the trachea regardless of challenge dose (callison et al., 2006) . treatment with qr448(a) may have lowered the amount of virus in the birds but by 5 days post-challenge when we collected samples, little or no differences in viral load were observed. examining birds at 5 days post-challenge is the usda recommended procedure (usda, aphis title 9, code of federal regulations http://www.access.gpo.gov/nara/cfr/waisidx 08/9cfrv1 08.html) for evaluating protection from ibv challenge. since challenge virus was detected in the trachea of the qr448(a) treated birds it is possible that clinical signs would have been observed in more of the treated birds if they were observed beyond 5 days post-challenge. it is also possible that fewer or no clinical signs would be observed in those birds beyond 5 days post-challenge, since a delay in clinical signs may represent additional time for the birds to mount a protective immune response to the challenge virus. protection of broiler chickens for up to 4 days post-treatment from clinical signs of disease but not from infection may provide an opportunity to induce some level of local immunity due to the presence of the challenge virus. although we did not measure local immunity in these studies, protecting birds from severe upperrespiratory disease with qr448(a) and at the same time allowing field viruses to infect and induce an immune response may provide a mechanism to specifically protect chickens from resident field virus(es) regardless of the virus type. transmission of arkansas type ibv vaccine from infected to contact control pen mates was examined and we observed 22% of the non-treated contact exposed birds positive for ibv whereas only 4% of the treated contact exposed birds were positive for virus at 14 days post-exposure. clearly qr448(a) treatment diminished transmission of ibv to contact exposed birds and the effect was statistically significant (p ≤ 0.03). to ensure that maternal antibodies did not play a role in this experiment, we vaccinated the birds at 2 weeks of age when maternal antibodies were no longer detected. previously reported studies have shown that arkansas type vaccine is the most prevalent virus in the commercial broilers and is capable of persisting in the flock (jackwood et al., 2005; mckinley et al., 2008; nix et al., 2000; van santen and toro, 2008) . vaccine virus was used in this experiment because colony houses (necessary to house the number of birds needed for the experiment) are not biologically secure, preventing us from using pathogenic isolates of ibv. although this experiment was designed to be a model for the prevention of spread of pathogenic ibv isolates, the data directly address an extremely important problem of arkansas vaccine virus persistence in commercial broiler flocks. persistence of ibv in a flock can cause a severe vaccine reaction and provides opportunity for the vaccine virus to mutate and revert to pathogenicity as well as change antigenically avoiding the immune response of the bird. a reduction in transmission afforded by treatment with qr448(a) is extremely important since it will limit ibv transmission in the flock, and likely lessen or even prevent persistence of arkansas vaccine virus in flocks. since qr448(a) is effective against the pathogenic mass41 strain of ibv it is expected that it will also limit transmission of pathogenic field strains of ibv in commercial broilers and thereby decrease the severity of the disease. this is the first report on the use of a mixture of all natural ingredients, specifically botanical oleoresins and essential oils, as a control measure for ibv. mass delivery of qr448(a) by spray is compatible with application in a commercial setting and it is effective against both mass and ark type ib viruses, indicating that it ought to be effective against ibv regardless of serotype. the effect appears to be more pronounced on cell free virus indicating that the activity is likely virucidal, which is important because it may also be effective against other enveloped respiratory viruses like avian influenza virus and newcastle disease virus in commercial poultry, as well as other coronaviruses in animals and humans. in addition, since virucidal compounds work by inactivating viruses, rather than targeting specific virus genes or proteins, it reduces the likelihood that resistance will develop from mutations during virus replication. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronaviruses in poultry and other birds infectious bronchitis crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants therapy of chronic hepatitis b: trends and developments evaluation of chemical and antiviral properties of essential oils from south american plants a laboratory manual for the isolation, identification, and characterization of avian pathogens 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overlapping specificities but distinct functions in viral replication key: cord-296831-wdpatr2z authors: matoo, javaid jeelani; bashir, khalid; kumar, ajay; krishnaswamy, narayanan; dey, sohini; chellappa, madhan mohan; ramakrishnan, saravanan title: resiquimod enhances mucosal and systemic immunity against avian infectious bronchitis virus vaccine in the chicken date: 2018-04-07 journal: microb pathog doi: 10.1016/j.micpath.2018.04.012 sha: doc_id: 296831 cord_uid: wdpatr2z adjuvant enhancing mucosal immune response is preferred in controlling many pathogens at the portal of entry. earlier, we reported that a toll-like-receptor 7 (tlr7) agonist, resiquimod (r-848), stimulated the systemic immunity when adjuvanted with the inactivated newcastle disease virus vaccine in the chicken. here, we report the effect of r-848 when adjuvanted with live or inactivated avian infectious bronchitis virus (ibv) vaccines with special emphasis on mucosal immunity. specific pathogen free (spf) chicks (n = 60) were equally divided into six groups at two weeks of age and immunized with either inactivated or live ibv vaccine adjuvanted with or without r-848. groups that received either pbs or r-848 served as control. a booster was given on 14 days post-immunization (dpi). r-848 enhanced the antigen specific humoral and cellular immune responses when co-administered with the vaccines as evidenced by an increase in the antibody titre in elisa and stimulation index in lymphocyte transformation test (ltt) till 35 dpi and increased proportion of cd4(+) and cd8(+) t cells on 21 dpi in the flow cytometry. interestingly, it potentiated the iga responses in the tear and intestinal secretions when used with both live and inactivated ibv vaccines. the combination of ibv vaccine with r-848 significantly up-regulated the transforming growth factor beta 4 (tgfβ4) transcripts in the peripheral blood mononuclear cells (pbmcs) than that of the respective vaccine per se. an enhanced secretory iga response is likely due to the up-regulation of tgfβ4, which is responsible for class switching to iga. in conclusion, co-administration of r-848 with inactivated or live ibv vaccine enhanced the systemic as well as mucosal immune responses in the chicken. avian infectious bronchitis (ib) is an acute, highly contagious disease of all age groups of chicken and affects primarily the respiratory system with possible infection of renal and reproductive systems [1, 2] . it is a disease of global importance and listed in the world organization for animal health (oie) diseases. infectious bronchitis virus (ibv), the causative agent of ib, belongs to the genus coronavirus, family coronoviridae, of order nidovirales [3] . vaccination with live attenuated and inactivated vaccines is the mainstay tool to control the ib [4] . as ibv enters the host through the mucosal surfaces and replicates in the epithelial cells, vaccines inducing mucosal immunity can prevent the entry of the pathogen before establishment of infection [5] . studies indicate that local administration of attenuated ibv vaccines is effective as compared to systemic administration implying that respiratory mucosal immunity is essential for protection [6] [7] [8] . in fact, an increase in the lachrymal-fluid iga levels in chickens increases the resistance against ibv infection [9] and re-infection [10] . further, in the inbred chicken lines, resistance to ibv was correlated with higher iga levels in the tear and saliva [11] . limited number of adjuvants is available with the capacity to enhance antigen specific mucosal immunity. conventional mucosal adjuvants like heat-labile enterotoxin (lt) from e. coli and cholera toxin (ct) from vibrio cholerae are toxic to the host [12] . owing to the immunostimulatory capacity, toll-like receptor (tlr) agonists are explored as an alternate and promising source for future adjuvant systems [13] . [14, 15] . emerging evidence supports the notion that the tlr agonists increase the secretory iga (siga) when used with the vaccine antigens [16] [17] [18] [19] . lps, a tlr4 agonist, when used with inactivated newcastle disease virus (ndv) vaccine significantly increased the mucosal and humoral immune responses [20] . flagellin [21] and combination of cpg and poly i:c [22] increased the siga titres along with systemic immune responses in chicken with avian influenza virus vaccine. resiquimod (r-848) (tlr7/8 agonist) and gardiquimod (tlr7 agonist) enhanced siga response besides cellular and humoral responses when used with virus like particles (vlps) based norwalk virus vaccine in the mice [17] . the response seen is equivalent to that seen when cholera toxin is used as adjuvant with the same antigen. co-delivery of norwalk vlps with gardiquimod (tlr7 agonist) or cpg odn (tlr9 agonist) produced strong systemic as well as mucosal immune responses in the mice [23] . recently, we reported the adjuvant potential of r-848 in the chicken when used with inactivated ndv vaccine [24] . however, the effect of r-848 on the mucosal immune response is not explored in the chicken hitherto. accordingly, we investigated the systemic as well as mucosal immune responses of live or inactivated ibv vaccine in the chicken when adjuvanted with r-848. specific pathogen free (spf) embryonated eggs were procured from venky's india private limited, pune, india and hatched at central avian research institute, izatnagar. birds were maintained following standard management practices and provided ad libitum sterile feed and water. the experiment was approved by the institute animal ethics committee. tlr7 agonist (resiquimod; r-848) was sourced from invivogen, california, usa. both live and inactivated massachusetts strain of ibv vaccines were purchased from the commercial sources. two week old spf chicks were immunized with live or inactivated ibv vaccines in the presence or absence of r-848 (table 1) with a booster given on 14 days post-immunization (dpi). ibv specific antibody levels were evaluated in the serum at weekly interval. levels of siga were checked in the tear and intestinal secretion of the experimental birds at weekly intervals after booster dose. cellular immune response was evaluated by lymphocyte transformation test (ltt) on 14, 21, 28 and 35 dpi and by flow cytometry on 21 dpi. expression of tgf β4 transcripts in the peripheral blood mononuclear cells (pbmcs) of the experimental birds was quantified at 24 and 48 h post-booster dose by quantitative real time polymerase chain reaction (qpcr). the qpcr analysis of tgf β4 transcripts was also done in the spleen samples on 21 dpi. birds of two week age were randomly divided into six groups (n = 10/group) and immunized withlive or killed ibv vaccine (table 1) . a booster was given at the same dose and route on 14 dpi and r-848 (50μg/bird) was given through intra-muscular route (i.m.). blood samples (n = 6/group on 7, 14, 21, 28 dpi and n = 3/group on 35 dpi) were collected to separate sera that was stored at −20°c for the detection of ibv specific antibody levels on 7, 14, 21, 28 and 35 dpi. tear samples (n = 6/group on 21, 28 dpi and n = 3/group on 35 dpi) were collected from the experimental birds as reported elsewhere [25] on 21, 28 and 35 dpi. briefly, a pinch of molecular grade sodium chloride crystals was sprinkled on either eye that induced lachrymation within 35-45 s. the tear was aspirated with a micropipette and stored in microcentrifuge tube at −20°c for iga assay. birds (n = 3/group) were humanely sacrificed on 21, 28 and 35 dpi and spleen, intestinal secretions were collected as reported earlier [26] . briefly, around 5 cm long duodenum, jejunum and ileum were collected from each bird and pooled; the parietal surface was washed with pbs. longitudinal strip of intestine was prepared and placed in a graduated tube to which equal volume of pbs (v/v) containing ethylene diamine tetra-acetate (edta) 50 mm, trypsin inhibitor 100 μg/ml and phenyl methane sulfonyl fluoride (pmsf) 0.35 mg/ml was added. the samples were vortexed briefly for 3-5 min before centrifugation at 8000 × g for 20 min at 4°c. the clear supernatant was aspirated and kept at −20°c until further use. the pbmcs were isolated from the experimental birds (n = 6/ group) at 24 and 48 h post-booster dose using ficoll hypaque (sigma, mo, usa) (1.077 g/ml) density gradient centrifugation as per the published protocol [27] . the ibv specific antibody in the serum (n = 6/group on 7, 14, 21, 28 dpi; n = 3/group on 35 dpi) was quantified using commercial ibv antibody test kit (idexx laboratories, usa) following the manufacturer's instructions. an antibody titre of > 396 or 2.598 log 10 was considered positive for ibv. the levels of iga both in the tear (n = 6/group on 21, 28 dpi; n = 3/group on 35 dpi) and intestinal secretion (n = 3/group) were measured using the commercial ibv antibody test kit (idexx laboratories, usa) with certain modifications using anti-chicken iga-hrpo (bethyl, usa) as the secondary antibody. cellular immune response in the experimental birds was assessed by lymphocyte transformation test (ltt) and flow cytometry as reported earlier [24] . blood was collected in a heparinized vial containing 20 iu/ml on 14, 21, 28 and 35 dpi for ltt. the pbmcs (n = 6/group on 14, 21, 28 dpi and n = 3/group on 35 dpi) were suspended in rpmi 1640 complete medium containing 10% fetal bovine serum (fbs) and 100 iu/ml penicillin, and 50 μg/ml streptomycin. cell viability was determined by trypan blue dye exclusion method. cell suspension was adjusted to 1 × 10 7 cells/ml and 100 μl of cell suspension was plated in each well of 96 well cell culture plate. rpmi 1640 medium (100 μl) with or without cona (20 μg/ml), ibv antigen (10 μg/well) was added to the wells in triplicate. the plates were incubated at 37°c, 5% co 2 for 72 h in a humidified chamber. at the end of incubation, mtt [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide; sigma, usa] 20 μl was added from the stock (5 mg/ml). the plates were reincubated at the same condition for another 4 h. culture supernatant table 1 immunization plan followed in the spf chicken. vaccine preparation route (100 μl) was discarded from each well and the formazan crystals were dissolved by adding dimethyl sulfoxide (amresco, usa) 100 μl to each well and optical density (od) was taken at 570 nm in a microplate elisa reader. blastogenic response was calculated by dividing the mean od of the stimulated well by the mean od of unstimulated well and expressed as stimulation index (si). flow cytometry analysis. the proportion of cd4 + and cd8 + t cell subsets in the pbmcs (n = 6/group) was measured on 21 dpi by flow cytometry. for analysis, 2 × 10 5 cells were stained with antichicken cd4/cd8 r-pe and cd3-fitc labeled monoclonal antibodies (abcam, usa) and kept overnight at 4°c in the dark. subsequently, the cells were washed with pbs containing 2% fbs and were not fixed. the aliquots of 1 × 10 4 cells were analyzed per sample by bd facs tm calibur instrument (bd biosciences, uk). the unstained cells served as the negative control. the pbmcs (1 × 10 6 ) (n = 6/group) were collected from the experimental birds as mentioned above at 24 and 48 h post-booster and one ml of ribozol™ (amresco, usa) was added. similarly, one ml of ribozol™ was added to spleen tissue collected following sacrifice (n = 3/group). total rna extraction was done by phenol : chloroform and isopropanol method and the purity was checked by absorbance at 260 and 280 nm in a nanodrop uv spectrophotometer. total rna was used for the preparation of cdna employing revertaid™ first strand cdna synthesis kit (thermo scientific, usa), following manufacturer's instructions. quantification of the tgf-β4 gene was done by quantitect sybr green qpcr kit (qiagen, ca, usa) on cfx96 real time system (bio-rad, ca, usa) following the published report [28] . β-actin served as the housekeeping gene. published primer sequences were used for βactin (f: 5′ tatgtgcaaggccggttt 3′, r: 5′ tgtctttctggcccat accaa 3′) [29] and tgf-β4 (f: 5′ cggccgacgatgagtggctc 3′, r: 5′ cggggcccatctcacaggga 3′) [30] genes. each sample was tested in triplicate on the same plate. expression of tgf-β4 was calculated relative to the β-actin gene and expressed as n-fold increase or decrease relative to the control. the data of real time pcr was calculated by 2 −δδct method [31] . each experiment was repeated twice independently and data from the first experiment was used for analysis. the treatment effect at each time point was assessed by one way analysis of variance (anova) with duncan's multiple range test as post hoc test to find the significance of pair-wise mean difference. the minimum level of significance was set at 95%. results are presented as mean ± se. statistical software spss™ 20.0 (ibm corp., usa) was used for analysis while graphpad prism version 5.0 was used for generating the graph. effect of r-848 on vaccination induced ibv antibody titre in the sera is presented in fig. 1 . the antibody titre in the pbs control and r-848 groups was consistently negative for ibv. there was no significant (p > 0.05) difference in the antibody response between the vaccinated and control groups on 7 dpi. only live ibv vaccine with or without r-848 induced significantly higher antibody response than that of the control group on 14 dpi. the combination of vaccine plus r-848 showed significantly higher (p < 0.05) antibody titre than that of the respective vaccine alone group after secondary immunization consistently (fig. 1) . effect of r-848 on vaccination induced iga response in the tear and intestinal secretion is presented in figs. 2 and 3 , respectively. vaccine, either live or inactivated, induced a significantly higher iga response than that of the control group after secondary vaccination (p < 0.01). co-administration of r-848 with live or inactivated ibv vaccine significantly increased the iga response in the tear and intestinal secretion from 21 dpi, which was maintained till 35 dpi as compared to the vaccine alone group (p < 0.01). the peak iga response in the tear was observed in the live vaccine plus r-848 group followed by inactivated vaccine plus r-848, live vaccine and inactivated vaccine groups. the iga response in the intestinal secretions was comparable with that of tear. the antigen specific lymphocyte proliferation following different treatment is depicted in fig. 4 . live as well as inactivated ibv vaccine significantly increased the si as compared to the control at each time point studied. further, co-administration of r-848 significantly potentiated the si as compared to the vaccine alone groups (p < 0.01). the si was maximum in the live vaccine plus r-848 group, which was 1.55 ± 0.022, 1.69 ± 0.021 and 1.76 ± 0.01, 1.89 ± 0.02 on 14, 21, 28 and 35 dpi, respectively (fig. 4 ). both the live and inactivated vaccines significantly increased the cd4 + and cd8 + t cells (%) as compared to the control (p < 0.05) as depicted in fig. 5 . co-administration of r-848 with either type of vaccine showed a significant increase in the cd4 + and cd8 + t cells (%) indicating a immunomodulatory role in the adaptive immunity. combination of r-848 and live vaccine showed the highest percent increase of 25.54 ± 1.54 and 17.43 ± 0.61, respectively in cd4 + and cd8 + t cells. the groups receiving vaccine along with r-848 showed significantly (p < 0.01) higher expression of tgf-β4 than that of the respective vaccine alone groups (fig. 6) . the highest tgf-β4 transcripts was observed in the live vaccine plus r-848 group, which was 4.07 ± 0.09 and 5.58 ± 0.60 folds higher than that of the control group at 24 and 48 h post-booster immunization, respectively. the response seen in the inactivated vaccine plus r-848 group was almost equal to the live vaccine alone group. expression of tgf-β4 transcripts was comparable in both spleen (data not shown) and pbmcs. resiquimod, a tlr7 agonist, is an imidazoquinoline compound with tremendous immunodulatory capacity. the antiviral activity of tlr7 agonists has been reported against genital warts, herpes genitalias and molluscum contagiosum [32] . tlr 7/8 agonists like imiquimod and r-848 are fda approved drugs for basal cell carcinoma, actinic keratosis and papilloma virus in the human [33] [34] [35] [36] . recently, we reported the enhanced antigen specific cellular as well as humoral immune responses in the chicken when resiquimod (r-848) was used with inactivated ndv vaccine resulting in complete protection against virulent ndv challenge [24] . since the adjuvant potential is likely to vary with the type of vaccine, we studied the effect of r-848 with ibv vaccine. to the best of our knowledge, this is the first report on the effect of r-848 in modulating the mucosal immune response in the chicken. humoral immune response plays an important role in ibv infection [37] . in the present study, antibody titre was highest in the spf chicken that received live vaccine with r-848 than other groups (fig. 1) . the live vaccine virus stimulates more vigorous immune response as they replicate in the host and simulate the natural infection. adjuvanted live ibv vaccine induced higher immune responses than live vaccine alone [38] , supports the concept of present study. further, the combination of r-848 withinactivated ibv vaccine showed higher antibody response than the vaccine alone group. these findings indicate the adjuvant capacity of r-848 with live as well as inactivated ibv vaccines in increasing the antibody response, which is supported by our earlier report on inactivated ndv vaccine in spf chicken [24] . in addition to antibodies, cell mediated immunity also plays an important role in immunity against ibv. transfer of lymphocytes from birds on day 10 post-ibv infection to naïve chicken completely eliminates the viral infection and clinical signs after challenge [39] and viral load was reduced in the lung by increasing ibv-specific cytotoxic t cells (ctls) in spleen [40] . in the present study, cell mediated immunity was analyzed by ltt as well as immunophenotyping. we found that spf chicken receiving live vaccine with r-848 mounted a strong antigen specific proliferation as compared to other groups (fig. 4) , which is supported by the concomitant increase in the proportion of cd4 + and cd8 + t cells (fig. 5) . similarly, an enhanced cellular immune response with inactivated ibv vaccine recorded in the present study is supported by the findings with inactivated ndv vaccine [24] . there is a need for adjuvants that increase antigen specific mucosal immune response to curtail the infection at the entry level [41] . despite the fact that the local ctls are essential for the virus clearance in early infection [42] , siga of lacrimal origin is a good indicator of protection in ibv infection [9, 43, 44] as mucosal antibody response resists reinfection [6] . r-848 enhanced the siga response in the tear (fig. 2) as well as intestinal secretions (fig. 3) when adjuvanted with live or fig. 1 . vaccination induced ibv antibody titre in the spf chickens in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with a booster 14 days later. antibody response was monitored in the serum samples by commercial elisa kit (idexx laboratories, usa) at weekly interval till 35 dpi (n = 6/ group on 14, 21, 28 dpi and n = 3/group on 35 dpi). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 5%. * indicate significant difference (p < 0.05) between the groups. $ ibv specific antibody titre > 2.598 log 10 was considered positive. dpi: day post-immunization. fig. 2 . vaccination induced ibv specific iga concentration in the tear of spf chicken in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with a booster 14 days later. iga response was monitored after booster at weekly interval till 35 dpi (n = 6/ group on dpi 21, 28 and n = 3/group on 35 dpi). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 1%. **indicate significant difference (p < 0.01) between the groups. dpi: day post-immunization. fig. 3 . vaccination induced ibv specific iga level in intestinal secretions of spf chicken in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with one more dose of the same preparations 14 days later. iga response was monitored after booster dose at weekly interval till 35 dpi (n = 3/group). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 1%. **indicate significant difference (p < 0.01) between the groups. dpi: day post-immunization. inactivated ibv vaccine. in support of the findings, it is reported that the administration of r-848 with vlps of norwalk virus enhanced the mucosal iga response in the mice [17] . to explain the impressive siga response, we studied the relative expression of tgf-β4 as it has a major role in the induction of iga class switching [45] . it is well known that tgf-β1, which is the orthologue of tgf-β4, is primarily responsible for class switching of b cells to produce iga in the mammals [46] [47] [48] [49] [50] . a significant increase in the relative copy number of tgf-β4 (fig. 6) suggests a role of r-848 in iga class switching in the groups that received live or inactivated ibv vaccine. in this study, the tgf-β4 transcripts were significantly higher in vaccine plus r-848 groups than that of the respective vaccine alone groups. activation of tlr7 by r-848 would initiate the signaling cascade through the myd88-dependent pathway, which might have resulted in the up-regulation of tgf-β4 transcripts [51] . studies by our group have shown that r-848 improves the vaccine response of ndv when adjuvanted [24] and has prophylactic potential against ibdv [29] . thus, the adjuvant effect of r-848 with ibv is needed to be tested following challenge studies. in conclusion, co-administration of r-848 with inactivated or live ibv vaccine enhanced the mucosal immunity by increasing siga which is likely mediated by tgf-β4. the potential of r-848 to enhance mucosal immunity has translational significance in poultry vaccination. fig. 4 . lymphocyte proliferation specific to ibv antigen in the pbmcs collected from the spf chickens following vaccination in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with a booster 14 days later. pbmcs (n = 6/group on 14, 21, 28 dpi and n = 3/group on 35 dpi) were collected and stimulated with ibv antigen to assess lymphocyte proliferation using mtt dye at weekly interval from dpi 14. the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 1%. **indicate significant difference (p < 0.01) between the groups. dpi: day post-immunization. 5 . cd4 + and cd8 + cells (%) in the pbmcs of spf chicken following ibv vaccination in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with a booster 14 days later. the pbmcs (n = 6/group) were collected from the birds on 21 dpi and analyzed by flow cytometry following addition of chicken specific monoclonal antibodies. the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 5%. *indicate significant difference (p < 0.05) between the groups. dpi -day post-immunization. fig. 6 . quantitative real time pcr analysis of tgf-β4 expression in the pbmcs of the spf chicken following vaccination in the presence or absence of r-848. birds of 2 week age were immunized with live or inactivated ibv vaccine in the presence or absence of r-848 (50μg/bird) with a booster 14 days later. the pbmcs (n = 6/group) were collected from the birds on 15 and 16 dpi (24 and 48 h post-booster) and the tgf-β4 transcripts were analyzed by real time pcr using β-actin as the house keeping gene and expressed as n-fold increase or decrease relative to the control following 2 −δδct method (34) . the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at 1%. **indicate significant difference (p < 0.01) between the groups. the authors declare that there is no conflict of interest with respect to the content of the manuscript. coronavirus avian infectious bronchitis virus an overview of infectious bronchitis virus in chickens virus taxonomy: classification and nomenclature of 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and tgf-β the authors thank the director, indian veterinary research institute for providing the facilities to carry out the work. key: cord-308950-bl83r4v3 authors: miguel, b.; pharr, g. t.; wang, c. title: the role of feline aminopeptidase n as a receptor for infectious bronchitis virus : brief review date: 2002 journal: arch virol doi: 10.1007/s00705-002-0888-1 sha: doc_id: 308950 cord_uid: bl83r4v3 feline aminopeptidase n (fapn) has been shown to serve as a receptor for feline, canine, porcine and human coronaviruses. our objective was to determine if fapn can serve as a receptor for infectious bronchitis virus (ibv). feline kidney cells that express fapn and hamster kidney fibroblasts that do not express fapn were inoculated with ibv and monitored for replication by indirect fluorescent assay and confocal microscopy and in chicken embryonated eggs. the results showed that the feline cells were permissive to ibv but the hamster cells were not. the hamster cells became permissive to ibv after transfection with a fapn cdna suggesting that the feline apn molecule plays a role in ibv entry. members of the family coronaviridae infect a wide range of hosts and have been classified into three groups. one major group (group i) was recently shown to use aminopeptidase n (apn) as its cell surface receptor [7, 9, 10, 11, 16, 21-23, 28, 43] . aminopeptidase n, also called cd13 in humans [31] , is a zinc metalloprotease. it is a 150-kda glycoprotein with zinc located in its globular region that is believed to be the active enzymatic site playing a major role in the cleaving of peptides [1, 2, 12, 15, 33] . apn is expressed on the plasma membranes of granulocytes [2, 36] , lymphocytes and monocytes [26, 30, 31, 35] . it is also expressed on non-hematopoetic tissue including fibroblasts [35] , synaptic membranes in the central nervous system [6] , epithelial cells from the renal proximal tubules and the intestinal brush border, and endothelial and epithelial cells of the respiratory tract [8, 12, 34, 35] . most recently apn has been isolated from chicken embryo yolk [32] and chicken intestine [14] . the feline aminopeptidase n has been shown to serve as a receptor for feline, canine, porcine and human coronaviruses in group i [38, 39] . the coronaviruses from group i cause disease only in their usual target host species. however, it has been reported that some strains of canine coronavirus (ccv) and human coronavirus 229e can infect other non-target species without causing disease [4, 5] . cats inoculated with human coronavirus 229e or ccv seroconverted and in many cases shed the virus with no signs of clinical disease [4, 5] . infectious bronchitis virus (ibv) is a member of the group iii of coronaviridae. this virus causes severe respiratory disease in chickens, and its receptor has not been identified. the objectives of this study were to: (1) determine if feline cells were permissive to arkansas 99 (ark 99) serotype of ibv, (2) evaluate if the ibv can replicate in feline cells, and (3) determine the contribution of apn to permissiveness of feline cells. a cell line derived from a normal kidney cortex of a 12-week-old female feline (felis catus) (crfk-ccl-94) was purchased from american type culture collection (manassas, va) and is henceforth designated as fek. the cells were cultured in dulbecco's modified eagle medium (dmem) (sigma chemical co., st. louis, mo) consisting of 10% fetal bovine serum (fbs), 100 iu/ml penicillin and 250 µg/ml streptomycin (sigma). the cells were incubated in 5% co 2 , 90% humidity at 37 • c until they were confluent. for the ifa assay, 4 × 10 4 cells/ml were seeded into 6-well tissue culture plates (becton dickinson labware, franklin lakes, nj) containing 18 × 18 mm coverslips. the cells were used once attachment had occurred (24 h). a cell line of normal hamster kidney fibroblasts (bhk-21) was used as control cells for the indirect immunofluorescence assay (ifa). the medium and culture conditions for bhk cells were identical to those used for fek cells. a field strain of ark 99 of ibv provided by dr. j. gelb jr. (dept. of animal and food sciences, university of delaware, newark, de) was propagated by passage in specific pathogen free (spf) 10-day-old embryonated chicken eggs (hy-vac, adel, iowa). the allantoic fluid was harvested 24 h after infection, clarified by centrifugation at low speed (1,075 × g for 15 min), and the pellet was discarded. the virus was collected from the supernatant and concentrated by overnight high-speed centrifugation (10,000 × g) at 4 • c. the supernatant was discarded and the pellet was resuspended in 1 ml of tne buffer (10 mm tris-hcl ph 7.4, 0.1 m nacl, 1 mm edta) [16] . the virus suspension was aliquoted and stored at −70 • c until used. for the calculation of 50% embryo infectious dose (eid 50 ) an aliquot was titrated in spf embryonated chicken eggs [40] . the feline aminopeptidase cdna (pbk-fapn) subcloned into the plasmid pbk-cmv vector (stratagene) was kindly provided by dr. andreas kolb, hannah research institute, ayr, scotland, uk. bhk-21 cells were transfected with pbk-fapn or pbk-cmv (control plasmid) using lipofectamine reagent (stratagene). transfected cells were seeded in 25-cm 2 flasks for 24 h, at which time the medium was changed. the cells incubated for an additional 48 h before evaluation by flow cytometry. to evaluate the permissiveness of cells to ibv, monolayers of transfected (pbk-fapn and pbk-cmv) bhk-21, non-transfected bhk-21 and fek on coverslips were infected with a 10 3.2 eid 50 of ark/ibv virus. the cells were washed, fixed in ethanol, rinsed again with pbs (ph 7.4) and incubated with a monoclonal antibody to the s1 glycoprotein of ark serotype ibv monoclonal (dr. s. a. naqi, dept. of microbiology and immunology, cornell university, ithaca, ny) at a 1:50 dilution for 45 min at 37 • c. the coverslips were washed in pbs three times for 5 min each and then incubated with a goat anti-mouse igg conjugated to fluorescein isothiocyanate (fitc) (southern biotechnologies associates inc., birmingham, al) at a dilution of 1:50 for 45 min at 37 • c. the coverslips were washed three times for 5 min in pbs, then mounted on microscope slides with mounting medium (dako corporation, carpinteria, ca). slides were viewed using epifluorescent microscopy (olympus america, inc., lake success, n.y.), and confocal microscopy (leica, heidelberg, germany). fek grown on 6-well tissue culture plates were infected with a 10 3.2 eid 50 of ark/ibv virus. the supernatant of these infected cell cultures was collected at 1, 2, 3 and 10 d post infection. the media in this last culture was collected twice, pooled and frozen at −80 • c until titrated. virus titer was calculated using the method of reed and munch described in [40] . expression of fapn on the surface of transfected bhk-21 cells was determined using a monoclonal antibody to human cd13 clone wm-47 (accurate antibodies, westbury, ny) that cross-reacts with the feline apn (personal communication with dr. k. v. holmes). bhk-21 cells were washed two times with phosphate buffered saline (pbs), incubated with anti-human cd13 or an isotype matched control, washed, and then incubated with fitc conjugated goat anti-mouse igg. an additional control included cells with the secondary antibody only. all incubations were done for 5 min on ice. after the final wash, all cells were suspended in 500 µl of pbs and analyzed by flow cytometry (calibur flow cytometry system, san jose, ca). ifa and confocal analyses of fek cells inoculated with ark/ibv demonstrated that the fek cells were susceptible to the virus. fluorescence was detected on the surface of inoculated cells and the accumulation of viral antigen in the cytoplasm, the site of replication for ibv, was detected (figs. 1a and 1e ). viral antigen was detected as soon as 1 h post infection (pi). the bhk-21 cells, which were shown to be non-permissive to ibv infection by ifa, were used as a negative control cell line (figs. 1b and if) . to determine if ibv could replicate in the fek cells, cell cultures were infected with ark/ibv and the supernatants from infected cells were collected at 1, 2, 3 and 10 d pi. the cell supernatants were titrated in chicken embryos and the eid 50 was calculated. the results indicated that ark/ibv does replicate in the fek cells in vitro. the eid 50 /ml values were 10 3.8 , 10 5.8 , 10 7.2 and 10 9.2 for the 1, 2, 3, and 10 d incubation, respectively. the pathological lesions, including dwarfed and hemorrhagic embryos with curled toes typically present in ibv infected embryos [40] were produced in spf embryonated chicken eggs infected with virus passaged in fek cells (fig. 2) . previous reports have shown fapn to be the molecule that allows entry of the coronavirus from other species into the feline cells [38, 39] . to test the hypothesis that the feline apn had a role in permissiveness of fek cells to ark/ibv, bhk-21 cells were transfected with pbk-cmv plasmid or pbk-fapn. surface expression of fapn was assayed by flow cytometry. the bhk-21 cells transfected with pbk-fapn were shown to express the fapn receptor on the cell surface (fig. 3) , and were permissive to ark/ibv (figs. 1c and 1g). viral antigen labeled with fluorescein was detected in the cell cytoplasm, and further examination of the cells with confocal microscopy also showed that the virus was intracytoplasmic and not membrane bound (fig. 1g ). however, fluorescence was not detected on the surface or in cytoplasm of bhk cells transfected with pbk-cmv (figs. 1d and 1h). this study showed that fek cells in culture were permissive to ark/ibv and the virus was capable of replicating within the cells. previous studies have shown that cats may become infected with coronavirus from other animal species and seroconvert without developing clinical signs of disease [4, 5] polymerases, and therefore may undergo recombination [3, 13, 17] in cats coinfected with different coronaviruses. the resulting recombinant viruses may have properties different from either parent, infect different hosts and have different tissue tropism, antigenicity and virulence, possibly resulting in the emergence of a new disease [38] . because feline cells can harbor coronaviruses from several species, this may explain why recombination of coronaviruses occurs somewhat frequently. cats may serve as a vehicle for the recombination event and may contribute to new ibv emerging serotypes. there have been more than 20 serotypes of ibv identified and new ibv variants continue to emerge. recombination has been experimentally proven to occur in ibv [19, 24, 25, 27, 29, 41] particularly in the hypervariable region of s1 [42] . this region of the s1 glycoprotein of ibv has also been postulated to be one of the major elicitors of the immune response [18, 20, 37] . consequently, current vaccines generally do not cross protect against the different serotypes, resulting in a serious problem in the control and prevention of this disease. bhk-21 cells are not naturally permissive to this virus but became permissive when transfected with the fapn cdna. this result indicates that the fapn plays a role in ibv entry. previous work demonstrated that fapn serves as the receptor for group i coronaviruses infecting feline cells [38, 39] . this is the first report indicating that ibv of the group iii coronaviruses also uses this receptor. while the fapn appears to play a role as a receptor molecule for the ark/ibv in feline cells, further investigation is needed to determine if the chicken apn plays a role in the entry of the ark/ibv in its natural host. role of intestinal brush border membrane aminopeptidase n in dipeptide transport metalloprotease activity of cd13/aminopeptidase n on the surface of human myeloid cells a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus experimental inoculation of cats with human coronavirus 229e and subsequent challenge with feline infectious peritonitis virus localization of aminopeptidase n and dipeptidyl peptidase iv in pig striatum and in neuronal and glial cell cultures interspecies aminopeptidase-n chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus biosynthesis of intestinal microvillar proteins. forskolin reduces surface expression of aminopeptidase aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev further characterization of aminopeptidase-n as a receptor for coronaviruses determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site aminopeptidase n is a marker for the apical pole of porcine thyroid epithelial cells in vivo and in culture evidence for variable rates of recombination 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229e receptor function of human aminopeptidase n molecular analysis of the coronavirus-receptor function of aminopeptidase n first experimental evidence of recombination in infectious bronchitis virus. recombination in ibv experimental evidence of recombination in coronavirus infectious bronchitis virus aminopeptidase n (cd13, ec 3.3.4.11.2) occurs on the surface of resting and concanavalin a-stimulated lymphocytes sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus molecular cloning and sequence comparison of the s1 glycoprotein of the gray and jmk strains of avian infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv expression of the aminopeptidase n (cd13) gene in the human t cell lines hut78 and h9 human myeloid plasma membrane glycoprotein cd13 (gp150) is identical to aminopeptidase n isolation and characterization of cdna encoding chicken egg yolk aminopeptidase ey structure and expression of aminopeptidase n an enhancer with cell-type dependent activity is located between the myeloid and epithelial aminopeptidase n (cd 13) promoters induction of aminopeptidase n/cd13 on human lymphocytes after adhesion to fibroblast-like synoviocytes, endothelial cells, epithelial cells, and monocytes/macrophages look at (1991) separate promoters control transcription of the human aminopeptidase n gene in myeloid and intestinal epithelial cells induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed in a recombinant baculovirus feline aminopeptidase n is a receptor for all group i coronaviruses feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i isolation and identification of avian pathogens, 4th edn fapn as a receptor for infectious bronchitis virus evidence of natural recombination within the s1 gene of infectious bronchitis virus experimental confirmation of recombination upstream of the s1 hypervariable region of infectious bronchitis virus human aminopeptidase n is a receptor for human coronavirus 229e we thank dr. andreas kolb key: cord-272305-eniovfwy authors: zhao, ye; cheng, jin-long; liu, xiao-yu; zhao, jing; hu, yan-xin; zhang, guo-zhong title: safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine date: 2015-10-22 journal: vet microbiol doi: 10.1016/j.vetmic.2015.07.036 sha: doc_id: 272305 cord_uid: eniovfwy infectious bronchitis (ib) is a highly contagious respiratory and urogenital disease of chickens caused by infectious bronchitis virus (ibv). this disease is of considerable economic importance and is primarily controlled through biosecurity and immunization with live attenuated and inactivated ib vaccines of various serotypes. in the present study, we tested the safety and efficacy of an attenuated predominant chinese qx-like ibv strain. the results revealed that the attenuated strain has a clear decrease in pathogenicity for specific-pathogen-free (spf) chickens compared with the parent strain. strain yn-inoculated birds had clinical signs of varying severity with 30% mortality, while the attenuated group appeared healthy, with less tissue damage. the attenuated strain also had relatively low tissue replication rates and higher antibody levels. the superior protective efficacy of the attenuated strain was observed when vaccinated birds were challenged with a homologous or heterologous field ibv strain, indicating the potential of the attenuated yn (ayn) as a vaccine. producing a vaccine targeting the abundant serotype in china is essential to reducing the economic impact of ib on the poultry industry. infectious bronchitis virus (ibv) belongs to the order nidovirales, family coronaviridae, and genus gammacoronavirus. infectious bronchitis (ib) is an important worldwide viral disease of poultry. this poses a major economic threat to the poultry industry because of poor weight gain and lost feed efficiency in broilers and reduced numbers and quality of eggs in layers. in addition, some virulent strains cause high mortality in young chickens due to renal disease. while chicks are the most susceptible, ibv affects chickens of all ages. despite a predilection towards the respiratory tract, ibv also infects a wide range of organs, including the kidneys, gastrointestinal tract, and oviduct, causing respiratory disease, interstitial nephritis, and decreased egg production (cavanagh and naqi, 2003) . clinical signs include coughing, sneezing, tracheal rales, and watery eyes (cavanagh, 2007; raj and jones, 1997) . lesions in infected birds include degeneration of renal epithelium, renal interstitial lymphoplasmacytic inflammation, and degeneration and necrosis of the ciliated respiratory epithelium. the main method of protecting chickens from ib is inoculation with both attenuated live and inactivated vaccines. although inactivated vaccines are cheaper and easier to administer, the poultry industry prefers to use live vaccines rather than inactivated ones, as they are more effective (huang and wang, 2006) . despite widespread vaccination in china and other countries, ib outbreaks still occur sporadically because of little or no cross protection between different ibv serotypes (cook et al., 2001; liu et al., 2009 ). specifically, a new ibv variant has been circulating in china since 1998 (wang et al., 1998) . this virus has been identified as the qx strain and has been primarily associated with various renal pathologies (terregino et al., 2008 zsofia et al., 2009 . phylogenetic analysis showed that the ibv isolates that clustered with qx were mainly chinese isolates. these results further indicated that ibv isolates that are prevalent in china were significantly evolutionarily distant from mass-type strains. however, mass-type vaccine strains (e.g., h52, h120, ma5, and w93) are mainly ibv vaccine strains typically administered in china today, which may not be able to provide efficient protection against field strains. other vaccine strategies including combination of mass type vaccine with 4/91, conn type etc., were attempted before and could get a broader protection than the mass type vaccine alone (cook et al., 2001 (cook et al., , 1999 . however, the 4/91 and conn type vaccine have not been licensed as commercial vaccines in china nowadays. thus, it will be necessary to rapidly develop new vaccines against the qx-like field strains. it has been shown that the spike glycoprotein (s) of coronavirus is a determinant of tissue tropism and virulence and a tiny change in the s gene may lead to vaccine failure cavanagh et al., 1986) . thus, until a universal vaccine can be developed, the ongoing determination of epidemic serotypes and production of new vaccines are key factors in controlling infectious bronchitis (jackwood et al., 2005) . early on, it was recognized that live-attenuated ib vaccines could be developed by reducing virus virulence by multiple serial passages in 10-11-day-old embryonated eggs, and this method is still widely applied today (cook et al., 2012; gelb and cloud, 1983) . commercial ib vaccines are developed by multiple passage of a field isolate in specific-pathogen-free (spf) embryonated eggs until the desired blend of non-pathogenicity and immunogenicity has been achieved (bijlenga et al., 2011; jackwood et al., 2003) . we have previously isolated and characterized a virulent ibv strain from 30-day-old vaccinated broiler chickens in the yunnan province of china. this isolate is genetically similar to most of the prevalent strains of ibv found in china, albeit with increased pathogenicity than previously characterized strains (feng et al., 2012) . to determine its utility as a vaccine, this yn strain was attenuated by 118 serial passages in 10-day-old spf embryonated chicken eggs. we tested the safety and efficacy of the attenuated strain in this study. the ibv qx-like strain yn (genbank accession no.: jf893452) originated from a h120 vaccinated broiler flock with respiratory and renal disease, with a death rate of 30% (feng et al., 2012) . the virus was purified and passaged by inoculating 10-day-old spf embryonated chicken eggs via the allantoic sac route. inoculated eggs were incubated for 40 h at 37 c and the allantoic fluid harvested for subsequent passages. eggs that died within 24 h of inoculation were discarded. at every 10th passage, eggs were examined using reverse transcription polymerase chain reaction (rt-pcr) for the presence of virus. the resulting attenuated yn strain was shorted as ayn and was titrated by inoculating 10-fold serial dilutions with phosphate-buffered saline (pbs) of the virus stocks into the allantoic sac of 10-day-old spf embryonated eggs. the embryo 50% infectious doses (eid 50 ) were determined according to the method of (reed and muench, 1938) . the homologous (yn strain) and heterologous (sd strain) challenged ibv strains were used for determine the protective efficacy of ayn. the heterologous challenged ibv sd strain was isolated from mass-type vaccinated chickens of shandong province in 2013. based on the phylogenetic analysis, it has the highest homology with lx4 (genbank accession number: ay189157), and has a 89.7% similarity with the yn strain. the eid 50 for strain yn and sd was 10 6.83 eid 50 /0.2 ml and 10 5.63 eid 50 /0.2 ml, respectively. one day or 5-week-old spf chickens were used to determine the safety and efficiency of ayn. all chickens were kept in isolators at china agricultural university throughout the experiment and the animal rearing facilities were approved by the beijing administration committee of laboratory animals under the leadership of the beijing association for science and technology (approval id syxk [jing] 2013-0013). all of the animals used in this study were cared for in accordance with established guidelines, and the experimental protocols were performed with the approval of the animal welfare and ethical censor committee at china agricultural university. a total of sixty 5-week-old spf white leghorn chickens were assigned to 3 groups of 20 chickens each. the chickens were maintained in isolators with positive pressure in air-conditioned rooms. of the three groups, two were inoculated with 200 ml yn or yn attenuated containing 10 5.0 eid 50 by intranasal and intraocular routes. the third group was maintained as a negative control. birds were housed separately in isolators for chickens with consistent conditions, and food and water were provided ad libitum. to determine the pathology produced by the two ibv strains, all birds were observed daily for 21 days post-inoculation (dpi) for clinical signs. clinical signs indicative of infection with ibv strains consisted of hunched posture, depression, reluctance to move, emaciation, diarrhea and soiled vent were given daily clinical scores: 0 for normal, 1 for mild depression, 2 for severe depressed, 3 for paralysis/prostration, and 4 for death. two chicks from each group were sacrificed at 3, 5, 7, 10 and 15 dpi. gross changes were noted and samples of trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius were collected for virus detection via real-time quantitative reverse transcription polymerase chain reaction (rt-qpcr) and in 10% neutral formalin for histopathological examination. necropsies were carried out, and external and internal abnormalities were recorded. lesions in the trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius were scored as 0 for no lesions, 1 for mild, 2 for moderate and 3 for severe lesions. mean lesion scores (mlss) were calculated for each group. blood samples from surviving chicks in each group were collected at the end of the experiment for antibody detection at 21 dpi. tissues collected above were routinely processed, embedded in paraffin wax, and cut into 5 mm sections. sections were stained with hematoxylin and eosin and examined for lesions using light microscopy. the slides were examined by light microscopy and the lesions were scored according to the severity of the observed lesions. absence of injury was classified as -, while mild, moderate and severe were classified as +, ++, and +++ respectively. duplicate tissue sections were prepared for immunohistochemistry (ihc) to detect viral antigen via the following protocol. briefly, 5 mm tissue sections were subjected to antigen retrieval and then incubated in 10% normal goat serum in pbs for 30 min to block nonspecific binding. slides were further incubated with chicken anti-ibv hyperimmune serum at 1:500 dilutions in pbs for 2 h followed by incubation with a horseradish peroxidaseconjugated rabbit chicken igg for 1 h. the reaction was visualized by the addition of 3,3-diaminobenzidine (dab, sigma, st.louis, mo, usa) for 15 min. finally, sections were counterstained with hematoxylin, air dried, and examined by light microscopy. the trachea was removed under strict aseptic conditions. the tracheal rings were cut into semicircles and quarter-circles. they were immersed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer, ph 5.5 and fixed for 2 h at 4 c. after primary fixation, samples were then washed with pbs, post-fixed in 1% osmium tetroxide, washed, and dehydrated in an increasing series of ethanol solutions. dehydrated pellets were embedded in an epoxy resin and sections were cut at 70 nm. finally, sections were placed on copper sieves and stained with uranyl acetate and lead citrate. sections were imaged using a jem-1230 tem microscope (jeol, tokyo, japan). tracheal samples were fixed as described above. after dehydration in an increasingly concentrated series of alcohol, samples were critical point-dried (polaron e3000) with carbon dioxide and mounted on aluminum stubs. the mounted samples were gold-coated (polaron e5100) and imaged using a jeol jsm-5310lv (jeol, tokyo, japan). total rna was extracted from trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius using trizol (invitrogen, carlsbad, ca, usa). cdna was obtained via reverse transcription as previously described [20] . cdna samples were analyzed using sybr green i real-time rt-qpcr to determine viral loads. primers were designed using primer premier 5.0 based on the conserved region of n gene of the yn strain (genbank accession no.: jf893452). the 20-ml pcr mixture was composed of 10 mlof power sybr green pcr master mix (applied biosystems, foster, ca, usa), 0.5 ml of each primer, 1 ml of template, and 8 ml of doubledistilled water. real-time pcr was performed on a 7500 system sds software (applied biosystems) using the following thermal cycles: a 10 min hot start at 95 c, followed by 40 cycles of denaturation at 95 c for 15 s, annealing at 55 c for 35 s and extension at 72 c for 40 s. all qpcr reactions were carried out in triplicate and repeated at least twice. a linear regression was determined plotting the logarithmic values of the number of copies of plasmid dna containing the insert against the cycle threshold (ct) values to convert ct values into copy numbers. the relative n gene expression was analyzed using graphpad prism version 6.0 (graphpad software inc., san diego, ca, usa). a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx laboratories, westbrook, me, usa) was used to measure ibv antibody levels according to the manufacturer's instructions. the viral titer was calculated using the formula provided by idexx. ninety 1-day-old spf chickens were divided into five groups of 18 birds as follows. group ayn-yn and ayn-sd were vaccinated . bars indicate mean ae sd. the significance was considered as follows: significant at p 0.05 (*), highly significant at p 0.01 (**), and very highly significant at p 0.001 (***). intranasally with 10 5.0 eid 50 ayn and challenged intranasally with homologous field strain yn or heterologous field strain sd at a dose of 10 5 eid 50 /bird at 21 days post vaccination, respectively. group control-yn and control-sd were left unvaccinated and challenged with yn or sd at the same time. group control-nc was maintained as a negative control. all birds were housed separately in isolators with consistent conditions, and food and water were provided ad libitum. following challenge, all birds were observed daily for clinical signs attributable to ib infection for two weeks. two birds from each group were killed humanely at 3 and 5 day postchallenge (dpc) for gross lesions observation and the evaluation of trachea ciliary activity. ten oral swabs from each group were randomly collected at 5dpc for the detection of virus shedding via rt-pcr. for pcr, 10 ml taq supermix (transgen, beijing, china) and 10 pmol of each primer were added to 100 ng cdna as template in a total of 20 ml reaction volume. pcr was performed at 95 c for 5 min, followed by 30 cycles of denaturation (95 c, 45 s), annealing (53 c, 45 s), and polymerization (72 c, 1 min), and the postpolymerization step was performed at 72 c for 10 min. a pair of primers (forward: 5 0 -ttttggtgatgacaagatgaa-3 0 ; reverse: 5 0 -cgcattgttcctctcctc-3 0 ), which amplify and detect a 403-bp fragment of the m gene of ibv was used in the procedure. amplified sequences were analyzed by 1.0% agarose gel electrophoresis. for evaluation of tracheal ciliostasis, three sections of the upper, middle and lower part of the upper, middle and lower part of the trachea respectively, nine rings per bird, were analyzed. the rings were placed in a petri dish containing eagle culture medium with 10% fetal bovine serum. they were then determined by inverted light microscope at a magnification of 400â, observing the degree of integrity and preservation of the ciliary movement of the tracheal epithelial cells. a score of 0 was given if the cilia in the complete tracheal section showed movement; a score of 1 was given if the cilia of 75-100% of the tracheal section showed movement; a score of 2 if the cilia of 50-75% of the trachea showed movement; a score of 3 if the cilia of 25-50% of the trachea section showed movement; and a score of 4 if the cilia of less than 25% of the trachea section showed movement or no movement at all. for each group, the average ciliostasis score was calculated. data were analyzed using unpaired t-test in graphpad prism version 6.0 for windows to obtain a statistical analysis of the differences between ayn and yn inoculated groups. in the case of the serology test and ciliary activity inhibition test, collected data were analysed using two-way anova to see whether there was any significant difference between the different groups. multiple comparison between the groups was performed using tukey's multiple comparisons test; the significance was considered as follows: significant at p 0.05 (*); highly significant at p 0.01 (**); and very highly significant at p 0.001 (***). chicks inoculated with the yn strain showed clinical signs as early as 2 dpi persisting until 7 dpi. diseased chicks showed signs of coughing, sneezing, tracheal and bronchiolar rales, listlessness, huddling, ruffled feathers, increased water intake, and slight watery diarrhea. three birds in the yn inoculated group died during the experiment, and the mortality reached 30% (fig. 1a) . no obvious clinical signs or deaths attributable to ibv were observed in either the control group or the yn attenuated group (fig. 1c) . at necropsy, lesions were detected in the respiratory, urinary, and digestive systems in chickens inoculated with yn, including punctate hemorrhages and catarrhal exudates in the throat and trachea (fig. 1f) . the kidneys were swollen with urate deposits frequently observed in the tubules and ureters (fig. 1i) . the lungs of several chickens had congestion and edema (fig. 1l) . digestive tract lesions consisted of thickening of the wall of the proventriculus and duodenum (figs. 1o and r), in some cases associated with mucosal congestion. the bursas had heavy exudates of mucus and hemorrhage (fig. 1u ). chickens inoculated with the attenuated strain had only occasional petechiae in the throat (fig. 1e) and a slight amount of mucus adhered to the bursa (fig. 1t) . no gross lesions were observed in any birds in the control group (figs. 1d, g, j, m, p, and s). number of birds with gross lesions and mean lesion scores at necropsy were showed in table 1 . the microscopic lesions were consistent with the gross lesions described above. moderate to severe lesions were predominantly found between 3 and 10 dpi and were more common in the chickens infected with the wild-type than the attenuated strain (fig. 2) . inflammatory cell infiltrates and varying degrees of epithelial desquamation of ciliated cells were detected in the trachea ( fig. 2b and c) , with hemorrhage and mucosa injury in some chickens. foci of necrosis and intense multifocal nephritis, interstitial congestion, with lymphoplasmacytic infiltration and tubular table 1 number of birds with gross lesions and mean lesion scores at necropsy. group the significance was considered as follows: significant at p 0.05 (*), highly significant at p 0.01 (**), and very highly significant at p 0.001 (***). a yn group infected with yn strain; ayn group infected with attenuated yn strain. there were no lesions in birds in the control group. b no. of birds with lesions out of two birds. c mean lesion scores out of two birds. dilation were scattered throughout the kidneys of yn-inoculated chickens ( fig. 2e and f) . proteinaceous material was detected in the tubules of a number of yn-inoculated chickens. congestion, hemorrhages, erythrocytes and lymphocytes were frequently detected in the bronchial and air capillary lumina of the lungs. a number of yn-inoculated chickens exhibited bronchiectasis ( fig. 2h and i) . in the proventriculus, lesions included loss of mucosal epithelium with inflammatory cell infiltration ( fig. 2k and l) . no histologic evidence of damage was detected in the duodenum and bursa (fig. 2n , o, q, and r). no ibv-related lesions were observed in the control birds ( fig. 2a , d, g, j, m, and p). histological scores in different organs of spf chicks inoculated with yn or ayn were showed in table 2 . the presence of ibv antigen was widely detected in all ibv target organs. the cytoplasm of epithelial cells of the mucosa and lamina propria of the trachea, air capillaries, renal tubules, proventriculus, duodenum, and bursa of fabricius all had immunoreactivity. more intense immunoreactivity was noted within the yn-inoculated group than in the attenuated group (fig. 3) . to further examine ibv replication in the respiratory tract, we observed the virus particles using transmission electron microscopy. as shown in fig. 4a and b, typical coronavirus particles were noted in the lumina of tracheas inoculated with each of the virus strains. sem examination of yn-infected tracheas revealed severe lesions in the respiratory mucosa caused by replication of the inoculated virus (fig. 4e, h, and k) . this was evidenced by severe erosion of the respiratory epithelium with associated inflammation. in the yn attenuated group, relatively mild tracheal damage was observed (fig. 4d, g, and j) . no lesions were observed in the tracheas of control birds (fig. 4c , f, and i). mean of the severity index from two chicks: -, no change; +, mild; + +, moderate; +++, severe. fig. 3 . immunohistochemical detection of ibv antigens at 7 dpi. black arrows indicate viral antigen immunoreactivity. scale bar = 50 mm. 3.1.6. viral genome copy number in tissues no viral dna was detected in tissues from groups before inoculation and in mock-infected chickens. table 3 shows results of viral dna testing from both infected groups. the proportion of positive samples in the group infected with the yn strain was significantly higher than that of the attenuated group. the time-dependent viral load levels in different organs of infected groups are shown in fig. 5 . the viral dna levels in the spleen, lung, and duodenum in the yn group peaked at 3 dpi and fig. 4. transmission (a-b) and scanning (c-k) electron micrographs of tracheal epithelium at 7 dpi. a, b: virus particles in the tracheal lumen. c, f, i: control epithelium (â600, â2.0k, and â20.0k, respectively). d, g, j: respiratory epithelium from yn attenuated infected chickens (â600, â2.0k, and â5.0k, respectively). e, h, k: respiratory epithelium from yn infected chickens (â600, â2.0k, and â5.0k, respectively). tissue tropisms in 35-day-old spf chickens. proventriculus lung tracheal duodenum kidney bursa yn attenuated 4/10 2/10 6/10 2/10 5/10 7/10 yn 6/10 4/10 8/10 6/14 6/10 8/10 subsequently gradually decreased. no virus was detected in the yn attenuated samples from similar time points. the maximum amount of viral dna was detected at 5 dpi in the tracheas of both groups and the proventriculus and bursa from the yn group. viral loads in the kidneys of both groups increased starting at 5 dpi and peaking at 7 and 10 dpi for the yn and attenuated strains, respectively. the proventriculus and lung samples from the yn attenuated group peaked at 7 dpi, and the duodenum and bursa of fabricius samples from the yn attenuated group had peak viral numbers at 10 dpi. all samples except those from the proventriculus contained more viral copies in the yn group. in addition, the number of copies of viral dna in the trachea and kidneys were significantly greater than other organs at the same time. antibody responses were measured using a commercial elisa kit (idexx laboratories). antibodies against ibv were not detected in any groups before inoculation (0 dpi) and were never detected in the mock-infected group. chickens in both infected groups fig. 5 . viral loads in samples after yn or yn attenuated strain infection. the significance was considered as follows: significant at p 0.05 (*), highly significant at p 0.01 (**), and very highly significant at p 0.001 (***). fig. 6 . survival percentage (a) and trachea ciliostasis scores (b) in chickens experimentally challenged with ibvyn and sd strains. bars indicate mean ae sd. the significance was considered as follows: significant at p 0.05 (*), highly significant at p 0.01 (**), and very highly significant at p 0.001 (***). seroconverted by 21 dpi and the mean titers induced by the yn and yn attenuated were 2587 and 5504, respectively. the differences in the mean titers between birds inoculated with the yn and yn attenuated strains were significantly different (p < 0.05) (fig. 1b) . birds in unvaccinated groups which challenged with the yn or sd strain showed clinical signs and death as early as 4dpc. diseased chicks showed signs of listlessness, huddling, ruffled feathers, increased water intake, and slight watery diarrhea. during the observation period, the mortality in group control-yn and control-sd were 28% and 67%, respectively (fig. 6a) . no obvious clinical signs or deaths attributable to ibv were observed in ayn vaccinated groups and group control-nc. at necropsy, all euthanized chickens showed the slight hemorrhage with serous catarrhal exudate in the trachea and typical kidney lesions characterized by obvious swelling and urate deposition in the tubules and ureters in group control-yn and control-sd. no gross lesions were observed in any birds in ayn vaccinated groups and group control-nc. inhibition of the ciliary activity was measured at 3 and 5 dpc in the trachea. the group control-yn and control-sd showed a maximum average ciliostasis score of 4, while the average ciliostasis score in group ayn-yn and ayn-sd were below 1 (fig. 6b) . the difference of ciliostasis was extremely significant between ayn vaccinated groups and unvaccinated groups (p < 0.0001). detected by rt-pcr method, virus shedding rate was 100% in group control-yn and control-sd at 5dpc, while the rates were 0 and 30% in group ayn-yn and ayn-sd, respectively. no virus was detected in the unchallenged group (control-nc). the best protection against challenge is achieved by a vaccine containing homologous strains (gelb et al., 1990) . as previously demonstrated in some reports, cross-protection is poor between different serotypes and genotypes of ibv strains. consequently, currently available vaccines cannot provide sufficient protection for heterologous challenge gelb et al., 2005; liu et al., 2006) . the s1 gene of ibv has serotype-specific and neutralization-specific epitopes. serotype evolution and the genetic diversity of ibv are monitored by analysis of the s1 gene. the majority of qx-like ibv isolates present in the field in china had poor similarity in the s1 part of the spike protein with vaccine strains, indicating the antigenic differences and large evolutionary distances between vaccine strains and ibv field strains in china (liu et al., 2006; zhao et al., 2014) . despite the widespread use of live attenuated ibv vaccine (mass serotype), such as strains h120, h52, and ma5, vaccinated chicken flocks usually fail to achieve complete protection against field virulent ibv challenge. the poultry industry has, in recent years, detected an increasing incidence of outbreaks related to qx-like ibv strains of different serotypes in many countries (abro et al., 2012; terregino et al., 2008; valastro et al., 2010; xu et al., 2007) . optimal vaccines against circulating ibv strains in china require attenuated vaccines designed from local strains in china. a previous ibv isolate (the yn strain) was passaged 118 times through spf chicken embryonated eggs. as a result of this process, the virus becomes more adapted to the embryo, reflected by more efficient replication and higher pathogenicity for the embryo (data not shown). this attenuated yn strain was inoculated via the oral or oculonasal route into spf chickens to compare the tissue tropism and pathogenicity to its parent strain. after infection, birds in the attenuated group showed no common clinical signs as those observed in birds inoculated with the yn strain. however, gross lesions, although much milder and in fewer organs than those in chicks infected with the yn strain, were still detectible at necropsy. histopathology indicated lower pathogenicity of the attenuated strain, which showed moderate inflammatory infiltration and epithelial degeneration. lesions caused by the yn strain were similar to those described in the previous report, which characterized it as a virulent qx-like ibv strain (feng et al., 2012) . in general, the systemic distribution of yn antigen was demonstrated by ihc staining and was most abundant in the respiratory and urinary systems. the epithelia of the trachea and alimentary tract of the yn-infected group were strongly immunoreactive to ibv antigens compared with the attenuated group. the presence of virus particles in both the infected groups post-inoculation indicates that a significant viral infection was delivered to the experimental spf chickens and suggests that ciliated respiratory tract cells play a significant role in viral replication. these lesions appeared to damage the host cell, leading to the loss of cellular functions and decreasing immunity, increasing the opportunities for secondary or multiple infections (davies et al., 2009 ). however, the increased antigen production and cilia loss in the yn strain supports it being more pathogenic than the attenuated strain. in terms of the real-time rt-qpcr examination, both strains were detected in respiratory and non-respiratory tissues, including the kidney, trachea, lungs, proventriculus, duodenum, and bursa of fabricius, indicating viral replication in these organs; however, this was relatively limited in the birds infected with the yn attenuated strain. we also observed that the viral rna levels in the tracheas of birds in both groups declined after the peak, then reached another small peak. loss of ciliated columnar epithelium, and, presumably, the associated viral replication in those cells, is a common lesion with ibv infection (callison et al., 2006; geerligs et al., 2011) . the small peak may be related to the recovery of the epithelial cells in the upper-respiratory tract after extensive damage at the early infection period. among the tissues examined by histopathology, the damage was most severe in the trachea and kidney, the primary target organs of ibv (uenaka et al., 1998) . the viral loads were highest in these two organs. therefore, the higher the viral load, the more severe the histologic damage, and the stronger the positive signal. the yn attenuated strain was able to induce a higher humoral antibody response following inoculation. this suggests that this strain may be useful in vaccination programs under field conditions to reduce the economic losses caused by qx-like ibv infections on commercial layer and broiler farms. information regarding vaccine efficacy against circulating infectious bronchitis virus strains of china will provide valuable knowledge for the poultry industry when considering vaccine types. therefore we invested the efficiency of the yn attenuated strain to determine if it could provide protection against the homologous and heterologous virulent strains. the results indicated that the strain ayn protected spf birds against morbidity and mortality from challenge with the highly virulent strains yn and sd. assessment of immunity to challenge with ibv is most commonly done by removal of trachea at 4 or 5 days after challenge followed by either quantification of ciliostasis cook et al., 1976) or detection of viral shedding. there was a clear decrease in ciliostasis scores in the yn attenuated vaccinated groups compared with the unvaccinated groups after challenge with homologous and heterologous virulent strains, indicating the yn attenuated strain could protect the respiratory tract efficiently. in addition, virus shedding in the infected chickens is a big challenge to the control of infectious bronchitis virus. it would encourage viral spread among chickens and support virus persistence. thus the reduction of virus excretion should be taken into consideration when choosing the vaccine type and program. in our experiment, a significant decrease was observed in the vaccinated groups after challenge. we also noticed that the virus shedding inhibition in yn challenged group was more significant than the sd challenged group, which indicating that the vaccine strain could provide a better protection against the challenge with homologous strains. the genetic mechanism responsible for loss of pathogenicity is still not well understood. the s1 gene is responsible for induction of protective immunity, and small differences in s1 contribute to poor cross protection . it has been shown that a number of amino acid residues in s1 contribute to ibv attenuation (cavanagh et al., 2005; liu et al., 2007) . besides the s1 subunit, the nucleocapsid protein can also induce protective immune responses in chickens (boots et al., 1992) . previous studies have shown that amino acid substitutions within the replicase gene may result in attenuation following serial passage in embryonated eggs (armesto et al., 2009 ). further genetic investigation will be necessary to determine the key mutations responsible for the attenuation in this sample. moreover, the stability of the vaccine and its tendency to revert should be investigated in the future study before administration in the field to ensure the security and reliability of this vaccine. the attenuated yn strain showed lower replicate ability and decreased pathogenicity than its parent strain, with an efficacious protection against homologous and heterologous field strains, indicating the potential to become an alternative vaccine candidate for controlling ib infections in china. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronavirus avian infectious bronchitis virus relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virusneutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection relationship between sequence variation in the s1 spike protein of 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virus (ibv) strain in china infectious bronchitis virus: immunopathogenesis of infection in the chicken a simple method of estimating fifty per cent endpoints pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes intracloacal infection with avian infectious bronchitis virus qxtype infectious bronchitis virus in commercial flocks in the uk isolation and identification of glandular stomach type ibv (qx ibv) in chickens isolation and identification of four infectious bronchitis virus strains in china and analyses of their s1 glycoprotein gene molecular characterization of an infectious bronchitis virus strain isolated from northern china in 2012 comparison of the pathogenicity of qx-like, m41 and 793/b infectious bronchitis strains from different pathological conditions this study was supported by beijing agriculture innovation consortium of poultry research system. the authors declare no conflict of interest. key: cord-303393-9zs3qqo4 authors: alsultan, musaed abdulaziz; alhammadi, mohamed ali; hemida, maged gomaa title: infectious bronchitis virus from chickens in al-hasa, saudi arabia 2015-2016 date: 2019-03-19 journal: vet world doi: 10.14202/vetworld.2019.424-433 sha: doc_id: 303393 cord_uid: 9zs3qqo4 aim: this study aimed to isolate some of the currently circulating infectious bronchitis virus (ibv) strains from some broiler chicken farms in al-hasa and to do some molecular characteristics of these strains. materials and methods: we collected 300 tissue specimens, including the trachea, bronchi, lungs, and kidneys from some four commercial chicken farms showing respiratory manifestations. we tested these tissue specimens by the real-time polymerase chain reaction (rt-pcr) and gel-based pcr. we selected some pcr positive samples for isolation in the embryonated chicken eggs (ece). we sequenced some pcr-positive samples and conducted phylogenetic analysis based on the obtained sequences. results: our molecular surveillance revealed that 31.6% of the tested specimens were ibv positive by pcr. we selected some positive specimens showing low ct values by the qrt-pcr for virus isolation by the ece. the infected eggs showed hemorrhage, dwarfing, and death in some cases after three passages in the ece. we sequenced some of the positive pcr specimens and used the obtained sequences to draw the phylogenetic tree based on the partial ibv-orf-1a, n, and s1 gene sequences. the phylogenetic trees based on the ibv-n and s1 gene sequences showed that the circulating ibv strains in al-hasa during 2016 was showing a high degree of identity to some strains from taiwan and italy. meanwhile, the grouping of these strains based on the ibv-s1 sequences revealed that the currently circulating ibv strains in al-hasa belonged to gr.i.7 along with strains from taiwan. conclusion: our results confirmed the continuous circulation of the ibv among the chicken population in al-hasa despite the intensive application of vaccines against this virus. infectious bronchitis virus (ibv) is a highly contagious respiratory viral disease of chickens of all ages. the ibv belongs to the order nidovirales, family coronaviridae, and genus gammacoronavirus [1, 2] . the ibv genome is a positive sense rna molecule with a high tendency to frequent changes and recombination. there is a high degree of genetic diversity among ibv strains. until recently, there was no available system for the definite grouping and identification of various ibv genotypes. one recent study used the combination between the phylogenetic analysis and the pairwise similarities on both the nucleotides and the amino acid levels to do fine mapping of the ibv genotypes [3] . this study used the full-length s1 gene sequences to develop a novel classification system for the ibv genotypes and lineages [3] . based on the comparison of the coding sequence of the s1 gene from 1652 ibv strains, they were able to categorize the currently circulating ibv strains into 6 genotypes and 32 lineages [3] . the ibv infection usually causes high economic losses among the poultry industry. it is quite possible to become endemic in the chicken industry in some regions of the world. the virus has wide tissue tropism, including the respiratory, digestive, renal, and reproductive systems of the affected birds. it may affect the oviduct and lead to low production and low-quality eggs or may cause severe renal complications and mortality among the affected birds [4] . the ibv-infected birds usually shed the virus in their body secretions such as respiratory and the gastrointestinal tract secretions. these birds may remain active shedders of the virus for up to several weeks post-infection [5] . secondary bacterial infections (escherichia coli and mycoplasma gallisepticum) always exaggerate the viral pathogenesis and auscultate the mortality rates among the infected chicken population [6] . the ibv infections continue to be a major problem to the poultry industry worldwide. in spite of the availability of several ibv vaccines, the virus continues to cause many outbreaks among the chicken farms in both broiler and layer settings [7] . efforts to control spreading of the ibv infections through vaccination resulted in some variable outcomes. many ibv strains and serotypes have emerged since its discovery >80 years ago; meanwhile, the misuse of the ibv vaccines complicates the evolution and emergence of new ibv strains [8] . one possible explanation for the emergence of new ibv strains is the poor proofreading capability of the viral rna polymerase. this resulted in high mutation rates alongside the viral genome. this leads to the emergence of new ibv strains occasionally [9, 10] . many ibv vaccines are commercially available, including inactivated, live attenuated, and recombinant. the live-attenuated vaccines are the most commonly used in all types of poultry; these provide a good immune response; however, there is a possibility of revert to virulence. meanwhile, the inactivated vaccines are usually administered to layers and breeder's chickens before the laying time as booster vaccines [11, 12] . the ibv infection in chicken was first reported in saudi arabia in 1984 in field samples by the realtime polymerase chain reaction (rt-pcr) using n primers [13] . another study reported the circulation of the ibv/4/91 serotype in saudi arabia in 2000 using the partial s gene [14] . several ibv genotypes were previously reported in saudi arabia, including ch/ck/ldl/971, is/720/99, isvariant2/98, and the d24 [15] . we recently reported the circulation of very virulent ibv strains in chicken farms from eastern saudi arabia [16] . several ibv variants and genotypes are currently circulating in the middle east, asia, and north africa such as iraq, egypt, libya, iran, and jordan [17] . furthermore, many outbreaks were reported in saudi arabia, especially in the central region of the country [15, 18, 19] . however, the full molecular characterization of these ibv strains and variants is not well reported yet. this study aimed for isolation and molecular characterization of the ibv circulating strains of the ibv in al-hasa in the eastern region of saudi arabia. this region is one of the major hubs for intensive poultry production in saudi arabia. we conducted this study according to the king faisal's university animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/59 (http://www.kfsh.med.sa/kfsh_website/ usersuploadedfiles%5cncbe%20regulations%20 english.pdf). the animal ethics committee of the king faisal's university approved this protocol. we conducted molecular surveillance for ibv on some chicken farms across al-hasa from november 2015 to april 2016. birds in these farms were suffering from acute respiratory signs and high morbidity and mortality rates. these outbreaks were mapped around three major cities in the al-hasa province (table-1), saudi arabia (al-hufuf, mahasen, and almubarez). 100 tissue specimens (trachea, lung, and kidney) representing four suspected ibv outbreaks were collected. these samples were stored at −80°c for further processing. briefly, 1 g per each tissue specimens was ground in a sterile mortar mixed with 9 ml of phosphate-buffered saline (pbs) and sterile sands. tissues were then centrifuged at 5000 rpm for 15 min. the supernatants were harvested and stored at −80°c until use. isolation of the currently circulating ibv strains from some poultry farms in al-hasa was carried out by the inoculation of the ece. we used 9-11 days' chicken embryos from native breeds (non-ibv vaccinated) and proved to be ibv-negative antibodies by elisa. the inoculum was prepared as follows; ibv-suspected tissue suspensions were centrifuged for 10 min at 5000 rpm. antibiotic mixture was added (penicillin and streptomycin) to each tissue suspension. we used 100 µl per each tissue suspension to inoculate through allantoic route into five embryonated eggs [20] . the inoculated eggs were incubated at 37°c. negative control pbs-inoculated eggs were done in parallel to the experimental infection of the ece. we observed the inoculated eggs daily by candling for or up to 3 days post-inoculation. any early deaths within 24 h post-inoculations were excluded due to the non-specific trauma-related death [21] . the total viral rnas were extracted from tissue using the qiaamp viral rna mini kit (qiagen cat. no. 52906 qiagen, germany] as per the manufacturer's recommendations. the eluted viral rnas were b=broiler, l=layer, rs=respiratory signs, lu=lung, t=trachea, k=kidney stored at −80°c for further testing. the positive control (the commercial live ibv-h120 vaccine for ibv, veterinary vaccine production centre, ksa) was processed in parallel to each batch of specimens as described above. the rt-pcr was performed using the ibv rt commercial kits (subang jaya, selangor de, malaysia) (qpcr/rt-qpcr kits kestrel bioscience llc, doc. no. cat# av1007011). the reactions were conducted as per the kit's instructions with some minor modifications. briefly, each reaction consisted of 2×rt-pcr master mix (10 µl, ibv ppm 1 µl, ppm iec 1 µl, nuclease-free water 3 µl, and 5 µl of rna template) in a total reaction volume of 20 µl. meanwhile, we used 5 µl of nuclease-free water as negative control. furthermore, a positive control reaction was carried out in parallel to each run. duplicate reactions were done per each specimen. the cycling parameters were 55°c for 10 min, then 95°c for 8 min, followed by 50 cycles at 95°c for 10 s, and 60°c for 1 min. we designed some primers to amplify the partial ibv-n gene. we designed the ibv-n primers based on the ibv-n (accession no: kt762154). the nucleotide sequences of the used ibv-n gene are ibv-nf (5'-cgctggagaatttcctcttg-3') and the ibv-nr (5'-ctagtccctagcagccatgc-3'). meanwhile, we used the following primers, ibv-s1f (5'-accggctgatggatggcat-3') and the ibv-s1 (5'-ttgcttacaaccaccctgtag-3'), to amplify the partial ibv-s1 gene. the rt-pcr reactions were conducted in a 25 µl reaction volumes using the one-step rt-pcr kit (qiagen, valencia, ca no. 210212). the reactions were conducted as per the manufacturer's instructions. the reaction mixture consists of 10 µl of 5×reaction buffer, 2 µl of each primer, 2 µl of enzyme mixture, 2 µl of dntp mixture (400 µm of each dntp), 3 µl of the rna template, and 4 µl of rnase-free water. reverse transcription was started at 50°c for 30 min; initial taq polymerase activation was performed at 95°c for 15 min, along with 40 cycles of denaturation at 94°c for 1 min, annealing at 55°c for 1 min, and then extensions at 70°c for 1 and 10 min. for visualization of the amplified pcr products, electrophoresis of 10 µl of each pcr products on 1% agarose gel was performed. this was done under the ultraviolet light and photographed by the bio-rad gel documentation system. 10 µl of per each pcr product was mixed with 3 µl of the loading buffer (blue/orange loading dye, roche). a 100-bp, ready-to-load dnamarker ladder was loaded in a separate, parallel lane. we processed a confirmed pcr-ece-p3-ibv isolate for sequencing by the illumina next-generation sequencing approach. we used this approach to decode the partial-length genome sequences of this ibv isolate as previously described [22] . we constructed the phylogenetic tree based on the obtained partial ibv-orf1-a, ibv-n, and s1 gene sequences. we constructed the tree using the multiple alignments of these sequences with other ibv sequences retrieved from the genbank done using the mega-7 package, and phylogenetic analysis was done by the maximum likelihood method using the best-fit model determination and at least 500 bootstrap replicates as previously described [23] . meanwhile, we used the mega-7 software to calculate the pairwise distances between our ibv-s1 sequence and the available sequences from the genbank. we applied the non-probability sampling strategy for our specimen collection with incidental assignment approach as previously described [24] . the ibv infection in chickens produces a wide range of clinical syndromes. some birds showed respiratory manifestation in terms of coughing, sneezing, and nasal discharges. necropsy examination of some ibv-infected birds revealed serous, catarrhal, or caseous exudate in the tracheal, nasal passage, sinus, and bronchi congestion (figure-1a) . the edema of the lungs, cloudy or fibrinous inflammation of the air sacs, occasionally contains a yellow caseous exudate. edema and swelling of the kidneys were observed in some birds (figure-1b) . we conducted molecular surveillance to check the prevalence of ibv across the al-hasa from october 2015 to april 2016. we investigated four ibv outbreaks across the al-hasa province by the rt-pcr (table-1 ). we tested 100 chickens' specimens from non-vaccinated chickens using tissue specimens from the trachea, lungs, and kidneys by the rt-pcr technique. our overall results showed that 19 pooled chickens' tissue specimens were positive of 60 tested samples (31.6%). simply, specimens from farm no: 8 is showing that 6 out of 12 specimens (50%) were positive. in the case of farm no: 4, 8 of 24 (33.33%) were ibv positive. however, three specimens of nine tested from one particular chicken farm were positive in case of farm no: 10. interestingly, only two specimens from poultry farm on the farm no: 9 were positive of 15 tested specimens (13.33%) ( table-2) . we already tested different tissue specimens, including the trachea, lungs, and kidneys, from some suspected ibv-infected birds. our results are showing that 11 tracheal tissue specimens of 20 were positive, while 6 lung specimens of 20 were positive; meanwhile, only two specimens from the kidneys were positive (table-3 ). we processed 15 specimens (trachea, lungs, and kidneys) after pooling from three ibv outbreaks. we did the propagation for three passages. the ibv isolation was successful only in case of two specimens. the first specimen was collected from an rt-pcr-ibvpositive chicken trachea collected from farm no. 7. the second specimen was collected from positive ibv-rt-pcr lungs collected from chicken farm no. 5 area (table-4 ). however, isolation from the inoculated kidney tissues from farm no. 3 was not successful (table-4 ). the ibv infection of the ece resulted in many pathological changes on the inoculated embryos such as dwarfing, congestion, hemorrhage, and death at different time points. no pathological changes, alteration, or death was reported in the negative control group of embryonated eggs in the three subsequent passages. we used three tissue suspensions (trachea, lungs, and kidneys) from three specimens representing three different ibv outbreaks in some chicken farms from al-hasa region. we followed up these three inoculums on the ece for three subsequent passages. the ibvinfected tracheal specimen caused death and induced some pathological changes in two embryos from the inoculated embryos in the three passages. however, one of them showed no pathological changes or death at the end of the experiment. meanwhile, the ibvinfected lung tissue specimens showed that only one embryo was affected per each passage. we used commercial available rt-pcr kits to confirm the identity of some suspected ibv-infected chicken tissues, as well as some fluids from the embryonated egg passages. we carried out the rt-pcr amplification for three representative specimens from the ece passages p-1-3. meanwhile, we 1 7 t 15 3 2 1 2 1 2 1 2 5 lu 15 3 1 2 1 2 1 2 3 3 k 15 3 -3 -3 -3 total 45 9 3 6 4 5 4 5 ibv=infectious bronchitis virus already included six original specimens from the ibvsuspected outbreaks from different farms, including some pooled tissue specimens (trachea, lungs, and kidneys). furthermore, we already included the ibv vaccine as a positive control of the reaction in addition to the kits positive control in the reactions. in addition to a negative control used a non-dna template. our results show that 6/9 (66.67%) of the three tested specimens representing embryonated eggs passages 1-3 were positive. three specimens from selected ibv specimens were positive, as shown in figure2. based on the reported ibv-orf1a sequences, these strains were closely related to ibv-l1148 strains (figure-3) . the phylogenetic tree based on the partial ibv-s1 genes revealed that these strains were closely related to other ibv strains from taiwan and italy (figure-4) . the evolutionary relationship analysis based on the ibv-s1 sequences confirmed the s1-based analysis (figure-4) . the phylogenetic analysis based on the generated ibv-n gene sequences clearly showed that the circulating strains were closely related to the ibv strain reported from taiwan and china (figure-5) . both the pairwise distance (table-5 ) and the phylogenetic analysis based on the generated ibv-s1 sequences of our isolates confirmed the blast results ( figures-4 and 5 ). the geographical distribution of the suspected outbreaks represents different regions across the al-hasa. some chicken farms were selected around the al-hasa region as targets for our study. some of these farms were reporting ibv outbreaks based on figure-4: phylogenetic analysis based on the partial s1 gene for the circulating infectious bronchitis virus (ibv) strains from some chicken farms in al-hasa region 2015-2016. phylogenetic tree of the obtained partial saudi ibv-s1 gene isolated from al-hasa region 2015-2016. the maximum likelihood phylogenetic tree was based on the partial ibv-s1 gene. the bootstrap is 1000. the reported local ibv sequences clustered together with other ibv strains from italy and taiwan. a red triangle marks the reported sequence in this study. the obvious clinical signs and post-mortem lesions, while birds in some others were apparently healthy. the clinical examination of the affected birds showed depression, ruffled feathers, loss of the body weight, respiratory rales, nasal discharge, and lachrymal discharge (data not shown). necropsy examination of some affected birds revealed congested trachea, caseous and bloody plugs at the tracheal bifurcations ( figure-1a) , congested lungs, and swollen kidneys ( figure-1b) in some cases. these clinical signs are very much typical of the ibv pattern of infection in poultry farms [25] . we conducted molecular surveillance for ibv among some ibv chicken outbreaks in al-hasa region 2014-2016. we selected some tissue specimens to be tested by the commercial ibv-rt pcr kits. our results are showing that six specimens of nine (66.67%) were ibv positive (table-4 ). we also selected some of these positive ibv specimens for the propagation and isolation of the currently circulating strains by the ece. we assessed the success of the ibv isolation in two different ways. first, the inoculated embryos showed pathological changes relevant to the standard ibv inoculation in the ece. these changes were in the form of congestion, dwarfing, hemorrhage, and death of the inoculated embryos. second, we were able to detect the ibv signal by doing rt-pcr assays on the ece tissues and fluids from different egg passages (data not shown). furthermore, we confirmed the propagation of the ibv on the eces by another method using the truncated ibv-n primers through the rt-pr technique. we conducted an ibv molecular surveillance among some selected chicken farms in al-hasa region by the rt-pcr technique using the conserved ibv-n primers. we tested 100 specimens from layers, and broiler non-vaccinated chicken's flocks represented four suspected ibv outbreaks across the al-hasa. these tissue specimens were collected from various organs of the affected birds such as the trachea, lungs, and kidneys. we did pooling five organs per each tube considered as one specimen. we found that 19/60 specimens (31.67%) were positive (table-4 ). our results also showed that 11/20 (55%) of the tested trachea were positive, while 6/20 (30%) lungs were positive. however, 2/20 (10%) tested kidneys were ibv positive (table-4) . we used the generated sequences of three independent genes (orf1a, s1, and n) in the ibv genome to do the phylogenetic analysis of the circulating strains in al-hasa region during 2016. we found great overlapping and consistency of the phylogenetic analysis based on the ibv-s1 and n genes. the reported sequences were closely related to other ibv strains reported in taiwan, italy, and china ( figures-3-5 ). this high identity to foreign ibv strains may be due to that saudi arabia does import chickens from different countries including uk, china, italy, and taiwan. however, the ibv-orf1a sequences revealed that these strains were related to other ibv strains belonging to the ibv-qx strain (l1148) (figure-3) . it is well known that there is a great diversity of s1 gene among ibv strains, which make it a strong candidate for the ibv classification [26] . furthermore, novel variations were reported among most of the ibv-n gene [27, 28] . this is suggesting the potential use of some ibv-s1 and maybe ibv-n genes for the classification of different ibv strains as described [3] . our results confirmed recent reports about the circulation of different ibv strains in the kingdom and the eastern region in specific [15, 29] . this indicates a wide distribution of ibv in the al-hasa in the kingdom in general. in spite of the massive application of ibv vaccines in chicken farms under study, ibv is circulating, and thus, the currently used ibv vaccines under field conditions did not provide enough protection against the ibv infection. another challenge in the context of this vaccination failure is the possibility of circulating of novel ibv strains that do not share any antigenic relationship with the currently used vaccines, and thus, protection cannot be achieved. we believe that ibv will continue to hit several chicken farms in the region and many other parts in the world. further studies are highly recommended to do thorough molecular characterization of the currently circulating and the possible emerging ibv strains; thus, protection was usually achieved through the preparation of the homologous relevant specific ibv vaccines. ibv continues to pose a great risk to the poultry industry in the al-hasa, saudi arabia. there is continuous circulation of the ibv between the broiler and layer chicken in this area. the currently circulating strains of the ibv belong to the gr.i-17 along with other genotypes from taiwan, china, and italy. table-5: pairwise distance analysis of partial s1 gene for the circulating ibv strains in some chicken farms in al-hasa region gi.16/it/kj941019 in vitro and in ovo expression of chicken gamma interferon by a defective 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avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens rapid detection and identification of avian infectious bronchitis virus variation in the spike protein of the 793/b type of infectious bronchitis virus presence of infectious bronchitis virus strain ck/ ch/ldl/97i in the middle east molecular characterization and phylogenetic analyses of virulent infectious bronchitis viruses isolated from chickens in eastern saudi arabia genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in egypt detection of variant infectious bronchitis viruses in broiler flocks in libya molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 isolation and propagation of coronaviruses in embryonated eggs infectious bronchitis disease. in: oie manual of diagnostic tests and vaccines for terrestrial animals. world organisation for animal health genetic characterization of the belgian nephropathogenic infectious bronchitis virus (nibv) reference strain b1648 mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets numerical taxonomy freeman. the principles and practice of numerical classification. freeman, san francisco pathogenicity study of iranian genotype of avian infectious bronchitis virus (ir-1) complete sequences of 3' end coding region for structural protein genes of turkey coronavirus novel variation in the n protein of avian infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan molecular survey and phylogenic analysis of infectious bronchitis virus (ibv) circulating among chicken flocks in riyadh province, saudi arabia mgh designed and performed the experiments, oversee the entire work, and performed data analysis and interpretation. muaa conducted the fieldwork, performed some laboratory experiments, and drafted the manuscript. moaa helped in the data analysis and reading of the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord-316525-uadfehr6 authors: zhang, x. w.; yap, y. l.; danchin, a. title: testing the hypothesis of a recombinant origin of the sars-associated coronavirus date: 2004-10-11 journal: arch virol doi: 10.1007/s00705-004-0413-9 sha: doc_id: 316525 cord_uid: uadfehr6 the origin of severe acute respiratory syndrome-associated corona-virus (sars-cov) is still a matter of speculation, although more than one year has passed since the onset of the sars outbreak. in this study, we implemented a 3-step strategy to test the intriguing hypothesis that sars-cov might have been derived from a recombinant virus. first, we blasted the whole sars-cov genome against a virus database to search viruses of interest. second, we employed 7 recombination detection techniques well documented in successfully detecting recombination events to explore the presence of recombination in sars-cov genome. finally, we conducted phylogenetic analyses to further explore whether recombination has indeed occurred in the course of coronaviruses history predating the emergence of sars-cov. surprisingly, we found that 7 putative recombination regions, located in replicase 1ab and spike protein, exist between sars-cov and other 6 coronaviruses: porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), bovine coronavirus (bcov), human coronavirus 229e (hcov), murine hepatitis virus (mhv), and avian infectious bronchitis virus (ibv). thus, our analyses substantiate the presence of recombination events in history that led to the sars-cov genome. like the other coronaviruses used in the analysis, sars-cov is also a mosaic structure. sars, a new disease characterized by high fever, malaise, rigor, headache and non-productive cough, has spread to over 30 countries with around 8% of mortality rate on average. sequence analysis of sars coronavirus (sars-cov) [17, 25] showed that it is a novel coronavirus [12] . anand et al. [1] reported a three-dimensional model of sars-cov main proteinase and suggested that there are a number of methods and software packages that have been developed for detection of recombination events in dna sequences. the performance of these methods has been extensively evaluated and compared on simulated and real data [23, 24] . in the present study we applied these methods to rna viruses. sars-cov and other 6 coronavirus genomes (sars-cov, ibv, bcov, hcov, mhv, pedv, tgev) were first aligned using clustalw [33] . sites with gaps were removed and a 25077-nt alignment was generated. subsequently, seven methods were employed to detect the occurrence of recombination (see corresponding reference in parenthesis for details of each method): bootscan [26] , geneconv [28] , dss (difference of sums of squares) [20] , hmm (hidden markov model) [8] , maxchi (maximum chi-square method) [19] , pdm (probabilistic divergence measures) [9] , rdp (recombination detection program) [18] . bootscan, maxchi and rdp are implemented in rdp software package, http://web.uct.ac.za/depts/microbiology/microdescription.htm. geneconv is implemented in the program, http://www.math.wustl.edu/∼sawyer/geneconv/. dss, hmm and pdm are implemented in topali software package, http://www.bioss.sari.ac.uk/software.html. basically default parameter settings were used in all the programs, except the following values: gscale = 1 (geneconv), internal and external references (rdp), window size = 300 and step = 10 (dss, hmm and pdm). after potential recombination events were identified by at least 3 methods above, separate neighbor joining trees were constructed for each putative recombination region to better evaluate the evidence for conflicting evolutionary histories of different sequence regions. all trees were produced with topali mentioned above. table 2 summarizes the results of bootscan analysis with 100% bootstrap support and significant p-value (<0.05 for uncorrected and mc corrected pvalue). two regions (13151-13299 and 16051-16449, position in alignment) are identified as putative recombination regions and all 6 coronaviruses are potential parents with sars-cov as potential daughter. geneconv detected 9 putative recombination events occurred in a wide range of positions 5941-24997 (in alignment) at a significant level p < 0.05 for two p-values: simulated p-value (based on 10,000 permutations) and blastlike bc ka p-value (table 3 ). all 6 coronaviruses are potential parents with sars-cov as potential daughter. maxchi identified 15 putative recombination events (table 4 , possible misidentification events are not retained). most of the breakpoints are significant at about 0.001 level; the position located in alignment spans from 3534 to 22840, but some beginning or ending breakpoints are not determined. similarly, 6 coronaviruses are potential parents with sars-cov as potential daughter. rdp revealed that 6 putative recombination events occur in the domain of alignment 5910-13334 (table 5) , with the uncorrected and mc corrected pvalue at less than 0.002 and 0.05 respectively. in this case, 4 coronaviruses (ibv, bcov, mhv and pedv) are potential parents with sars-cov as potential daughter. figure 1 shows the dss profiles of putative breakpoints between sars-cov and other coronaviruses (dotted line indicates the 95 percentile under the null hypothesis of no recombination): sars-cov, ibv, bcov and mhv (fig. 1a) , sars-cov, mhv, pedv and tgev (fig. 1b) , sars-cov, ibv, hcov and tgev (fig. 1c ). there are about 6 different breakpoints (significant peaks): 13614 and 16085 (fig. 1a) , 11008 and 12850 (fig. 1b) , 12805, 13614 and 16444 (fig. 1c) . hmm plots for sars-cov, ibv, bcov and hcov (fig. 2 ) revealed that the putative breakpoints are at about position 5500 and 19000. there is a clear transition from state 1 (sars-cov grouped with ibv) (fig. 2a) into state 3 (sars-cov grouped with hcov) (fig. 2c) . the region between 5500 and 19000 is noisy, and at this moment no information can be provided by hmm. figure 3 shows the results of pdm analysis performed on sars-cov and other coronaviruses (dotted line indicates the 95% critical region for the null (fig. 3c, d) , 1393, 6111, 16624, 19859 and 20802 (fig. 3e, f) . posada [23] suggested that one should not rely too much on a single method for recombination detection. here we consider the regions identified by at least 3 methods as putative recombination regions. the results are summarized in table 6 . seven putative recombination regions span a range of positions in sars-cov phylogenetic trees constructed by using putative recombination regions and nonrecombination regions identified by above techniques are shown in figure 4 . the left panels stand for non-recombination regions and the right panels for recombination regions. we compared each row of figures and found that the phylogenetic tree in the left panel (non-recombination region) had very different topology when compared to the phylogenetic tree in the right panel (recombination region), which indicates that recombination has occurred. for example, in fig. 4a , 7 coronaviruses are divided into 4 groups: group 1 for tgev, hcov and pedv, group 2 for bcov and mhv, group 3 for ibv, and group 4 for sars-cov, consistent with marra et al. [17] ; while in fig. 4b, 7 coronaviruses are divided and sars-cov, suggests that sars-cov is most closely related to bcov and mhv, which is consistent with a recent report [29] . at the same time, sars-cov is also most closely related to tgev (fig. 4d) and ibv (fig. 4f) . thus, phylogenetic analysis substantiates the presence of recombination events in the history that led to the sars-cov genome. in this study, seven recombination detection methods and phylogenetic analyses were performed on sars-cov and the six coronaviruses identified by blast (ibv, bcov, hcov, mhv, pedv and tgev). these techniques successfully identified recombination events in bacteria and viruses [2, 3, 6, 21, 26, 39] . our analysis concurred to suggest the occurrence of recombination events between ancestors of sars-cov and these 6 coronaviruses. indeed, pairwise alignment showed that many segments of high homology with ibv, bcov, hcov, mhv, pedv and tgev do exist in sars-cov genome, table 7 exhibits the segments with length >20 nt and identiy >80%, and fig. 5 shows the mosaic structure of the region 14930-15908 in sars-cov genome based on the segments with length >50 and identity >80%. of course, the other coronaviruses used in the analysis are also mosaic structures, for more sequence similarities exist among them than with sars-cov. it is noted that all the sequence comparisons in this study are based on nucleotide sequences. while the protein sequences in sars-cov are largely different from those in the known three groups of coronavirus [17] , such as, for s protein, the identity is: 25.9% for sars-cov and bcov, 21.7% for sars-cov and hcov, 21.5% for sars-cov and ibv, 25.6% for sars-cov and mhv, 20.6% for sars-cov and pedv, 19.4% for sars-cov and tgev. although sars-cov is close to bcov, mhv, tgev and ibv, the corresponding protein, replicase 1a, is still different: with identity 27.4% for sars-cov and bcov, 24.8% for sars-cov and ibv, 32.2% for sars-cov and mhv, 25.0% for sars-cov and tgev. naturally, we should take into account the role of convergent evolution, which would bear its mark on the viral genome. the recombination events that we witnessed in sars-cov are present in six different viruses, suggesting sequential horizontal transfers and progressive adaptation to new hosts cells or animals. indeed because viruses need both receptors to permeate host cells and resist the immune response of the host, their outer layer proteins are submitted to an extremely strong selection pressure that may restrict considerably the possible variations of the corresponding proteins (and accordingly of the corresponding genome pieces of sequences). it is nevertheless remarkable that, despite the inclusion of all possible types of viruses in our sample set (as well as shuffled genomes from the viruses we have identified as relevant) we find a more or less single category of viruses as similar to sars-cov. this suggests that even if the contribution of convergent evolution is important, this happened on a more or less common phylogenetic background, suggesting several steps of recombination followed by fine adaptation. in this context, we would like to suggest that ancestors of pedv, mhv or both are the most plausible origin of sars-cov. guan et al. [7] based on phylogenetic techniques and bootscan recombination analysis stavrinides and guttman [32] indicated that the replicase of sars-cov was a mammalian-like origin, the m and n proteins have an avian-like origin, and the s protein has a mammalian-avian mosaic origin. while in the present study we used phylogenetic analysis and 7 recombination detection methods, including the powerful methods of maxchi and geneconv among 14 methods studied (simplot (bootscan), geneconv, homoplasy test, pist, maxchi, chimaera, phypro, plato, rdp, recpars, reticulate, runs test, sneath test, triple) [23, 24] , to conduct whole genomewide recombination analysis. we identified seven putative recombination regions, which encompass, in terms of proteins involved, replicase 1a, replicase 1b and the spike glycoprotein. stavrinides and guttman [32] primarily inferred the occurrence of recombination qualitatively, but did not identify the precise recombination region in the protein involved (the s protein is an exception, they identified a recombination region in s protein, located between nucleotides 2472 and 2694 of the s protein, i.e. between nucleotides 23963 and 24185 of the sars-cov genome, basically covered by the last recombination region for s protein (table 6) ). most importantly, each of our recombination regions is identified by at least 3 methods, because one should not rely too much on a single method, as suggested in [23] . in general, we believe two studies lead to the overall conclusion: the evolution of sars-cov has involved recombination. the recombination event in the replicase is related to the fact that the rna polymerase of coronaviruses utilize a discontinuous transcription mechanism to synthesize mrnas. the viral polymerase must jump between different rna templates regularly during positive-or negative-strand rna synthesis and depending on the rejoining sites, the resultant rna recombination will be either homologous or nonhomologous. this is the copy-choice model of recombination in rna viruses [13, 27, 31, 34] . the recombination event in s protein is certainly important since this allows the virus to alter surface antigenicity and escape immunesurveillance in the animals, thus adapting to a human host. the existence of sars-cov-like viruses (99.8% homology to human sars-cov) in several wild animals in a live animal market in guangdong [7] indicated that interspecies transmission among the human and animal sars-cov-like viruses had occurred. the mutation analysis of sequence variations among these isolates will help identify the genetic signature of sars virus strains when a sufficient amount of sequence data is available. the very fact that several species of animals are affected does not allow one to trace directly the origin of the virus as endemic in one of these species, but, rather, might be indicative that animals and men might have been contaminated by a virus from a common origin, presumably located in animal food present in local markets in the guangdong province. investigating a wide variety of animal coronaviruses, especially in relation to rodents, birds, snakes and farm animals, would be interesting with regard to the origin of the sars-cov that caused disease in humans. finally, a challenging question arises. what is the molecular basis of recombination in sars-cov? many requirements are needed for recombination to occur: (1) two coronaviruses can infect a host simultaneously and continue to replicate without interference with each other; (2) sufficient nucleotide identity between these genomes is essential for genome-switching to occur during rna replication; (3) the proteins arising from recombination must be functional; (4) the recombinant virus must have some selective advantage for its survival. that is, the recombination that creates a successful "new" coronavirus is probably a rare event. so, we must stress that the potential recombination events in sars-cov, identified in the present study, are most likely "old" events, which may represent the events that occurred thousands of years ago. although the recent findings indicated that sars-cov did exist in a number of wild animals [7] , we have not yet determined where these sars-cov-like virus strains come from. coronavirus main proteinase (3cl pro ) structure: basis for design of anti-sars drugs testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype e full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from thailand evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes infectious bronchitis virus: evidence for recombination within the massachusetts serotype the heterosexual human immunodeficiency virus type 1 epidemic in thailand is caused by an intersubtype (a/e) recombinant of african origin isolation and characterization of viruses related to the sars coronavirus from animals in 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sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice regulation of transcription of coronaviruses recombination in sars-cov flood of sequence data yields clues but few answers evidence of natural recombination within the s1 gene of infectious bronchitis virus evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus experimental confirmation of recombination upstream of the s1 hypervariable region of infectious bronchitis virus widespread intra-serotype recombination in natural populations of dengue virus author's address: dr we wish to thank the hong kong innovation and technology fund for supporting the present research. ending in length identity match percent source sars sars (%) 10063 10109 47 41 88 mhv 10609 10641 33 30 91 tgev 12821 12854 34 31 92 hcov 13844 13879 36 32 89 bcov 13845 13879 35 ibv 14808 14835 28 26 93 hcov 14913 14947 35 31 89 hcov 14933 15070 138 112 82 bcov 14982 15091 110 89 81 ibv 14986 15055 70 64 92 mhv 15062 15093 32 29 91 hcov 15123 15173 51 43 85 tgev 15210 15232 23 22 96 pedv 15210 15238 29 27 94 bcov 15210 15253 44 40 91 ibv 15417 15482 66 57 87 bcov 15417 15457 41 37 91 ibv 15420 15479 63 55 88 mhv 15611 15682 72 64 89 pedv 15624 15670 47 42 90 hcov 15633 15672 40 35 88 tgev 15729 15770 42 40 96 mhv 15765 15817 53 46 87 hcov 15852 15908 57 49 86 mhv 17088 17125 38 35 93 ibv 17688 17714 27 25 93 tgev 17757 17800 44 39 89 pedv 17783 17809 27 25 93 hcov 18558 18577 20 20 100 pedv 18771 18847 77 65 85 tgev 18784 18833 50 44 88 hcov 19102 19132 31 29 94 ibv 19113 19132 20 20 100 hcov 19146 19252 107 87 82 mhv 19201 19252 52 45 87 ibv 19206 19253 48 44 92 bcov 19396 19420 25 24 96 mhv key: cord-296364-7rp60d2m authors: youn, soonjeon; leibowitz, julian l.; collisson, ellen w. title: in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication date: 2005-02-05 journal: virology doi: 10.1016/j.virol.2004.10.045 sha: doc_id: 296364 cord_uid: 7rp60d2m molecular clones of infectious bronchitis virus (ibv), derived from the vero cell adapted beaudette strain, were constructed, using an in vitro assembly method. in vitro transcribed rna from a cdna template that had been constructed from seven cdna fragments, encompassing the entire genome of ibv, was electroporated into bhk-21 cells. the cells were overlaid onto the susceptible vero cells and viable virus was recovered from the molecular clone. the molecularly cloned ibv (mibv) demonstrated growth kinetics, and plaque size and morphology that resembled the parental beaudette strain ibv. the recombinant virus was further manipulated to express enhanced green fluorescent protein (egfp) by replacing an open reading frame (orf) of the group-specific gene, orf 5a, with the egfp orf. the rescued recombinant virus, expressing egfp (gibv), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the mibv. after six passages of gibv, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed egfp. direct sequencing of rt-pcr products derived from cells infected with the plaque-purified virus, which had lost expression of egfp, confirmed loss of the egfp orf. the loss of egfp expression (δ5a ibv) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. this study demonstrates that the 5a orf is not essential for viral multiplication in vero cells. infectious bronchitis virus (ibv), a coronavirus, continues to be one of the most economically important pathogens in the poultry industry. coronaviruses are enveloped viruses with positive sense, 5vcapped and 3vpolyadenylated rna genomes, that range from 27.6 to 32 kb (lai and cavanagh, 1997) . two thirds of the coronavirus genome encodes the replicase activity, including a viral rna-dependent rna polymerase (rdrp), helicase, and viral proteinases. the remaining one third of the genome encodes the structural proteins and small group-specific orfs (lai and cavanagh, 1997) . ibv has four essential structural proteins, the three membrane proteins, the spike (s), integral membrane (m), and small envelope (e) proteins, and a phosphorylated, nucleocapsid (n) protein. the s protein interacts with cellular receptors and induces cell and viral membrane fusion (bosch et al., 2003) . n proteins, which interact with viral genomic rna, forming ribonucleocapsid (rnp) complexes, have been associated with replication and transcription (baric et al., 1988; compton et al., 1987; robbins et al., 1986) . the e and m proteins are localized in er-golgi intermediate compartment and play critical roles in viral budding (de haan et al., 1998; fischer et al., 1998) . coronaviruses are classified into three distinct groups by antigenic cross-reactivity and nucleotide sequence analysis (fields, 1996) . the group i coronaviruses include human coronavirus 229e, transmissible gastroenteritis virus (tgev), feline coronavirus, and feline infectious peritonitis virus. the group ii coronaviruses include human coronavirus oc43, murine hepatitis virus (mhv), and bovine coronavirus. ibv belongs to the group iii coronaviruses, all of which are avian specific. severe acute respiratory syndrome coronavirus (sars cov) respiratory disease in humans that resembles ibv in poultry may be distantly related to group ii coronaviruses (snijder et al., 2003; stavrinides and guttman, 2004) . coronavirus genomes also encode group-specific orf that have been reported to express in infected cells (lai and cavanagh, 1997; liu and inglis, 1992; liu et al., 1991) . functions of these genes are not well understood, however, deletion of combinations of several group-specific genes in mhv resulted in in vivo attenuation (de haan et al., 2002) . although the functions of the four ibv group-specific orf, 3a, 3b, 5a, and 5b are not known, a vero cell passaged ibv mutant with a truncated 3b was viable in vitro and in vivo (shen et al., 2003) . reverse genetic systems for human coronavirus 229e, tgev, mhv, sars cov, and ibv have been developed using three distinct methodologies. bacterial artificial chromosomes (bac) and vaccinia virus vectors, both of which can accommodate the large coronavirus genomes, have been utilized to carry a full-length cdna clone of the genome (almazan et al., 2000; casais et al., 2001; gonzalez et al., 2002; thiel et al., 2001) . a third system relied on the in vitro assembly of a modest number of cloned cdnas. instead of cloning the entire genome of tgev into vectors, yount et al. (2000) amplified the viral genome into several fragments by rt-pcr and the cloned amplicons were ligated through unique restriction sites for in vitro assembly of the entire genome. this strategy was further modified to construct an infectious cdna clone of mhv with their bno see'mq technology, in which restriction endonuclease sequences were incorporated into amplicons, such that upon enzyme treatment, the endonuclease sites were eliminated prior to in vitro ligation. the assembled cdna product was used as template for in vitro transcription to generate genomic rna (yount et al., 2002) . more recently, this strategy was used to construct a reverse genetic system for the sars cov (yount et al., 2003) . in the current study, molecular clones of ibv were generated using the in vitro assembly of cdna fragments as a template for transcription of full-length genomic rna. we further have demonstrated that the 5a orf can be eliminated or replaced by the egfp orf while maintaining virus viability. therefore, this gene is not essential for viral replication and its orf may potentially be used as a site for heterologous gene delivery. cdna was constructed that encompassed the entire ibv genome the cloning strategy for a full-length ibv-beaudette construct is illustrated in fig. 1a . five rt-pcr fragments were produced that cumulatively amplified the entire genome of the vero cell adapted beaudette ibv strain. amplicons, overlapping by about 10 nucleotides, were designated a through e and the fragment lengths were as follows; a, 2269 nts; b, 6356 nts; c, 6895 nts; d, 5030 nts; and e, 7058 nts (fig. 1b) . however, consistent with the report that these regions were toxic or unstable in bacteria, difficulties arose in cloning the b and c fragments into bacterial plasmid based cloning vectors (casais et al., 2001) . therefore, the b and c fragments were each cloned as two smaller fragments b1 and b2, and c1 and c2. fragments a through c2 were cloned into the psmart vector and fragments d through e2 were cloned into pcr-xl-topo cloning vectors. in order to determine the consensus sequence of the genome, two to five independent clones of each amplicon were sequenced using a panel of internal primers. clones with consensus sequences were used for in vitro assembly of full-length cdna template. there were several sequence differences, distributed throughout the entire genome of ibv, between our standard cell adapted ibv strain and the published beaudette sequence (table 1 ). the standard parental strain of ibv beaudette used for these studies had a five nucleotide insertion at position 25,414 between the m and 5a orfs, resulting in a genome size of 27,613, instead of 27,608 nt. the sequence differences were confirmed by sequencing three independent clones and direct sequencing of rt-pcr products. a nucleotide insertion was found in an intergenic sequence of our strain compared to the original published sequence. in addition, an e amplicon with a single unique nucleotide change of a to g at position 25,793 nt, resulting in amino acid change from q to r, was used as a marker for the molecular clone. full-length cdna was in vitro assembled with amplicons conforming with our established consensus sequence. to increase full-length in vitro ligation efficiency, ordered ligation reactions were used. the appropriately sized ligation reaction products were purified from 1% agarose gels before further ligating with neighboring inserts (fig. 1c) . a full-length genomic transcript of ibv was produced in vitro using t7 rna polymerase with the t7 rna promoter, incorporated at the 5vend of fragment a. the final transcript product was verified by northern blot analysis using a probe against 3v utr (data not shown). any transcript of a size comparable to viral genomic rna with a detectable 3vutr presumably contained the entire genome of ibv because only the a fragment had a t7 rna promoter and thus could initiate transcription. the transcripts from the full-length cdna (along with the n transcript alone), total cellular rna from ibv infected vero cells (positive control), and pbs (negative control) were electroporated into non-permissive bhk-21 cells, which were then cultured with vero cells. three days after transfection, typical cytopathic effects (cpe), including syncytia formation, were seen in cells transfected with the positive control. supernatant from the cells, which were overgrown by 3 days after transfection, were harvested and passaged into fresh vero cells. one day after passage, the positive control showed cpe. two days after passage, cells transfected with the transcript of the full-length ligated cdnas showed cpe. cpe was never observed in the negative control cells. we were unable to rescue virus from full-length ibv rna transcript without co-transfection of n transcript, confirming the possible essential role of n transcript as described by previous reports (casais et al., 2001; yount et al., 2000 yount et al., , 2002 . the virus generated by the molecular cdna clone was referred to as mibv. the derivation of mibv from the ligated cdna was confirmed by rt-pcr sequence identification of the marker mutation of a g at nt 25,793. mibv was further characterized after three rounds of plaque purification. mibv formed plaques which were indistinguishable in size and morphology from standard beaudette cell adapted viral plaques (figs. 2a and b) . the growth kinetics of mibv and the standard virus were also compared and found to be similar (fig. 3) . the reverse genetic strategy we adapted for ibv was used to evaluate whether a small group-specific gene was necessary for the survival of ibv in cell culture. furthermore, the replacement with a foreign orf would be a first step in developing constructs of ibv as expression vectors. because it has been shown that the expression level of coronavirus genes can be modified by their transcription regulatory sequences (trs), rather than add an additional trs, a reporter gene was used to replace most of the 5a orf, thus maintaining the 5a trs sequence. based on results with mhv, we hypothesized that ibv group-specific genes would not be necessary for viral replication in cell culture. the 5a orf was used rather than the 3a, 3b region which contains an internal ribosomal entry site (ires) needed for expression of the essential structural, e protein (le et al., 1994) . the egfp orf was used to replace the 5a orf, beginning at the start codon, while the 3v end of 5a orf, that included the stop codon, was kept intact because this region overlaps with the 5b orf. pcr mutagenesis and the bno see'mq methodology were used to replace orf5a with egfp. the cloning strategy used to replace most of the 5a orf with egfp (d5a/egfp) is illustrated in fig. 4a . three regions, including the egfp orf (fig. 4b, lane 1) , and sequences between a region upstream of 5a, including orf m (fig. 4b, lane 2) , a region downstream of 5a through the 3v utr (fig. 4b , lane 3), were pcr amplified using primers that incorporated the bsmbi restriction enzyme recognition sequence (table 2) . these three pcr products were cloned into the pstblue cloning vector and the inserts were excised from the vectors with the bsmbi restriction enzyme. the e amplicon was replaced with the d5a/egfp amplicon and the full-length infectious cdna template was assembled and transcribed as described previously. three days after electroporation into bhk cells and coculturing with vero cells, cpe was observed in the positive control and in the cells transfected with the gibv rna transcript (fig. 5a ). the cells that showed cpe by gibv infection also expressed egfp which was easily detected under a uv microscope (fig. 5b) . interestingly, some cells expressed egfp before even showing virus infection induced morphological changes. following nucleotide sequencing analysis, it was confirmed that no unintentional mutations were incorporated into the amplicons used to construct the recombinant gibv. therefore, not only is 5a expression not essential for viral replication but also it is possible to replace the 5a with a functional foreign orf. a-g -22,441 c-g l-v a according to our vero-adapted ibv derived sequence. the entire sequence has been submitted to genbank. b left nucleotide according to the previously published sequences and the right nucleotide from our vero cell-adapted derived strain. c left amino acid from the previously published sequences and the right amino acid from our vero cell-adapted derived strain. -indicates that there was no amino acid difference. to confirm the stability of the recombinant gibv, the virus was subsequently passaged in vero cells. egfp expression was detected in each of seven sequential passages. gibv was further characterized by comparing plaque morphology and one step growth curves. compared to our standard virus and the virus from the cloned mibv, gibv-generated plaques were much smaller in size (fig. 6a) . the growth of gibv was similar to the standard ibv with maximum growth at 14 h post-infection. however, gibv grew to a 10-fold lower titer compared with the standard beaudette ibv or mibv (fig. 3) . beyond passage five in vero cells, the plaque assays of the gibv stocks began to demonstrate plaques similar in size to those formed by our standard virus. viruses derived from these plaques had lost the ability to express egfp. to explain the phenomena, the region of egfp insertion/5a replacement from several plaque purified viruses was rt-pcr amplified and compared with rt-pcr products of mibv and the gibv expressing egfp (forming smaller plaques). the rt-pcr product of the latter was smaller than the gibv and mibv (fig. 7a ). in addition, the region across the insertion site of egfp orf for ten gibv derived strains that had lost egfp expression was amplified by rt-pcr. sequencing of the rt-pcr products confirmed that these stains had either partially or completely lost the egfp orf. the 10 strains examined could be categorized into four distinct groups, depending on the deletion pattern of the egfp orf (fig. 7b) . the 5b orf of all was maintained and completely intact while the start codon at the insertion site of the egfp was deleted in all of these phenotypic revertants. interestingly, the size of plaques with the 5a deletions, including a revertant that had completely lost the egfp orf, was comparable to plaques generated by our standard beaudette cell-adapted beaudette virus (fig. 6b ). when compared with the standard virus, growth kinetics and maximum titer of the revertants with the 5a deletion was similar to the standard cell adapted beaudette virus (fig. 3) . molecular clones of several coronaviruses have been constructed using three different methodologies, vaccinia virus vectors and bac as cloning systems, and the in vitro assembly strategy. in this study, we constructed an infectious cdna clone of ibv and recombinant ibv expressing egfp instead of 5a protein, using in vitro assembly which is less labor intensive and allows for easy manipulation, such as the introduction of targeted mutations. we chose to use highly attenuated strain of ibv, the vero cell-adapted beaudette us strain as the cdna backbone because we hope to eventually use ibv as a gene transfer vector, as well as to elucidate mechanisms of pathogenesis and generate new appropriate vaccines. contrary to the difficulties stated in a previous report (casais et al., 2001) , our study has demonstrated that the in vitro assembly method used to generate infectious rna from in vitro assembled cloned cdnas of tgev, mhv, and sars coronavirus also can be applied to ibv. this is also the first report that the group iii coronavirus group-specific 5a gene is not essential for viral replication. based on studies with mhv and tgev, in which deletion of multiple group-specific genes resulted in attenuation of virulence, group-specific genes of ibv are reasonable candidates for viral attenuation and foreign gene replacement (de haan et al., 2002; sola et al., 2003) . however, it has been shown that these small orf, including 5a, are conserved among the various strains of ibv (brooks et al., 2004) . therefore, it is likely that 5a is important, if not critical, for in vivo replication. further studies will determine the in vivo role of 5a in pathogenesis and whether its deletion results in attenuation. gibv produced smaller sized plaques compared to our standard parental virus. although many recombinant viruses have been constructed to express egfp or gfp without known reports that egfp is toxic to virus replication, egfp was shown in at least one study to induce cellular apoptosis (liu et al., 1999) . in the current study, it is possible that infected cells died by apoptosis because of high expression of egfp prior to producing maximum amounts of virus viral replication. this is consistent with the finding that we could detect expression of egfp before cells showed cpe. alternatively, manipulation of orf 5a could affect orf 5b expression. it has been assumed that gene 5b is expressed by leaky scanning mechanism (liu and inglis, 1992) . the egfp orf contained an appropriate kozak sequence and may have reduced the expression of 5b. therefore, reduction in 5b expression could potentially have been associated with the observed change in viral replication. gibv phenotypic revertants, which lost the egfp orf, maintained an intact 5b orf, and regained standard beaudette cell adapted virus plaque size, growth kinetics and titer. based on sequence analysis, four distinct deletion events were indicated. it has been shown that the gfp gene has homology with the mhv trs and hinders expression of gfp because of abortive transcription (fischer et al., 1997) . however, we do not favor this explanation because, contrary to fisher et al., we had sufficient expression of egfp, with recombinant virus during the first several passages, to be easily detectable and no sequence similarity could be identified in or around the deleted region between the ibv trs and the egfp gene. non-homologous recombination events during ibv fig. 6 . egfp deletion revertant of gibv showed comparable plaques to standard ibv or mibv. after six serial passages of gibv, heterogeneoussized plaques were observed. the arrow indicates typical gibv plaques with the smaller size compared to mibv and the arrow head indicates a larger plaque from the gibv stock that was comparable to the standard cell adapted ibv, and plaques purified from the larger plaques in a are shown in panel b. replication likely accounts for the deletion of a gene, such as egfp, that is not necessary for the viral replication. it is not clear whether the foreign gene itself or its specific location in the viral genome causes this spontaneous deletion event. furthermore, we cannot conclude whether deletion of the egfp gene, under the control of the 5a trs, is a general phenomenon of recombinant ibv or a result of egfp toxicity. coronaviruses have several attractive traits as gene delivery systems, including their unusually high packaging capacity, exclusive replication in cytoplasm, excluding any chance of viral genome incorporation into host chromosomes, and the ability to express multiple genes which has been shown by thiel et al. (2003) . the stability of a foreign orf replacing the 5a orf should be further examined with genes that encode non-toxic proteins, in order to determine the replication status and pathogenesis of these recombinant viruses in vivo. furthermore, this methodology provides with a convenient reverse genetic tool for correlating the pathogenesis with the group-specific genes of ibv. a vero cell-adapted strain of ibv beaudette us, from american type culture collection (manassas, va) was plaque purified three times before use. the virus was propagated in an african green monkey kidney vero cell line, obtained from viromed laboratory (minnetonka, mn) and maintained in dulbecco's modified eagle medium (dmem) containing 5% fetal bovine serum (fbs) supplemented with penicillin g (100 units/ml) and streptomycin (100 ag/ml). the baby hamster kidney cell line, bhk-21, was fig. 7 . gibv phenotypic revertant had lost the egfp orf. the absence of the egfp orf was confirmed by rt-pcr and nucleotide sequencing of the region corresponding to nucleotides 25,162 to 25,613 of the standard cell adapted ibv. compared to gibv, the gibv revertant produced a smaller rt-pcr product, smaller than the e amplicon, suggesting that the revertant had lost the egfp and 5a orfs. (a) rt-pcr and pcr products of the region corresponding to nucleotides 25,162 to 25,613 of gibv, the phenotypic 5agfp deletion revertant and the control, e amplicon. the m lanes represent the dna molecular size marker; lane 1, rt-pcr product of gibv expressing egfp; lane 2, the rt-pcr product of the phenotypic revertant that had lost expression of egfp, and 3, as a control, the pcr product from the e fragment. (b) alignment of the nucleotide sequences of the region spanning the 5a/egfp orf of d5agfp viruses identified four distinct types of egfp deletions. italicized nucleotides and the positions starred below the sequence alignment represent conserved sequences. every mutant lost the egfp start codon but maintained the 5b start codon (underlined). also obtained from viromed and maintained in dmem containing 10% fbs supplemented with antibiotics as described for vero cells. total cellular rna was extracted from ibv-beaudette infected vero cells with trizol reagent (invitrogen, carlsbad, ca), according to the manufacturer's directions. reverse transcription was performed with superscript ii (invitrogen) and reverse direction primers p1r, p2r, p3r, beaussr, and gnrt (table 2) , containing bsmbi and sapi restriction endonuclease recognition sequences. the ibv sequence, as reported in genbank (accession number m95169), was used for primer design and nucleotide sequencing. each dna fragment was amplified from cdna templates by pcr using expand long polymerase (boehringer mannheim biochemical). in order to transcribe rna using t7 rna polymerase, the t7 rna promoter sequence was incorporated into the t7p1f primer. pcr primer pairs used to amplify genomic regions are listed in table 2 . pcr amplification of cdna fragments was performed using the following conditions; denaturation at 94 8c for 2 min, 10 cycles at 94 8c for 30 s, 55 8c for 30 s, and 68 8c for 2-6 min depending on the size of the fragment and 18 cycles of 94 8c for 30 s, 55 8c for 30 s, and 68 8c for 2-6 min (with an additional 20 s for each subsequent cycle). the pcr products were isolated from agarose gels and cloned into pcr-xl-topo (invitrogen) or psmart (lucigen, middleton, wi) vectors, according to the manufacturer's directions. two to four independent clones of each amplicon were isolated and sequenced by using specific primers and the abi dye termination sequencing method. a consensus sequence was determined by comparison of direct pcr product sequencing from each independent clone and amplicons, encoding the consensus sequence of each region of ibv, were cloned using standard recombinant dna techniques (sambrook et al., 1989) . the entire ibv n orf, including the 3v utr, was amplified by rt-pcr from total cellular rna extracted from ibv infected vero cells, and the rt-pcr product was cloned into a transcription vector, pgem-3zf(+) (promega, madison, wi). the egfp orf (f fragment) was pcr amplified from plegfp-n1 (clontech laboratories, inc., palo alto, ca), using the gfpsf and gfper primer pair, and cloned into pstblue ( table 2 ). the region upstream of the 5a orf (g fragment) was pcr amplified using the e amplicon as a template with the 5asr and bmf primer pair and the product cloned into pstblue (novagen, darmstadt, germany), referred to as 5as. the region downstream of the 5a orf (fragment h which included the 3vutr and polyadenylated tail) was pcr amplified using the e amplicon and the 5aef and gnrt primer pair before cloning into the pcr-xl-topo vector (referred to as 5ae). the g fragment was prepared by digesting 5as with xhoi and bsmbi restriction enzymes. the f fragment was digested from the vector with bsmbi. the g and f fragments were ligated into the 5ae amplicon which was digested with xhoi and bsmbi 5v -cgtctctaagcgtatacgcccaccca-3v a as described in fig. 1 . b as derived from the sequence of our vero cell-adapted, laboratory beaudette strain. c the t7 rna polymerase recognition sequences shown in bold. underlined are bsmbi restriction enzyme recognition sequences. restriction enzymes. replacement of 5a sequences with egfp orf was confirmed by sequencing of the d5a/egfp amplicon. each amplicon, having the consensus sequence, was prepared from an overnight bacterial culture. plasmid was isolated and digested with the indicated restriction endonucleases according to the manufacturer's directions. briefly, the ibv a amplicon was digested with xhoi and treated with calf intestine alkaline phosphatase (cip), before digesting with the sapi enzyme. the ibv e amplicon was digested with ecori, cip treated and digested with bsmbi. the b1, b2 and c1 amplicons were digested with bsmbi at 55 8c and then digested with sapi at 37 8c. the c2 and d amplicons were digested with bsmbi. inserts of each amplicon were resolved by electrophoresis on 1% agarose gels containing crystal violet and isolated with qiaquick gel extraction kit (qiagen inc., valencia, ca) according to the manufacturer's directions. one picomole of each cdna insert was ligated in an ordered reaction (a + b1, b2 + c1, c2 + d, e1 + e2 or ed5a/egfp + e2). the appropriately sized ligation reaction products were purified from 1% agarose gels and further ligated with neighboring inserts for 4 h, pooled and ligated overnight (fig. 1c) . the final ligation product was purified by extracting with a phenol/chloroform/isoamyl alcohol (25:24:1) mixture and precipitated with ethanol before using as a template for in vitro transcription reactions. full-length transcripts of the ibv cdna constructs were generated in vitro using the mmessage mmachine t7 ultra kit (ambion, austin, tx) according to the manufacturer's direction with certain modifications. the in vitro transcription reaction was performed at 37 8c for 2 h in 20 al reaction mixtures, supplemented with 3 al of 30 mm gtp, resulting in a 1:1 ratio of gtp to capping analog. a similar reaction was performed for the n transcript using a 1:3 ratio of gtp and capping analog. before electroporation, the transcripts were treated with dnase i and analyzed by 1% denaturing agarose gel (containing 2.2m formaldehyde) electrophoresis. the identity of the full-length in vitro transcript was confirmed by northern blot analysis using a probe against 3vutr of ibv. after bhk-21 cells were grown to subconfluence (80%), treated with trypsin, and washed with cold depc treated pbs twice, they were resuspended in depc-treated pbs at a concentration of 10 7 cells/ml. rna transcripts were added to 400 al of the bhk-21 cell suspension in microfuge tubes on ice, gently pipetted and transferred to electroporation cuvettes. three consecutive electrical pulses of 850 v at 25 af were given, using the electro-cell manipulator 600, btx (genetronics, inc., san diego, ca). the transfected bhk-21 cells were diluted 1 to 20 with complete dmem in 100 mm cell culture petri dishes and co-cultured with 2 â 10 6 vero cells/dish. triplicate wells of vero cells in six-well plates were infected with virus at a multiplicity of infection (m.o.i.) of 2 to 3. the cells were harvested every 4 h for 24 h and stored at à80 8c until they were used for quantification by titration. the cells were three times frozen and thawed, and 10-fold serially diluted in dmem without serum. two hundred microliters of each dilution were inoculated into six-well plates for an hour, washed with pbs and overlaid with dmem, containing 2% fbs, 0.8% agarose, before incubating at 37 8c for 3 days. the plaques were counted after removing the agarose overlay from the cells and staining the monolayer with crystal violet. engineering the largest rna virus genome as an infectious bacterial artificial chromosome interactions between coronavirus nucleocapsid protein and viral rnas: implications for viral transcription the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex comparisons of envelope through 5b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes reverse genetics system for the avian coronavirus infectious bronchitis virus in vitro replication of mouse hepatitis virus strain a59 coronavirus particle assembly: primary structure requirements of the membrane protein the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host coronaviridae: the viruses and their replication analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by insertion of an intron the molecular biology of coronaviruses distinct structural elements and internal entry of ribosomes in mrna3 encoded by infectious bronchitis virus identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus a polycistronic mrna specified by the coronavirus infectious bronchitis virus is green fluorescent protein toxic to the living cells? rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay molecular cloning: a laboratory manual emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity mosaic evolution of the severe acute respiratory syndrome coronavirus infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus multigene rna vector based on coronavirus transcription strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus we thank dr. ralph baric for providing valuable advice in developing the in vitro assembly procedure for ibv. this work was supported in part by grants from the u.s. poultry and egg association (297) key: cord-305079-foifc8ch authors: zhou, ying shun; zhang, yi; wang, hong ning; fan, wen qiao; yang, xin; zhang, an yun; zeng, fan ya; zhang, zhi kun; cao, hai peng; zeng, cheng title: establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 date: 2013-02-22 journal: vet microbiol doi: 10.1016/j.vetmic.2012.08.013 sha: doc_id: 305079 cord_uid: foifc8ch infectious bronchitis virus (ibv) strain h120 was successfully rescued as infectious clone by reverse genetics. thirteen 1.5–2.8 kb fragments contiguously spanning the virus genome were amplified and cloned into pmd19-t. transcription grade complete length cdna was acquired by a modified “no see’m” ligation strategy, which employed restriction enzyme bsa i and bsmb i and ligated more than two fragments in one t4 ligase reaction. the full-length genomic cdna was transcribed and its transcript was transfected by electroporation into bhk-21 together with the transcript of nucleocapsid gene. at 48 h post transfection, the medium to culture the transfected bhk-21 cells was harvested and inoculated into 10-days old spf embryonated chicken eggs (ece) to replicate the rescued virus. after passage of the virus in ece five times, the rescued h120 virus (r-h120) was successfully recovered. r-h120 was subsequently identified to possess the introduced silent mutation site in its genome. some biological characteristics of r-h120 such as growth curve, eid50 and ha titers, were tested and all of them were very similar to its parent strain h120. in addition, both r-h120 and h120 induced a comparable titer of ha inhibition (hi) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with mass41 ibv strain. the present study demonstrated that construction of infectious clone from ibv vaccine strain h120 is possible and ibv-h120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach. avian infectious bronchitis virus (ibv), the causative agent of infectious bronchitis (cavanagh, 1997; cavanagh et al., 1999) , is a gammacoronavirus in the family coronaviridae (casais et al., 2005; saif et al., 2008; tidona and darai, 2011) . ibv is worldwide distributed and difficult to control because of the existence of multiple serotypes and variants of the virus (cavanagh, 2007) . ibv is an enveloped, unsegmented, positive sense ssrna virus and has a genome of approximately 27.6 kb in length (casais et al., 2001 (casais et al., , 2003 hodgson et al., 2006) . similar to other coronaviruses, the 5 0 two thirds of the ibv genome encodes two polyproteins, pp1a and pp1ab, and the latter is an extension product of pp1a as a result of a à1 frameshifting (almazan et al., 2004; brierley et al., 1989) . the remaining one third of the genome encodes the structural proteins and group-specific orfs, including spike glycoprotein (s), envelope protein (e), membrane veterinary microbiology 162 (2013) infectious bronchitis virus (ibv) strain h120 was successfully rescued as infectious clone by reverse genetics. thirteen 1.5-2.8 kb fragments contiguously spanning the virus genome were amplified and cloned into pmd19-t. transcription grade complete length cdna was acquired by a modified ''no see'm'' ligation strategy, which employed restriction enzyme bsa i and bsmb i and ligated more than two fragments in one t4 ligase reaction. the full-length genomic cdna was transcribed and its transcript was transfected by electroporation into bhk-21 together with the transcript of nucleocapsid gene. at 48 h post transfection, the medium to culture the transfected bhk-21 cells was harvested and inoculated into 10-days old spf embryonated chicken eggs (ece) to replicate the rescued virus. after passage of the virus in ece five times, the rescued h120 virus (r-h120) was successfully recovered. r-h120 was subsequently identified to possess the introduced silent mutation site in its genome. some biological characteristics of r-h120 such as growth curve, eid50 and ha titers, were tested and all of them were very similar to its parent strain h120. in addition, both r-h120 and h120 induced a comparable titer of ha inhibition (hi) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with mass41 ibv strain. the present study demonstrated that construction of infectious clone from ibv vaccine strain h120 is possible and ibv-h120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach. ß 2012 elsevier b.v. all rights reserved. protein (m) and nucleocapsid protein (n), which are essential for replication of the virus (armesto et al., 2009; fang et al., 2007; youn et al., 2005) . in addition, ibv also encodes a set of accessory proteins of unknown function that may be absent in some strains and not essential for virus replication in vitro (armesto et al., 2009; casais et al., 2005; hodgson et al., 2006; youn et al., 2005) . the reverse genetic system for ibv was firstly established using vaccinia vector (casais et al., 2001) . subsequently, a more elegant protocol to obtain viral infectious rna was developed. in this system, full-length viral cdna was assembled in vitro by orderly ligating viral genomic cdna fragments and directly used as dna template for reverse transcription of viral infectious rna. the new technique was successfully applied to the studies on the role of accessory genes in viral replication (youn et al., 2005) , virulence determinant of beaudette strain (fang et al., 2007) and the relationship between s gene and tissue tropism of the virus casais et al., 2001 casais et al., , 2003 casais et al., , 2005 youn et al., 2005) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were based on vero cell-adapted beaudette strain, which was considered to be poorly immunogenic and never used as a vaccine strain (geilhausen et al., 1973) . therefore, application of the reverse genetics techniques to other strains including vaccine strains could improve the technique on modification of viral genome and provide a powerful tool for novel vaccine development. h120, an attenuated live vaccine strain of massachusetts (mass) serotype, was originally obtained by serial passage of strain h that was isolated in the netherlands in 1956 in embryonated chicken eggs up to the 120th passage (bijlenga et al., 2004) . in recent 50 years, h120 was considered to be one of the safest vaccine strains and used worldwide as a primary vaccine in broilers, breeders, and future layers. the complete genome of h120 was sequenced in our previous study (zhang et al., 2010) . in this study, we describe the in vitro assembly and recovery of an infectious clone of ibv-h120, the biological and immune characteristics of the rescued h120 virus (r-h120), and the potential to use r120 as a candidate of vaccine vector in the future vaccine development. the ibv strains, h120 and mass41, obtained from china institute of veterinary drug control (ivdc), were propagated in the allantoic cavities of the 10-day-old specific pathogen-free (spf) embryonated chicken eggs (ece), and the allantoic fluid was harvested 36 h post inoculation. bhk-21 cells were maintained in dmem containing 10% fbs, 100 u/ml penicillin and 100 mg/ml streptomycin. viral rna was extracted from h120-infected allantoic fluid with trizol 1 reagent (invitrogen, carlsbad, ca) according to the manufacturer's directions. reverse transcription was performed with superscript iii (invitrogen). the ibv-h120 sequence (genbank, accession number fj888351) was used for primer design and nucleotide sequencing. each dna fragment was amplified from cdna templates by pcr using kod plus polymerase (toyobo, japan). pcr primer pairs used to amplify genomic regions are listed in table 1 . pcr amplification of cdna fragments was performed in the following conditions: denaturation at 94 8c for 5 min, 25 cycles of 94 8c for 30 s, 55 8c for 30 s, and 72 8c for 2-6 min depending on the size of the products and final extension at 72 8c for 10 min. the pcr products were isolated from agarose gels and ta-cloned into pmd19-t vector (takara, japan), according to the manufacturer's directions. the entire nucleocapsid (n) orf, including the 3 0 -utr, was amplified by rt-pcr from total rna extracted from h120-infected allantoic fluid, and the rt-pcr product was also cloned into pmd19-t vector. in order to transcribe rna by using t7 rna polymerase, t7 promoter sequence was incorporated to 5 0 end primers of f1 and n-3 0 fragments. in addition, a silent nucleotide change of a to t at position 20,132 nt was incorporated in to f10f primer as a molecular marker. to determine the consensus clone, two to four independent clones of each amplicon were sequenced by sangon biological engineering technology & services co., ltd. each amplicon that represents the consensus sequence of ibv-h120 was then released from cloning vector by restriction enzyme digestion and recovered for construction of the full-length ibv genome in vitro. briefly, the plasmid carrying f1 amplicon was digested with sal i and nci i, and treated with calf intestine alkaline phosphatase (ciap: takara), before digesting with the bsmb i; the plasmid with f13 amplicon was digested with xho i, ciap treated and digested with bsmbi; the plasmids carrying amplicons f2 and f10 were digested with nci i and bsmb i; and the other plasmids inserted amplicon f3, f4, f5, f6, f7, f8, f9, f11 and f12, were digested with bsai. all the digested plasmids were separated on 0.8% agarose gels and the bands corresponding to each amplicon were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc., valencia, ca). the cdna amplicons were ligated orderly with t4 dna ligase as following: (1) ligation of the amplicons into four fragments (f1 + f2 + f3, f4 + f5 + f6, f7 + f8 + f9, and f10 + f11 + f12 + f13) in equal mole ratio and recovering the appropriately sized four fragments from 1% agarose gels and (2) ligation of the four fragments into full-length ibv genome (fig. 1) . the final ligation product was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.4% agarose gel. full-length transcript was generated in vitro using mmessage mmachine 1 t7 kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, 30 ml of transcription reaction with a 1:1 ratio of gtp to cap analog was sequentially incubated at 37 8c for 120 min. a similar reaction was performed for the n transcripts, which were generated by using a linearized pmd19-t-n-3 0 containing the ibv n gene and the 3 0 -utr region as templates. a 1:2 ratio of gtp to cap analog was used for the transcription of ibv n gene (fang et al., 2007; youn et al., 2005) . in the same time, bhk-21 cells were grown on monolayers to 80% confluence, treated with trypsin, washed with cold depc-treated pbs twice, and resuspended in depc-treated pbs at a concentration of 10 7 cells/ml. rna transcripts were added to 600 ml of bhk-21 cell suspension in microfuge tubes on ice, gently pipetted and transferred to electroporation cuvettes. three consecutive electrical pulses of 850 v at 25 af were given, using the gene pulser xcell tm electroporation system (bio-rad). the transfected bhk-21 cells were incubated at 37 8c for 48 h in dmem supplemented with 10% fbs (invitrogen). after 48 h, the virus containing medium was collected and inoculated into the 10-day-old spf embryonated chicken eggs. after 5 passages in ece, allantoic fluid was harvested according to the previous description (cavanagh, 1983) . to differentiate the rescued viruses from the parental viruses, viral rnas were extracted from allantoic fluid (r-h120 and parental h120) as described previously. rt-pcr was performed with selected primer pairs (5 0 -aatataagacagagcacaag-3 0 , 5 0 -ctgtcatacaaagcagcactaca-3 0 ) to amplify the fragment consisting of the silent nucleotide change of a to t at position 20,132 nt. the pcr fragment was cloned and sequenced to check the silent mutation site. to determine 50% egg infection dose (eid50) of the r-h120, serial 10-fold dilutions (10 à1 -10 à9 ) of the amplified virus were inoculated into the 10-day-old spf ece. for each dilution, 0.2 ml of virus solution was injected into each egg and four eggs were used for each dilution. the parental strain h120 was used as a positive control. the eid50 calculation was based on reed and muench (1938) method. r-h120 virus hemagglutination antigen was prepared by using the filtrate from a clostridium welchii to treat the 100-fold concentration. ha antigens were doubly diluted in a range of 1:2-1:2048 and titrated with 0.75% chicken erythrocytes. parental h120, spf allantoic fluid (free ibv) and pbs were used as controls. to determine the growth kinetics of the rescued virus r-h120, the viral titre (eid50) were tested. in this procedure, the parental strain h120 was a postive control. briefly, 0.1 ml of virus 10 3.0 eid50 titre of r-h120 and h120 were inoculated into the allantoic cavities of 10-days old embryonated eggs, and the allantoic fluid of six eggs from each group were harvested at the time points of 12, 24, 36,48, 60 and 72 h post inoculation and pooled for the determination eid50, were carried out as described by the office of international des epizooties. all the assays were run in triplicates and the eid50 of each virus were calculated according to standard curve. 2.5. immunization characteristics of r-h120 as a vaccine vector candidate 2.5.1. immunization of chickens with r-h120 to determine the immunization characteristics of r-h120 virus, 60 chickens of 7-day-old were randomly divided into three groups and raised separately. r-h120 virus at 10 3.0 eid50/ml (group 1) was injected intramuscularly to both quadriceps; h120 virus at 10 3.0 eid50/ml (group 2) was injected in the same way; and 0.5 ml pbs (group 3) was used as control injection. all groups were boosted with an equivalent dose on 14 days post the initial inoculation. serum samples were taken weekly from the three groups of immunized chickens on 7, 14, 21, 28, and 35-day posthatch for haemagglutination inhibition (hi) assay. the hi tests were carried out as described by the office of international des epizooties (oie, 2008) . the maximum dilution of each serum sample that caused ha inhibition (hi) was recorded as the endpoint of dilution and the hi titers were determined as the geometric mean titer (gmt) of log 2 . the immunized chickens were challenged with 10 3 eid50 of ibv strain mass41 in 0.1 ml by the nasal ocular route on the 36th day after the hatching. chickens were examined daily for 2 weeks for the clinical symptoms such as coughing, sneezing, dyspnea or death. dead chickens were necropsied to confirm that the death was due to ibv infection. the chickens in each group were euthanized on 14 days post challenge. kidney and lung tissues were collected individually from either the dead or euthanized chickens and virus in these tissues was detected by rt-pcr. 2043 bp, 1965 bp, 2020 bp, 2107 bp, 1985 bp, 1921 bp, 1559 bp, respectively assembly of the 13 fragments into four 1/4 full-length cdna fragments (fa: f1 + f2 + f3 (7.2 kb), fb: f4 + f5 + f6 (7 kb), fc: f7 + f8 + f9 (6 kb), and fd: f10 + f11 + f12 + f13 (7.6 kb)), then assembly of the four 1/4 full-length cdna fragments into a full-length cdna clone, and in vitro transcription of the full-length transcripts. thirteen 1.5-2.8 kb fragments spanning the entire ibv genome, designating f1-f13, were obtained by rt-pcr and all the fragments do have the same size as the predicated from h120 sequence. to facilitate the assembly of the fulllength cdna in vitro, each fragment has been ranged between two restriction enzymes in h120-ibv genome, e.g., bsmbi or bsai, either at the 5 0 or 3 0 ends of each fragment (fig. 1) . the amplified pcr fragments were recovered from agarose gel and all of them were successfully cloned into pmd19-t cloning vector. determined by sequencing, the consensus clones including those that contains the correctly introduced sequences such as t7 promoter at 5 0 -end of f1 and poly(a) 30 at 3 0 -end of f13 were chosen to construct the full-length cdna clone. the 13 cdna fragments were prepared by digestion of the corresponding plasmids with assigned restriction enzymes such as bsmbi and bsai and recovery from agarose gel. the full-length cdna was assembled by orderly ligation of the purified fragments in vitro, which in turn was used as the template for in vitro transcription. coronavirus n gene transcripts were shown to enhance the recovery of the rescued virus from the in vitro-synthesized full-length transcripts (casais et al., 2001; youn et al., 2005; yount et al., 2000 yount et al., , 2002 yount et al., , 2003 , therefore, n-gene orf was also prepared for transcription in vitro (fig. 2a3) . the full-length transcript together with the n transcript was electroporated into bhk-21 cells and cytopathic effects (cpe) including cell clustering and falling-off were observed on two days post transfection. to amplify the rescued virus, the supernatant from transfected cell culture was harvested and inoculated into allantoic cavities of embryonated chicken eggs for replication of the rescued virus. after 5 passages in ece, typical embryo lesions such as curling, stunting and dwarfing were observed, which indicated the successful rescue of h120 virus from infectious rna. finally, the rescued virus was designated as ibv r-h120. the recovery of r-h120 from the ligated cdna was confirmed by rt-pcr amplifying and sequencing the genomic region that contained molecular marker to distinguish r-h120 from h120. as expected, a nucleotide change at 20,132 nt was identified in r-h120 genome. the virus titer and growth curve of r-h120 were also estimated and compared with the parental strain h120. the results showed that (1) r-h120 was pathogenic to chicken embryos and the eid50 titer was comparable to its parental virus h120 (fig. 3a) ; (2) the tested ha titers of r-h120 was almost equal to that of h120 (fig. 3b) ; and (3) like the parental strain h120, r-h120 could replicate in ece cells and both viruses had very similar growth pattern (fig. 3c) . the above experimental data indicated that the rescued virus shared several essential properties with its parental virus h120. 3.4. serum hi antibody assay in chicken after immunizing the h120 and r-h120 to estimate the immunogenicity of the rescued virus, an important factor for using it to develop vaccine, r-h120 and h120 viruses were immunized to two groups of chickens and the induced serum antibody was titrated. the dynamic changes of hi antibody titer following the inoculation of r-h120 and h120 were observed (table 2 ) and no statistic difference of hi antibody titer was found between r-h120 and h120 groups. however, a significant difference was fig. 3 . biological characteristic of the r-h120. (a) the eid50 of the r-h120,the parental strain h120 was the positive control, spf allantoic fluid (free ibv) and pbs were the negative controls. (b) ha titer of the r-h120, parental strain h120 was the positive control, spf allantoic fluid (free ibv) and pbs were the negative controls. (c) comparison of the replication kinetics of r-h120 and h120 in ece cell. the rescued virus h120 (r-h120 and parent h120 (0.1 ml of 100 eid50) were inoculated into the allantoic cavities of 10-day old embryonated eggs, and the allantoic fluid of six eggs from each group was harvested at the time points of 12, 24, 36, 48,60 and 72 h post inoculation and pooled for the determination of eid50 in ece. detected between pbs and virus inoculated groups (p < 0.05). three groups of chickens were challenged with the virulent strain mass41 to test if the rescued virus could protect the immunized animals from ibv infection. on the 5th day post challenge, chickens started to show clinical signs of infection or death. the data shown affected chickens in each group after challenge, such as mortality rate and protection rate, were listed in table 3 . the chickens injected with pbs alone were not protected from ibv infection and developed cough, nasal discharge and dyspnea. the death rate in the group injected with pbs reached to 85%, but it was only 5% for both groups 1 and 2. to evaluate the level of protection, shielding virus in the collected lung and kidney samples were estimated by rt-pcr. the results showed that 15% of the chickens in groups 1 and 2 had virus shielding in their lungs and/or kidney in compared with 100% in control group (pbs). the results suggested that r-h120 and h120 both provide protection to immunized chickens from virulent ibv challenge. through different methodologies, vaccinia virus vectors or bac as cloning systems and the in vitro assembly strategy, reverse genetics systems of ibv and other coronaviruses (tgev, hcov 229e, mhv-a59, and sars-cov) have been constructed (almazá n et al., 2000; casais et al., 2001; yount et al., 2002 yount et al., , 2003 . for the purposes of studying the mechanisms of pathogenesis, or building transfer vectors, so far, casais et al. (2001) , youn et al. (2005) and fang et al. (2007) established the reverse genetic system for vero cell-adapted beaudette strain. after that, this reverse genetic system for vero cell-adapted beaudette strain are used to research the pathogenicity, tissue tropism and functionality of accessory gene of the ibv (armesto et al., 2009 (armesto et al., , 2011 britton et al., 2006; casais et al., 2003; hodgson et al., 2006) . previous reverse genetic system set for coronavirus was assembly of a genomic length cdna from 6 to 8 contiguous cdna subclones in size 3-7 kb (fang et al., 2007; youn et al., 2005) . although this segmentation strategy can decrease the number of cdna fragments and is easier to link to full-lengthen cdna, it is difficult to get the longer pcr product with correct sequence and clone these cdna fragments. in addition, the recombined plasmid containing larger fragments may be harmful to host bacteria, resulting some of them could not replicate in escherichia coli host cells. in order to solve these problems, certain modifications for assembling of ibv genomic rna in vitro were made to improve the efficiency of generating full-length cdna. in this study, two improvements for assembly of full-length viral genome were applied. at first, the ibv genome was cloned as thirteen 1.5-2.8 kb fragments. the shortened length of cdna fragments made them easier to clone, with less sequence mutation in each fragment and more stable in bacteria. secondly, three or four fragments were ligated in one t4 dna ligase reaction to reduce the number of recovery times which was negative correlated to dna recovery rate from agarose gels. the genome of the avain coronavirus was established to provide a tool not only for the mechanism studies but also for the development of new vaccines (matthijs et al., 2005; ziebuhr, 2005) . in this study, we were established a reverse genetics system based on h120 strain of ibv, a worldwide used attenuated live vaccine strain of massachusetts (mass) serotype. in china, h120 is always considered as the first vaccine selection. compared with the vero cell-adapted beaudette strain, h120 can be developed as the vaccine vector to express other antigens since it has a stronger immunogenicity than that of beaudette strain, although the latter was a widely used model of ibv on reverse genetics study and never considered as a vaccine strain (geilhausen et al., 1973) . according to the present data, the biological and immune data within a column with different letters differ significantly (p < 0.05); the same letters means no significance. protective effects in 4-week-old spf chickens immunized with r-h120 and h120 against challenge of ibv mass41 strain. characteristics of r-h120 including the eid50 and ha titer, growth curve and protection of immunized animals from challenge were all very similar to its parental strain h120. the same situation was found in other covs such as ibv beaudette strain (fang et al., 2007) , mhv (yount et al., 2002) , tgev (yount et al., 2000) , ndv (hu et al., 2009 ) and avian influenza virus (hoffmann et al., 2002; jackson et al., 2008) . it might imply that h120 was a good strain for the development of engineering avian vaccines. in addition, the 27.6 kb genome of h120 as a vaccine vector can be recombined with larger pathogenic antigen gene compared with 15 kb newcastle disease virus vaccine vector, which was a vector used presently to express different pathogenic antigen genes as vaccine to protect the immunized animals from correspondence pathogen's challenge (dinapoli et al., 2007; nakaya et al., 2001; veits et al., 2006) . it would make the procedure of constructing reverse genetics system labor intensive if a continuous cell line could not be used in infectious rna transfection and rescued virus replication. in our study, the continuous cell line, bhk-21, was applied to generating modified virus after electroporation, indicating that bhk-21 can be utilized to rescue virus from infectious rna although bhk-21 was never used to replicate h120 virus before. at present, most ibv strains replicate in primary cells like ck and cek and do not adapt to continuous cell lines (casais et al., 2003) . therefore, in vitro assembly strategy is applicable to ibv strains that were not adapted to the commonly used continuous cell lines through the established approach in the present study. it is interesting to find that the rescued virus r-h120 and h120 viruses cannot efficiently replicate in bhk21 and make the cells emerge obvious cpe, but r-h120 do rescue from bhk21 cell. in next step, the s gene of h120 may be modified by reverse genetics to find out bhk21 adapted h120 that may reveal the information on cell tropism of the virus. in conclusion, the present study demonstrated that the rescued virus r-h120 has similar biological and immunogenic characteristics with its parental strain h120 and it would be possible to develop novel avian vaccine through reverse genetics approach by modification or recombination of the present vaccine virus h120. engineering the largest rna virus genome as an infectious bacterial artificial chromosome the nucleoprotein is required for efficient coronavirus genome replication the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot genes 3 and 5 of infectious bronchitis virus are accessory protein genes gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism reverse genetics system for the avian coronavirus infectious bronchitis virus coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides nidovirales: a new order comprising coronaviridae and arteriviridae coronavirus avian infectious bronchitis virus longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells the pathogenesis of virulent and avirulent avian infectious bronchitis virus neither the rna nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication eightplasmid system for rapid generation of influenza virus vaccines a vaccine candidate of attenuated genotype vii newcastle disease virus generated by reverse genetics a new influenza virus virulence determinant: the ns1 protein four cterminal residues modulate pathogenicity effect of ibv-h120 vaccination in broilers on colibacillosis susceptibility after infection with a virulent massachusetts-type ibv strain recombinant newcastle disease virus as a vaccine vector manual of diagnostic tests and vaccines for terrestrial animals a simple method of estimating fifty per cent endpoints coronaviruses of domestic livestock and poultry: interspecies transmission, pathogenesis, and immunity the springer index of viruses newcastle disease virus expressing h5 hemagglutinin gene protects chickens against newcastle disease and avian influenza in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h120 the coronavirus replicase key: cord-309623-2ngr682l authors: han, xiaoxiao; tian, yiming; guan, ru; gao, wenqian; yang, xin; zhou, long; wang, hongning title: infectious bronchitis virus infection induces apoptosis during replication in chicken macrophage hd11 cells date: 2017-07-26 journal: viruses doi: 10.3390/v9080198 sha: doc_id: 309623 cord_uid: 2ngr682l avian infectious bronchitis has caused huge economic losses in the poultry industry. previous studies have reported that infectious bronchitis virus (ibv) infection can produce cytopathic effects (cpe) and apoptosis in some mammalian cells and primary cells. however, there is little research on ibv-induced immune cell apoptosis. in this study, chicken macrophage hd11 cells were established as a cellular model that is permissive to ibv infection. then, ibv-induced apoptosis was observed through a cell viability assay, morphological changes, and flow cytometry. the activity of caspases, the inhibitory efficacy of caspase-inhibitors and the expression of apoptotic genes further suggested the activation of apoptosis through both intrinsic and extrinsic pathways in ibv-infected hd11 cells. additionally, ammonium chloride (nh(4)cl) pretreated hd11 cells blocked ibv from entering cells and inhibited ibv-induced apoptosis. uv-inactivated ibv also lost the ability of apoptosis induction. ibv replication was increased by blocking caspase activation. this study presents a chicken macrophage cell line that will enable further analysis of ibv infection and offers novel insights into the mechanisms of ibv-induced apoptosis in immune cells. infectious bronchitis virus (ibv) can cause avian infectious bronchitis, an acute and highly infectious disease of chicken. ibv is a member of the family coronaviridae and genus coronavirus. it is a single stranded positive sense, enveloped rna virus 27-32 kb in length [1, 2] . like some other members of the coronavirus family, ibv mainly causes upper-respiratory tract disease. ibv is characterized by nephritis, proventriculitis and reduction in both laying rate and egg quality in infected chickens [3] . vaccination is an effective prevention measure, but ibv's ability to mutate has decreased vaccine protection [4] [5] [6] . in order to develop better prevention and control measures, the interactions between host and ibv needs to be better studied. almost all wild-type ibv strains are only able to proliferate in embryonated chicken eggs or primary chicken embryo kidney cells. the beaudette strains were used previously to study the resistance of ibv to the antiviral state induced by type i interferon (ifn) [7] , induction of apoptosis through endoplasmic reticulum stress in vero cells by ibv infection [8] and activate autophagy by ibv nonstructural protein (nsp) 6 [9] . however, there have been limited studies of the interactions between ibv infection and immune cell apoptosis. here, the beaudette strain was used to study the mechanism of ibv infection. apoptosis is a form of programmed cell death that results from the activation of intracellular self-destruction biochemical programs [10] . the activation of caspases (a family of cysteine protease) is a significant regulatory event in the apoptosis process [11] . caspase cascades are triggered by both extrinsic and intrinsic signals to mediate the cell apoptosis [12] . the relationship between cell apoptosis and virus infection is complex. cell apoptosis induced by virus may cause tissue damage, especially in the immune and nervous systems, suggesting that apoptosis is a pathogenic mechanism in virus-induced disease. at the same time, apoptosis of infected cells can directly interfere with viral replication, and immune cells can engulf apoptotic cells to prevent inflammation [13, 14] . previous studies demonstrated that ibv induced apoptosis in cultured mammalian cells and primary cells [6, 15, 16] . however, there is limited information about the apoptosis signaling pathways induced by ibv infection in immune cells. some studies indicated that ibv can transform certain elements of the innate immune system to promote secondary bacterial infections, and macrophages, as an important component of the innate immune system, may play a role in this process [17] . a nephropathogenic ibv strain (b1648) can productively replicate in blood monocytic cells, and the infected cells may act as carrier cells to play a crucial role in cell-associated viremia and the dissemination of virus to the internal organs [18] . some viruses have been shown to induce apoptosis in macrophage, like human immunodeficiency virus (hiv)-1 [19] , chikungunya virus (chikv) [20] and influenza virus [21] . therefore, additional study is required to investigate the functional roles of macrophages in ibv infection to help understand the mechanistic details of immune responses during virus infections [22] . in this report, we used chicken macrophage hd11 cells considered an accurate representation of primary avian macrophages [23, 24] . the hd11 cells were identified and characterized as a novel model that is permissive to ibv infection. the molecular and morphological variations in ibv-infected cells revealed that cell apoptosis was induced by ibv infection and appeared to activate caspase-8 by the fas/fas ligand (fasl)-mediated signaling pathway and to activate caspase-9 by the b-cell lymphoma 2 (bcl-2) family-mediated signaling pathway. apoptosis required viral replication in ibv-infected cells. the chicken macrophage hd11 cells were kindly provided by prof. xin-an jiao (jiangsu key laboratory of zoonosis, yang zhou university, yang zhou, jiangsu province, china). hd11 cells were cultured in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut, usa) supplemented with 10% fetal bovine serum (fbs) (gemini bio-products, west sacramento, ca, usa), 100 u/ml penicillin and 100 µg/ml streptomycin (hyclone, logan, ut, usa) at ph 7.2 and were kept at 37 • c with 5% co 2 . the vero cell-adapted ibv beaudette strain (p65) [25] used in the current study was kindly provided by prof. shi-qi sun (state key laboratory of veterinary etiological biology, lanzhou veterinary research institute, chinese academy of agricultural sciences, gansu province, china). traditional ibv strain m41, vaccine ibv strain h120 and virulent ibv strain sabik2 [26] were housed in our laboratory (viruses were propagated in specific pathogen free (spf) 10 days old embryonated chicken eggs). susceptibility of hd11 cells to ibvs was measured by morphological changes, growth curves using 50% tissue culture infective doses (tcid 50 ) and indirect immunofluorescence assay (ifa). here, tcid 50 were applied to hd11 cells to quantitate virus titers as described previously [27] . hd11 cells were cultured in 96-well plates, and ten-fold dilutions of the virus were prepared in dmem supplemented with 2% fbs. cultured cells were infected with the virus and then observed daily for cytopathic effects (cpe). in order to evaluate the virus growth kinetics in hd11 cells, the cells were infected with ibv at 10 multiplicity of infection (moi). the infected cells were collected at the indicated time points and analyzed using tcid 50 assay. a cell counting kit-8 (cck-8) assay (beyotime, haimen, jiangsu province, china) was utilized to identify the viability of cells as described previously [28] . hd11 cells were cultured in 96-well plates and infected with ibv at different moi (0.1, 0.5, 1, 5, and 10 moi) for specific lengths of times. in parallel, a negative control was set up. the cells were incubated with 10 µl/well cck-8 solution (beyotime, haimen, jiangsu province, china) and allowed to react for 2 h at 37 • c. the absorbance was detected using a microplate reader (model 680, bio-rad, hercules, ca, usa) at 450 nm. the negative control was set at 100%, and the treated samples were calculated according to the following formula: survival rate (%) = optical density (od) of the treated cells/od of the negative control × 100. hd11 cells were pre-incubated in 96-well plates and infected with ibv at 10 moi. to assess apoptosis, the condensed and fragmented nuclei were observed using hoechst 33342 staining (keygen biotech, nanjing, jiangsu province, china). at the specified time points, the cells were immobilized with 4% paraformaldehyde (keygen biotech, nanjing, jiangsu province, china) for 30 min and then incubated with hoechst 33342 (keygen biotech) in the dark for 15 min. the typical apoptotic morphological changes were observed using a fluorescence microscope (olympus ix71, olympus, tokyo, japan) with uv excitation at 350 nm. hd11 cells were grown overnight to 75% density in 96-well plates and were then infected with ibv at an moi of 10. after the appearance of typical cpe, the cells were immobilized with 4% paraformaldehyde for 30 min. a mouse polyclonal antibody against the ibv nucleocapsid (n) protein (1:200 dilution, prepared in our laboratory) was added, followed by incubation for 1 h at 37 • c. next, the cells were treated with a fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg (1:200, transgen biotech, beijing, china) for 1 h at 37 • c. the specimens were viewed with an olympus ix71 fluorescence microscope (olympus) with the appropriate excitation and emission wavelengths for fitc (490 nm and 525 nm, respectively). to identify the apoptotic rate, the percentage of cells undergoing apoptosis was determined by an annexin v-fitc apoptosis detection kit (absin, shanghai, china). hd11 cells were cultured in 6-well plates and infected with ibv at 10 moi. cells were harvested and washed three times with phosphate-buffered saline (pbs) at the indicated times. the cells were centrifuged at 500 g for 5 min and then suspended in 500 µl of binding buffer containing 5 µl fitc-conjugated annexin v antibody and 5 µl propidium iodide (pi). the mixture was incubated at room temperature for 15 min in the dark. the cells were detected by flow cytometer (bd biosciences, san jose, ca, usa) within an hour. the activities of caspase-3, -8, and -9 were detected by colorimetric assay kit (keygen biotech). the cells were incubated with lysis buffer, and the concentrations of protein were detected by bicinchoninic acid (bca) protein assay reagent (vazyme biotech, nanjing, jiangsu province, china). the protein (200 µg/sample) was treated with caspase-3, -8, and -9 substrate for each sample at 37 • c for 4 h. samples were read by a microplate reader (model 680, bio-rad) at 405 nm. total rna was isolated using trizol agent (invitrogen, carlsbad, ca, usa), and each rna sample was reverse-transcribed to complementary dna (cdna) by primescript™ rt reagent kit (takara, dalian, liaoning province, china). cdna was used for quantitative real-time polymerase chain reaction (qrt-pcr) analysis. the sets of primer pairs of apoptotic regulating genes are listed in table 1 [29] . for qrt-pcr reactions, the 25 µl reaction mixture included 2 µl cdna, 12.5 µl sybr premix ex taqtm ii (takara), 1.0 µl of forward and 1.0 µl of reverse primer and 8.5 µl rnaase-free water (takara). reaction conditions were 95 • c for 3 min followed by 44 cycles of 95 • c for 10 s, the specific melting temperature (tm) of a primer pair for 30 s, and then 95 • c for 10 s, and 72 • c for 10 s, using a bio-rad iq5 thermal cycler (bio-rad). β-actin was selected as a reference gene. the expression fold changes were calculated using the 2 −∆∆ct method [30] . table 1 . sequences of chicken primer pairs used for quantitative real-time polymerase chain reaction (qrt-pcr). fas all data are expressed as the mean ± standard error of the mean (sem) from three independent experiments performed in triplicate. the statistical analyses were conducted using student's t-test in graphpad prism version 5 (graphpad software, san diego, ca, usa). a p-value < 0.05 was considered significant, and a p-value < 0.01 was considered highly significant. to determine whether ibvs replicate in the chicken macrophage cell line, four ibv strains (beaudette, m41, h120 and sabik2 strains) were utilized in this study to test the infective processes in hd11 cells. for the m41, h120 and sabik2 strains, the infected cells were blindly passaged five times, and cpe was not observed. the growth curve using tcid 50 and ifa showed that replication of the three ibv strains in hd11 cells was severely restricted, and no significant replication was observed. however, the hd11 cells were highly permissive for the propagation of the attenuated ibv beaudette strain. the results showed that hd11 cells could be infected by ibv beaudette in passage one. first, morphological changes of ibv beaudette-infected hd11 cells were observed. after infection with ibv beaudette at 10 moi, cpe appeared in hd11 cells at 24 h post-infection (h.p.i.) and were evident at 36 h.p.i. when compared with the mock infection ( figure 1a ). the normal hd11 cells could also be re-infected with the culture supernatant from the virally infected cells. the susceptibility of hd11 cells to ibv beaudette infection was evaluated by growth kinetics. the growth kinetics of the virus were observed upon infection at an moi of 10 in hd11 cells. the virus titers increased until reaching the maximal level of 10 6.875 tcid 50 /ml ( figure 1b ). ibv beaudette replication in hd11 cells was also studied by performing an immunofluorescence assay. the production of fitc-stained virus was observed by 24 h.p.i. in contrast, mock-infected hd11 cells showed no fluorescence ( figure 1c ). infection of hd11 cells with ibv beaudette caused cell death in a time-and dose-dependent manner, as tested by cck-8 assay. (figure 2a ). the infected cells showed chromatin condensation and nuclear fragmentation. after 36h of infection, large amounts of apoptotic bodies were observed in hd11 cells ( figure 2b ). the rate of apoptotic cells was measured by flow cytometry. the rate of apoptosis significantly increased at 12 h.p.i. in virus-infected cells when compared with the mock infection of hd11 cells with ibv beaudette caused cell death in a time-and dose-dependent manner, as tested by cck-8 assay. (figure 2a ). the infected cells showed chromatin condensation and nuclear fragmentation. after 36 h of infection, large amounts of apoptotic bodies were observed in hd11 cells ( figure 2b ). the rate of apoptotic cells was measured by flow cytometry. the rate of apoptosis significantly increased at 12 h.p.i. in virus-infected cells when compared with the mock-infected cells ( figure 2c ). these findings indicated that apoptosis was induced by ibv beaudette infection in hd11 cells. viruses 2017, 9, 198 6 of 14 infected cells ( figure 2c ). these findings indicated that apoptosis was induced by ibv beaudette infection in hd11 cells. activation of the caspase proteinases is a significant event in the occurrence of apoptosis. the activity of caspases that play important roles in the activation of the apoptosis pathway was activation of the caspase proteinases is a significant event in the occurrence of apoptosis. the activity of caspases that play important roles in the activation of the apoptosis pathway was investigated in this study. when hd11 cells were infected with ibv beaudette at 10 moi, the levels of caspase-3, -8, and -9 were significantly increased from 8 h.p.i. and then increased further over time ( figure 3a ). to further identify the function of caspase-3, -8, and -9 in the apoptotic pathway, we measured the viability of infected-cells incubated with specific inhibitors of caspase-3, -8, and -9 (z-devd-fmk, z-ietd-fmk, and z-lehd-fmk; keygen biotech, nanjing, jiangsu province, china). the data revealed that cell viability was significantly increased by the specific inhibition of caspase-3, -8, and -9 ( figure 3b ). to confirm the function of caspase-8 and caspase-9 to activate caspase-3, the inhibitory efficacy of the caspase-8 or caspase-9 inhibitors on caspase-3 activity was also determined. when cells were pretreated with the caspase-8 or caspase-9 inhibitor, the activity of caspase-3 was significantly decreased in cells, and more significantly decreased when the two inhibitors were added together ( figure 3c ). these results revealed that caspase-3 activation and ibv beaudette-induced apoptosis may be triggered via both extrinsic and intrinsic pathways. viruses 2017, 9, 198 7 of 14 investigated in this study. when hd11 cells were infected with ibv beaudette at 10 moi, the levels of caspase-3, -8, and -9 were significantly increased from 8 h.p.i. and then increased further over time ( figure 3a ). to further identify the function of caspase-3, -8, and -9 in the apoptotic pathway, we measured the viability of infected-cells incubated with specific inhibitors of caspase-3, -8, and -9 (z-devd-fmk, z-ietd-fmk, and z-lehd-fmk; keygen biotech, nanjing, jiangsu province, china). the data revealed that cell viability was significantly increased by the specific inhibition of caspase-3, -8, and -9 ( figure 3b ). to confirm the function of caspase-8 and caspase-9 to activate caspase-3, the inhibitory efficacy of the caspase-8 or caspase-9 inhibitors on caspase-3 activity was also determined. when cells were pretreated with the caspase-8 or caspase-9 inhibitor, the activity of caspase-3 was significantly decreased in cells, and more significantly decreased when the two inhibitors were added together ( figure 3c ). these results revealed that caspase-3 activation and ibv beaudette-induced apoptosis may be triggered via both extrinsic and intrinsic pathways. caspase-8 has an important effect on apoptosis that is mediated by fas/fasl. the activity of caspase-8 was increased in the ibv beaudette-infected hd11 cells. this implied that apoptosis is induced by ibv beaudette infection through the fas/fasl pathway. to investigate this further, the expression levels of fas and fasl were detected in ibv beaudette-infected hd11 cells by qrt-pcr. the data revealed increased gene expression of fas and fasl over time ( figure 4a) . furthermore, the the data are shown as the mean ± sem, * p < 0.05, ** p < 0.01 versus ibv infection alone. (c) the effect of initiator caspase-8 or -9 on the activation of caspase-3: 20 µm of each caspase inhibitor was utilized to pretreat cells for 2 h. then, the treated and untreated cells were infected with ibv at 10 moi for 36 h. caspase-3 activity was detected using a colorimetric assay kit. data are shown as the mean ± sem, * p < 0.05, ** p < 0.01 versus virus infection alone. caspase-8 has an important effect on apoptosis that is mediated by fas/fasl. the activity of caspase-8 was increased in the ibv beaudette-infected hd11 cells. this implied that apoptosis is induced by ibv beaudette infection through the fas/fasl pathway. to investigate this further, the expression levels of fas and fasl were detected in ibv beaudette-infected hd11 cells by qrt-pcr. the data revealed increased gene expression of fas and fasl over time ( figure 4a) . furthermore, the members of the bcl-2 family are generally distributed on the surface of mitochondria, and their activation may regulate the intrinsic apoptosis pathway. to test this, the expression levels of bcl-2 and bcl-2-associated x protein (bax) were quantified by qrt-pcr in ibv beaudette-infected hd11 cells. the results showed the mrna levels of bcl-2 were obviously downregulated from 24 h.p.i. and declined over time. conversely, the mrna levels of bax were upregulated from 4 h.p.i. and continuously increased until 48 h.p.i ( figure 4b) . moreover, the activation of caspase-9 was partly inhibited in ibv beaudette-infected cells pretreated with the inhibitor of caspase-8 ( figure 4c ). this suggested that caspase-9 activation was affected by the blocking of caspase-8 activity. taken together, these findings suggested that the fas/fasl-mediated signal contributes to the activation of caspase-8. additionally, bcl-2 and bax might play important roles in regulating the activation of caspase-9. the activation of caspase-8 can also affect the extrinsic apoptosis pathway in ibv beaudette-infected cells. viruses 2017, 9, 198 8 of 14 members of the bcl-2 family are generally distributed on the surface of mitochondria, and their activation may regulate the intrinsic apoptosis pathway. to test this, the expression levels of bcl-2 and bcl-2-associated x protein (bax) were quantified by qrt-pcr in ibv beaudette-infected hd11 cells. the results showed the mrna levels of bcl-2 were obviously downregulated from 24 h.p.i. and declined over time. conversely, the mrna levels of bax were upregulated from 4 h.p.i. and continuously increased until 48 h.p.i ( figure 4b) . moreover, the activation of caspase-9 was partly inhibited in ibv beaudette-infected cells pretreated with the inhibitor of caspase-8 ( figure 4c ). this suggested that caspase-9 activation was affected by the blocking of caspase-8 activity. taken together, these findings suggested that the fas/fasl-mediated signal contributes to the activation of caspase-8. additionally, bcl-2 and bax might play important roles in regulating the activation of caspase-9. the activation of caspase-8 can also affect the extrinsic apoptosis pathway in ibv beaudette-infected cells. following incubation with z-ietd-fmk for 2 h, the cells were infected with ibv beaudette for 24 h. caspase-9 activity was detected using a colorimetric assay kit. data are shown as the mean ± sem. * p < 0.05 versus virus infection alone. to determine whether apoptosis plays a pivotal role in inhibition of virus replication, the virus titers of untreated cells or those treated with caspase inhibitors were detected by tcid 50 . the results showed that the caspase-3 inhibitor could increase the titer of ibv beaudette, but did not show obvious effects on caspase-8 and -9 inhibitor-treated cells ( figure 5a ). to test whether the ability of virions to enter cells was crucial to apoptosis, endosomal acidification was blocked by nh 4 cl to prevent the viruses from being released [31] . the virus titer was significantly decreased with nh 4 cl treatment ( figure 5b ). compare with non-treated cells, the rate of apoptotic cells was also decreased in nh 4 cl-treated cells when infected with ibv beaudette ( figure 5c ). next, the uv-treated virus was used to test whether apoptosis induction required virus replication. when the virus was subjected to uv treatment, the virus titer could not be detected ( figure 5d ). consistently, the rate of apoptotic cells was dramatically decreased in cells infected with uv-inactivated virus, when compared with the uv-untreated virus ( figure 5e ). in conclusion, apoptosis induction required viral replication in ibv beaudette-infected cells. viruses 2017, 9, 198 9 of 14 to determine whether apoptosis plays a pivotal role in inhibition of virus replication, the virus titers of untreated cells or those treated with caspase inhibitors were detected by tcid50. the results showed that the caspase-3 inhibitor could increase the titer of ibv beaudette, but did not show obvious effects on caspase-8 and -9 inhibitor-treated cells ( figure 5a ). to test whether the ability of virions to enter cells was crucial to apoptosis, endosomal acidification was blocked by nh4cl to prevent the viruses from being released [31] . the virus titer was significantly decreased with nh4cl treatment ( figure 5b ). compare with non-treated cells, the rate of apoptotic cells was also decreased in nh4cl-treated cells when infected with ibv beaudette ( figure 5c ). next, the uv-treated virus was used to test whether apoptosis induction required virus replication. when the virus was subjected to uv treatment, the virus titer could not be detected ( figure 5d ). consistently, the rate of apoptotic cells was dramatically decreased in cells infected with uv-inactivated virus, when compared with the uv-untreated virus ( figure 5e ). in conclusion, apoptosis induction required viral replication in ibv beaudette-infected cells. compared with other coronaviruses, ibv is not easily adapted to cell culture. several mammalian cell lines and primary cells have been previously revealed to be permissive to ibv infection. some strains of ibv can replicate and produce cpe in primary chicken embryo kidney cells. ibv holte and beaudette-42 strains can proliferate in the bhk-21 cell line [32] , and the beaudette strain of embryo-culture ibv has adapted to vero cells [33] . however, previous studies of chicken immune cells and the pathogenesis of ibv focused on primary immune cells separated from the blood or spleen [18, 34] . in this study, hd11 cells, a chicken macrophage cell line, were shown to be susceptible to ibv beaudette. additionally, the ibv beaudette-infected cells produced infectious virus progeny with a high virus titer. morphological assessment of the cells during ibv beaudette infection showed that cpe can be observed after 24 h.p.i. the virus growth kinetics for hd11 cells also showed peak viral titers at 36 h.p.i. immunofluorescence was used to identify and analyze virus infection, and strong fluorescence signals were observed in the ibv beaudette-infected cells. based on these results, the chicken macrophage hd11 cells will serve as an essential tool for future studies of ibv infection. apoptosis is an important part of the antiviral host response. however, some viruses actively trigger this process to facilitate their replication [13] . infection with coronavirus induced apoptosis in various cell types. transmissible gastroenteritis virus (tgev)-induced apoptosis in pk-15 cells was dependent on viral replication [31] . porcine hemagglutinating encephalomyelitis virus (phev) induced apoptosis through a caspase-dependent pathway in pk-15 cells [35] . severe acute respiratory syndrome (sars) coronavirus membrane (m) and nucleocapsid (n) proteins can induce apoptosis in hpf cells [36] . according to previous studies, apoptosis occurs in response to ibv infection in vero cells, df1 cells and chicken embryo kidney cells [16, 37] . this study is the first demonstration that ibv induces apoptosis in chicken macrophage hd11 cells. the ibv beaudette-infected hd11 cells exhibited typical characteristics of apoptosis including the condensation of the cell nucleus, reduction of cell viability, and an increased rate of apoptotic cells. caspases are a family of cysteine-catalyzed proteases that cleave aspartic acids. the triggering of caspase cascades plays indispensable roles in apoptosis and can be induced by many viruses. there are two major signaling pathways that contribute to caspase activation: death receptor and mitochondrial pathways [38] . some viruses have exhibited cell apoptosis that is mediated by fas/fasl signaling as a reaction to viral infection, such as hepatitis c virus (hcv) [39] and dengue virus (denv) [40] . cell apoptosis can be induced by some viruses by regulating the levels of bcl-2 family members, such as sars coronavirus [41] and epstein-barr virus (ebv) [42] . our results showed that the activation of caspase-8 in ibv beaudette-infected cells was regulated by fas and fasl. the results also showed that activation of caspase-9 in ibv beaudette-infected cells was regulated by decreased expression of bcl-2 and increased expression of bax. the caspase-3 activation and virus-induced apoptosis might be triggered through both extrinsic and intrinsic pathways. in most cases, cell apoptosis induced by virus is a process of interaction between extrinsic and intrinsic pathways. the activation of caspase-8 was inhibited by z-ietd-fmk, and the activation of caspase-9 was not completely eliminating by blocking caspase-8 activity, suggesting that the activation of caspase-8 is not the only pathway to activate caspase-9, requiring further research. ibv are known to induce apoptosis through caspase-dependent pathway [15] and intrinsic-dependent pathway regulated by bcl-2 family proteins [16] in vero cells. in this study, these two pathways were demonstrated that play an important role in ibv beaudette-infected hd11 cells. additionally, this is the first report that the extrinsic pathway regulated by fas/fasl was activated in ibv-induced apoptosis. improved knowledge of the mechanisms by which ibv activates the extrinsic and intrinsic apoptotic pathways will help to better understand the pathogenic properties of epidemic ibv strains in the host. some viruses can induce cell apoptosis through viral replication. uv-inactivated bhv-1 and tgev could not induce apoptosis, or the nh 4 cl-pretreated cells prevented the appearance of apoptosis [31, 43] . some other viruses can induce apoptosis without viral replication, such as vaccinia virus and vesicular stomatitis virus [44, 45] . here, infection with uv-inactivated ibv beaudette or treatment of hd11 cells with nh 4 cl reduced virus apoptosis induction, indicating that ibv beaudette -induced apoptosis in hd11 cells depends on viral replication. this finding is similar to that of a previous study showing that uv-inactivated ibv lost the capacity to induce apoptosis in mammalian cells [16] . we tested whether caspase activation is needed for ibv replication in cells. the finding revealed that treatment with the caspase-3 inhibitor can increase the virus titer in ibv beaudette-infected cells, suggesting that the caspase inhibitor might increase the survival time of cells to promote replication. from a therapeutic standpoint, available drugs controlling apoptosis could be used to limit ibv spreading [46] . the innate immune response is the first line of defense against viruses, and macrophages are an important component of this system. some viruses have evolved strategies to induce apoptosis to enhance the production of virus progeny and promote dissemination to neighboring cells with limited host immune/inflammatory responses. the presence of apoptotic cells may also lead to the mobilization and initiation of innate immune defenses [47] . previous studies have shown that virus-induced apoptosis of macrophage has an important impact on virus infection. porcine reproductive and respiratory syndrome virus (prrsv) stimulates anti-apoptotic pathways in macrophages early in infection, and these prrsv-infected macrophages die by apoptosis late in infection [48] . chikv infection induces apoptosis and enhances expression of major histocompatibility complexes (mhcs) and co-stimulatory molecules and interleukin (il)-6 and monocyte chemoattractant protein (mcp)-1 production in macrophages [20] . however, little is known about ibv-induced immune cell apoptosis. it has been reported that phagocytic cells may play a crucial role in dissemination of virus to the blood circulation and internal organs. therefore, establishment of this macrophage system of ibv beaudette infection and determination of the apoptotic mechanism might be proof of principle for ibv infection in the host. in conclusion, chicken macrophage hd11 cells were established for attenuated ibv strain beaudette infection. ibv beaudette induced cell apoptosis through caspase-8 activation mediated by fas/fasl and caspase-9 activation mediated by bcl-2/bax. in addition, ibv beaudette replication was essential to apoptosis induction, and ibv beaudette replication increased when caspase activation was blocked. based on these findings, this study has shown the establishment of a chicken macrophage cell line that will facilitate the further analysis of ibv infection. additional studies are required to clarify the detailed molecular mechanisms underlying ibv-induced apoptosis. an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv coronavirus avian infectious bronchitis virus the long view: 40 years of infectious bronchitis research recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity vaccination against infectious bronchitis virus: a continuous challenge recombinant infectious bronchitis virus (ibv) h120 vaccine strain expressing 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interaction with the bcl-x l protein epstein-barr virus interactions with the bcl-2 protein family and apoptosis in human tumor cells bovine herpes virus type 1 induces apoptosis through fas-dependent and mitochondria-controlled manner in madin-darby bovine kidney cells vesicular stomatitis virus induces apoptosis primarily through bak rather than bax by inactivating mcl-1 and bcl-x l dose-dependent lymphocyte apoptosis following respiratory infection with vaccinia virus caspase inhibitors: viral, cellular and chemical recent progress in studies of arterivirus-and coronavirus-host interactions porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages the authors declare no conflict of interest. key: cord-298920-1lc2xf7u authors: bello-perez, melissa; sola, isabel; novoa, beatriz; klionsky, daniel j.; falco, alberto title: canonical and noncanonical autophagy as potential targets for covid-19 date: 2020-07-05 journal: cells doi: 10.3390/cells9071619 sha: doc_id: 298920 cord_uid: 1lc2xf7u the sars-cov-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (covid-19). previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. the precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. in turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are fda-approved for the treatment of other diseases), is usually detrimental to viral replication, including sars-cov-2. recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. still, some potential therapeutic targets within this pathway, such as rab9 and its interacting proteins, look promising considering current knowledge. thus, the combinatory treatment of covid-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection. . classification of the subfamily orthocoronavirinae [1, 5, 9] . alphacoronavirus ( coronaviruses are classified into four genera based on genomic features: alphacoronavirus and betacoronavirus, which infect mammals (including humans), and gammacoronavirus and deltacoronavirus, which mostly infect birds, but also mammals. among the five subgenera of betacoronavirus, sars-cov-2 together with sars-cov belongs to the sarbecovirus subgenus [1, 5] , which has been the subject of extensive study due to its high incidence and lethality in humans. although sars-cov-2 is phylogenetically closer to some bat sars-like coronaviruses [10, 11] , it shares approximately 80% of its genomic identity and homologous gene organization with sars-cov [6, 11] , which led to their classification within the same species, sars-related coronavirus [1] . therefore, to some extent, our knowledge on the former sars-cov may be applicable to this new virus. the surface glycoprotein s mediates receptor binding and membrane fusion during the virus entry [7] . sars-cov and sars-cov-2 s proteins (hereafter termed s cov and s cov2 , respectively) share 76% amino acid sequence identity, which represents a high value considering it is one of the most exposed and thus variable proteins within the virus family [6, 11, 12] . furthermore, such an identity is much higher in relevant functional regions because s cov2 conserves the typical elements necessary for its function: the receptor binding domain (rbd) in subunit 1 (s1), the fusion peptide (fp), the heptad repeats (hr) 1 and 2, the transmembrane domain (tm) and the cytoplasmic domain (cp) in s2 [6] . additionally, despite minor differences, s cov and s cov2 molecular structures, and their changes to adopt the fusion-competent conformation, are homologous, and 20 out of 22 n-glycosylations in s cov2 are conserved in s cov [12, 13] . in fact, early studies have reported that both s cov and s cov2 (albeit s cov2 with a little more affinity [12, 13] ) recognize angiotensin-converting enzyme 2 (ace2) as the cellular surface receptor that mediates the viral entry into the host [11] [12] [13] . thus, in view of the high degree of homology shared with sars-cov, it is probable that sars-cov-2 may also enter cells by clathrin-dependent as well as by clathrin-and caveolae-independent endocytosis pathways, and then engage the endocytic pathway [14] [15] [16] . high ratios of protein sequence identity were also found for the 16 coronaviral nsps between both sars-covs. these values vary between 68 (nsp2) and 100% (nsp13), and half of them are over 95% (nsp5, 7-10 and [12] [13] [14] . nsp6, which is widely addressed in the present work, shares an 88% identity between both viruses [6] . all nsps play important roles in viral replication and transcription processes (for instance, nsp5, protease; nsp7, primase; nsp12, rna-dependent rna polymerase and nsp13, helicase). in particular, nsp6 is a transmembrane protein that complexes with nsp3 and nsp4 and is implicated in the formation of er-derived double-membrane vesicles (dmvs) during coronavirus replication [17] . such a high level of conservation in most nsps from sars-cov and sars-cov-2 suggests close molecular structures and homologous functions, and thus parallel/comparable replication cycles. such observations are of great importance in helping to identify the molecular pathways interacting with and responding to a particular viral infection, because the previous acquired knowledge on their modulation might offer strategically important therapeutic advantages. indeed, except for rare unknown or scarcely studied molecular pathways, an array of pharmacologically characterized drugs intended for modulating each route of interest are approved for treating certain diseases, and these can be expanded with corresponding repositioning. based on the literature, the macroautophagy (hereafter referred to as autophagy) pathway might be a promising target to tackle sars-cov-2 infection. autophagy is a highly conserved eukaryotic process of cytoplasmic degradation that is activated, among others, under conditions of starvation and endoplasmic reticulum (er) stress [18, 19] . this mechanism maintains cellular homeostasis and requires the orchestration of a variety of molecules and dynamic membrane rearrangements to achieve cells 2020, 9, 1619 4 of 18 complete autophagic flux. briefly, in the canonical pathway of starvation-induced autophagy (see figure 1 for a graphical description and abbreviations), the process begins with the generation of a sequestering compartment. this process involves the induction of an omega-shaped subdomain of the er membrane (termed an omegasome) that evolves to form the phagophore. the latter is a transient structure, which consists of a double-membrane sheet that expands until it closes in on itself, wrapping recyclable cellular material and forming the autophagosome. still bordered by a double membrane, the autophagosome may fuse with an acidic late endosome to form a single-membrane amphisome. finally, the autophagosome or amphisome fuses with the lysosome to form the autolysosome, inside which the enzymatic degradation of the cargo occurs under acidic conditions. as expected, each phase of the process is supported and regulated by the sequential recruitment and action of numerous proteins (figure 1 ), which are usually termed as autophagy related (atg). the initiation of autophagy is regulated by the ulk1 and class iii phosphatidylinositol 3-kinase (ptdins3k) complexes (under the negative regulation of mtor), which create domains that are enriched in the lipid ptdins3p. these domains recruit various proteins including the proteolytically processed form of map1lc3/lc3 (termed lc3-i) and mediate its conjugation to the lipid phosphatidylethanolamine (to generate lc3-ii). this recruitment is essential for phagophore closure, and therefore the formation of the autophagosome in the canonical autophagy pathway. a complex including atg16l1 specifies the site of lc3 lipidation [18] [19] [20] [21] [22] . and endoplasmic reticulum (er) stress [18, 19] . this mechanism maintains cellular homeostasis and requires the orchestration of a variety of molecules and dynamic membrane rearrangements to achieve complete autophagic flux. briefly, in the canonical pathway of starvation-induced autophagy (see figure 1 for a graphical description and abbreviations), the process begins with the generation of a sequestering compartment. this process involves the induction of an omega-shaped subdomain of the er membrane (termed an omegasome) that evolves to form the phagophore. the latter is a transient structure, which consists of a double-membrane sheet that expands until it closes in on itself, wrapping recyclable cellular material and forming the autophagosome. still bordered by a double membrane, the autophagosome may fuse with an acidic late endosome to form a single-membrane amphisome. finally, the autophagosome or amphisome fuses with the lysosome to form the autolysosome, inside which the enzymatic degradation of the cargo occurs under acidic conditions. as expected, each phase of the process is supported and regulated by the sequential recruitment and action of numerous proteins (figure 1 ), which are usually termed as autophagy related (atg). the initiation of autophagy is regulated by the ulk1 and class iii phosphatidylinositol 3-kinase (ptdins3k) complexes (under the negative regulation of mtor), which create domains that are enriched in the lipid ptdins3p. these domains recruit various proteins including the proteolytically processed form of map1lc3/lc3 (termed lc3-i) and mediate its conjugation to the lipid phosphatidylethanolamine (to generate lc3-ii). this recruitment is essential for phagophore closure, and therefore the formation of the autophagosome in the canonical autophagy pathway. a complex including atg16l1 specifies the site of lc3 lipidation [18] [19] [20] [21] [22] . the autophagy process manages to also be selective through the use of specific receptors that link various ligands to the autophagic machinery through receptor binding to lc3 on the concave side of the phagophore. in the case of microbes, this is often achieved by ubiquitin-tagging the cargoes via the action of a group of e3 ligase-family proteins. among other types of selective autophagy, xenophagy specifically targets intracellular pathogens for their degradation and further integration into both innate and adaptive immune responses [21, [23] [24] [25] [26] . conversely, in response to this cellprotective autophagy, several different families of viruses, including coronavirus, have adapted by evolving a large variety of strategies to escape and/or to benefit via the inhibition and/or stimulation of autophagy at different stages of the process [23, [27] [28] [29] . thus, the identification of these interaction points might bring the opportunity to disrupt the viral replication cycle at specific stages by targeting selected steps of autophagy. autophagy is regulated by three protein complexes: ulk1, comprising of ulk1, atg13, rb1cc1/fip200 and atg101; class iii ptdins3k, comprising of atg14, becn1, pik3r4/vps15 and pik3c3/vps34 and atg16l1, comprising of atg16l1, atg5 and atg12. under starvation conditions, mtor is inactivated allowing ulk1 complex formation, and activation of the ptdins3k, which creates the ptdins3p-rich regions on the surface of the omegasome. wipi proteins recognize these domains and recruit the atg16l1 complex, which facilitates lipidation of lc3-i to form lc3-ii. receptors such as sqstm1/p62 bind to ubiquitinated cargo and lc3-ii to facilitate selective autophagy. cytoplasmic cargo includes damaged mitochondria, organelles, proteins, nucleic acids, intracellular bacteria, etc. expansion of the phagophore through membrane addition sequesters a portion of the cytoplasm and upon closure forms the autophagosome. these autophagosomes are decorated with rab7, which leads to the fusion with lysosomes to form the autolysosomes, where the cargo is degraded. the endocytic pathway (used by some viruses) and autophagy converge, resulting in the formation of an amphisome, which also fuses with lysosomes to form autolysosomes. the pink color indicates acidic compartments. abbreviations: ampk, amp activated protein kinase; bcl2, bcl2 apoptosis regulator; becn1, beclin 1; lamp2, lysosomal associated membrane protein 2; map1lc3/lc3, microtubule associated protein 1 cells 2020, 9, 1619 5 of 18 light chain 3; mtor, mechanistic target of rapamycin kinase; pe, phosphoethanolamine; pik3c3/vps34, phosphatidylinositol 3-kinase catalytic subunit type 3; pik3r4/vps15, phosphoinositide-3-kinase regulatory subunit 4; ptdins3k, phosphatidylinositol 3-kinase; ptdins3p, phosphatidylinositol-3-phosphate; ptk2/fak, protein tyrosine kinase 2; rab7, rab7, member ras oncogene family; rb1cc1/fip200, rb inducible coiled-coil 1; sqstm1/p62, sequestosome 1; ulk1, unc-51 like autophagy activating kinase 1; wipi1/2, wd repeat domain, phosphoinositide interacting 1/2; zfyve1/dfcp1, zinc finger fyve-type containing 1. the autophagy process manages to also be selective through the use of specific receptors that link various ligands to the autophagic machinery through receptor binding to lc3 on the concave side of the phagophore. in the case of microbes, this is often achieved by ubiquitin-tagging the cargoes via the action of a group of e3 ligase-family proteins. among other types of selective autophagy, xenophagy specifically targets intracellular pathogens for their degradation and further integration into both innate and adaptive immune responses [21, [23] [24] [25] [26] . conversely, in response to this cell-protective autophagy, several different families of viruses, including coronavirus, have adapted by evolving a large variety of strategies to escape and/or to benefit via the inhibition and/or stimulation of autophagy at different stages of the process [23, [27] [28] [29] . thus, the identification of these interaction points might bring the opportunity to disrupt the viral replication cycle at specific stages by targeting selected steps of autophagy. the most apparent hint suggesting a connection between autophagy and the coronavirus replication cycle is the hallmark presence of dmvs in both cases [23, 27, 28] . coronavirus induce characteristic rearrangements of the er membranes towards the generation of connected dmvs and convoluted membranes. altogether, this cytosolic reticulovesicular system works as a scaffold for viral rna synthesis, and presumably protects the viral elements from the host's defense mechanisms [17, 30] . the precise functions and dynamics of these structures are not fully understood, but several viral nsps have been implicated [30] . among them, various in vitro studies highlight the crucial role of nsp6, one of the viral replicase proteins, to subvert the autophagic machinery for the generation of the dmvs [31] . evidence shows that coronavirus nsp6s induce the formation of these membrane rearrangements from the er [31, 32] . so far, only sars-cov nsp6 has been reported to partially colocalize with released lc3-positive dmvs [31] . the initial mechanism underlying such activation remains unknown, although it has been shown for ibv nsp6 that it is not mediated by the inhibition of the signaling pathway of mtor (the major autophagy suppressor [33] ) [31] , in contrast to a recent study on pedv nsp6 [34] . neither is it due to the activation of the nad-dependent deacetylase sirt1 (sirtuin 1; an mtor-independent inducer of autophagy [35] ) [31] . alternatively, er stress, which also triggers an autophagic process involving the unfolded protein response (upr; see figure 2 for a graphical description and abbreviations) [19, 36] , is induced by ibv infection via ern1/ire1 [37] , one of three unfolded protein sensors in upr signaling pathways, and a cellular autophagic response that has been reported as a prosurvival mechanism for er stress [38] . interestingly, just ern1, but not its downstream effectors xbp1 and mapk/jnk, is required for the induction of autophagy in ibv-infected cells, although its silencing does not inhibit ibv replication [39] . regarding the other two known pathways of the upr (figure 2) , their corresponding main regulators, atf6 and eif2ak3/perk, are not modulated by ibv infection, and their silencing has no effect on ibv-induced autophagy [39] . in line with these results, cottam et al. (2011) [31] , also showed that ibv, mhv and sars-cov nsp6s do not exert any significant effect on either the activation of xbp1 from the ire1 signaling pathway or the expression of the proapoptotic transcription factor ddit3/chop/gadd153, from the eif2ak3/perk pathway. regarding the other two known pathways of the upr (figure 2 ), their corresponding main regulators, atf6 and eif2ak3/perk, are not modulated by ibv infection, and their silencing has no effect on ibv-induced autophagy [39] . in line with these results, cottam et al. (2011) [31] , also showed that ibv, mhv and sars-cov nsp6s do not exert any significant effect on either the activation of xbp1 from the ire1 signaling pathway or the expression of the proapoptotic transcription factor ddit3/chop/gadd153, from the eif2ak3/perk pathway. in relation to the previously mentioned results, it is worth noting that the activation of the ern1-mapk signaling pathway in response to er stress has been described to induce both autophagy [38] and cell death [40] . in this sense, despite the fact that the eif2ak3/perk pathway of the upr appears to be irrelevant in coronavirus-induced autophagy, ibv induces apoptosis, and benefits from it, via the eif2ak3/perk and eif2ak2/pkr activation of ddit3/chop, which in turn suppresses the mapk/erk pathway [41] . this mapk/erk pathway together with the mapk/jnk pathway, figure 2 . diagram of the er stress unfolded protein response (upr) pathways triggering autophagy. er stress can activate autophagy through three different upr branches: eif2ak3/perk, ern/ire1 and/or the atf6 signaling pathway. eif2ak3/perk induces autophagy by activating the atg16l1 complex through atf4 or by inducing ddit3/chop expression, which indirectly causes becn1 dissociation from bcl2. ern/ire1, through mapk/jnk, mediates the phosphorylation of bcl2, which causes its dissociation from becn1. the xbp1 branch enhances the formation of lc3-ii. the atf6 pathway also induces autophagy by inhibiting phosphorylation at the akt-mtor pathway. abbreviations: akt/pkb, akt serine-threonine kinase; atf4/6, activating transcription factor 4/6; ddit3/chop/gadd153, dna damage inducible transcript 3; eif2ak3/perk, eukaryotic translation initiation factor 2 alpha kinase 3; ern/ire1, endoplasmic reticulum to nucleus signaling 1; mapk/jnk, mitogen-activated protein kinase and xbp1, x-box binding protein 1. in relation to the previously mentioned results, it is worth noting that the activation of the ern1-mapk signaling pathway in response to er stress has been described to induce both autophagy [38] and cell death [40] . in this sense, despite the fact that the eif2ak3/perk pathway of the upr appears to be irrelevant in coronavirus-induced autophagy, ibv induces apoptosis, and benefits from it, via the eif2ak3/perk and eif2ak2/pkr activation of ddit3/chop, which in turn suppresses the mapk/erk pathway [41] . this mapk/erk pathway together with the mapk/jnk pathway, mediate noncanonical autophagy via the regulation of the bcl2-interacting protein becn1 (beclin 1) [42] . moreover, it has been reported that mers-cov cell culture infections induce substantial changes in the phosphorylation of relevant elements of not only the mapk/erk pathway (also known as the ras-raf-map2k/mek-mapk/erk pathway), but also the class i phosphoinositide 3-kinase (pi3k)-akt-mtor pathway [43] , which is also involved in apoptotic processes [40, 44] . remarkably, treatment with specific inhibitors of these two pathways and eif2ak2/pkr, which have also been described to modulate autophagy, inhibit mers-cov infection [43] . along these lines, it was also demonstrated recently that skp2 (s-phase kinase associated protein 2), which is activated by akt1, cells 2020, 9, 1619 7 of 18 promotes becn1 degradation and the inhibition of autophagy, and in turn that skp2 suppression, and thus autophagy activation, inhibits mers-cov infection [45] . within this context, some coronaviruses have evolved an additional mechanism to prevent apoptosis, as well as the host's type i interferon (ifn) immune response, consisting of antagonizing the ifn-inducible oas (2'-5'-oligoadenylate synthetase)-rnasel (ribonuclease l) pathway and thus blocking rnasel activity, i.e., the cleavage of viral and host single-stranded rna, and subsequent cell death. to this end, the rnasel activator 2',5'-oligoadenylate is degraded by means of the cyclic phosphodiesterase activity of several betacoronaviral accessory proteins [46, 47] . paradoxically, it is described that rnasel triggers autophagy in response to viral infections [48] via the mapk/jnk pathway [49] ; however, as mentioned previously, coronavirus already modulate autophagy by means of ern1 with, apparently, no need of mapk/jnk [39] . major controversy arises as to whether the lc3-containing vesicles induced during ibv [31, 39, 50] , mhv [31, 50, 51] , mers-cov [45] , sars-cov [31, 50] and pedv [34] infections are actually autophagosomes. such an effect has been demonstrated to be mediated by the viral nsp6s in some of these cases [31, 34, 41, 50] , and appears to be driven via an omegasome intermediate [31, 50] , similar to that seen in canonical starvation-induced autophagy [52] . along this line, the generation of these coronavirus-induced autophagosomes requires the ptdins3p-enrichment of the er membrane outer leaflet, and the recruitment of zfyve1/dfcp1 (a key protein in omegasome formation), wipi1/2, atg5 and lc3-ii (all components of the autophagic machinery), and sqstm1/p62 (a receptor protein for selective autophagy) [31, 50, 51] . conversely, the omegasomes, autophagosomes and autolysosomes in nsp6-expressing cells undergo a different maturation process from those induced by, for instance, just starvation. cottam et al. (2014) [50] described that ibv, mhv and sars-cov nsp6s, and ibv infection, generate significantly smaller-diameter autophagosomes (ã� â�¤ 0.5 âµm) in comparison to the usual ones (ã�: about 1 âµm). further assays performed with ibv nsp6 in this regard show that its expression limits the expansion of both omegasomes and phagophores, even when they are induced by either starvation or inhibition of the mtor kinase. in this same work, because they demonstrate that ibv nsp6 does not prevent the fusion of autophagosomes and lysosomes (as was also recently reported for ibv infection [39] ), it is suggested that the reduced size of these autophagosomes limits their capacity to fuse with multiple lysosomes, generating smaller autolysosomes as a result. interestingly, it is also shown that nsp6 inhibits the recruitment of mtor to the surface of lysosomes, which may affect the activity of the final autolysosome. a converging work recently reported that mers-cov infection actually blocks autophagy at the autolysosome formation stage via nsp6 and the accessory proteins 4b and 5. this finding is still consistent with previous data because, as a consequence, this block increases the total number of early-stage autophagic vesicles and reduces the autolysosome ratio therein [45] , as it occurs even after treatment with the late autophagy blockers chloroquine or bafilomycin a 1 [53] [54] [55] . such an effect would benefit the viral replication in several ways: by preventing the maturation of endosomal and autophagic vesicles, and thus their potentially excessive degradative capacity with regard to viral elements at basically all the stages of their replication cycle, and providing them with all this new machinery for their replication in safe conditions [14, 15, 17, 30, 56] . consistent with this scenario, there are several supporting lines of evidence in which coronaviruses or their nsp6s (among others) induce not only the initiation of an autophagic process in the host, but also its blockade at a late stage of the process. for instance, there is the presence of common autophagy markers in vesicular structures from coronavirus infected-or nsp6-expressing cells ( table 2 ). the accumulation of lc3 and increased conversion to lc3-ii [34, 39, 57, 58] , the accumulation cells 2020, 9, 1619 8 of 18 of becn1 [58] and the increased degradation of sqstm1/p62 [57, 58] also occurs. it is then not surprising that the reduction or abolition of these events when an essential autophagic element is blocked, such as the proteolytic cleavage, allows the conversion of lc3-i to lc3-ii [31] . neither is it when such elements are chemically inhibited, such as pik3c3 with wortmannin to abolish the formation of the omegasome and thus the following autophagic processes such as lc3 recruitment and conversion [31, 57] ; or silenced, as seen for the example with atg5 [31, 34, 39] . however, the regulation of the canonical pathway of autophagy does not explain other wide-perspective observations, even considering the great differences that may exist among all the experimental systems used, i.e., different coronavirus genera/lineages and cell lines. the most contradictory fact, given the close interaction between coronaviral replication and autophagy pathways, is that the knockout of genes encoding autophagy-essential proteins such as atg5, atg7, becn1 or lc3 di not abolish the replication in cell culture of the coronaviruses tested; actually, for many of them, replication was unaffected or increased ( table 3 ). the lack of these elements also does not prevent the generation of the er pleiomorphic interconnected vesicular structures required for viral replication, although they were analyzed only in a few of these studies on mhv [51, 59, 61] . another surprising finding is the only partial colocalization of autophagosomes (lc3-labelled in most studies) with viral replication elements that massively accumulate in infected cells (table 2 ). in this sense, snijder et al. (2006) [62] described complete separation of lc3 and sars-cov nsp3 subunits in vero-e6 infected cells. all these facts together suggest an alternative autophagy pathway involved in coronavirus replication. in this sense, reggiori et al. (2010) [59] propose that coronaviruses induce er-membrane rearrangements by manipulating the alternative er-associated degradation pathway, specifically the selective clearing process of er degradation enhancing alpha-mannosidase like protein 1 (edem1). edem1, probably together with other er chaperones, is stored in vesicles termed edemosomes that are guided out of the er by means of a copii complex coat-independent mechanism, and delivered to endosomal compartments for disposal. in this work, it is suggested that mhv hijacks this pathway to promote the edem1-independent formation of viral dmvs coated with nonlipidated lc3 (lc3-i), which is essential for viral replication. by this approach, it made sense that atg5 [31, 39, 45, 51, 57, 58, 60, 61] and atg7 [57, 59] , which are involved in lc3-i processing to lc3-ii [59, 61] , are found dispensable for such a task. however, as discussed by the authors of the work, another route with these characteristics had just been revealed and might be implicated; atg5-and atg7-independent autophagy [63] . in 2009, nishida et al. [63] described that certain autophagosomes may result from late endosomes and the trans-golgi without participation of atg5, atg7 and lc3 conversion. additionally, its chemical blockade by brefeldin a (bfa) suggests that the initial steps of this alternative autophagy pathway might involve the fusion of vesicles in the er/cis-golgi region [64] , because this compound mediates the inhibition of adp ribosylation factor 1 (arf1), a gtpase from the ras superfamily, which recruits coat proteins for the vesicular trafficking between both organelles [65] . thus, atg5-and atg7-independent autophagy establishes an endocytic pathway-golgi route able to potentially interact with the replication cycle of (mostly enveloped) viruses at multiple stages/processes: (1) their entry by endocytosis and fusion of viral and host membranes; (2) the processing of the viral glycoprotein carbohydrate moieties that requires their transfer from the er to the cis-golgi; (3) their intracellular transport; (4) replication; (5) assembly or (6) egress by means of host vesicular scaffolds. as we know from many other viruses, not only the canonical but also the alternative autophagy pathways (or some of their elements) are hijacked and subverted for their replication [23, 66] , and bfa possesses antiviral activity in many cases [67] [68] [69] [70] , including coronaviruses [71] . for the elucidation of the particular contribution of each autophagy pathway to coronavirus replication cycles, further studies will be required including the use of modulators such as bfa together with the monounsaturated fatty acid oleate [64] ; bfa does not affect canonical autophagy but it inhibits both the atg5-and atg7-independent [63, 72] and the becn1-independent process [73, 74] , whereas the becn1-independent pathway is only induced by oleic acid [74] . additionally, it will be necessary to add new members to the list of autophagy markers such as rab9 for assessing the activity of the atg5-and atg7-independent pathway [63] , which mediates the trafficking of late endosomes to the trans-golgi [75] . the interplay between coronaviruses and autophagy is very complex and not completely understood. during a coronavirus infection, autophagy is both a cellular response mechanism and a viral replication tool. in fact, coronaviruses can both induce and inhibit autophagy with interactions at multiple levels within a narrow action area limited by apoptosis and the ifn response. other representative examples of this complexity are that, although autophagy activation inhibits tgev replication [57] , a proviral mitochondria-selective autophagy is induced in tgev-infected cells [76] , or that pedv induces autophagy and benefits from it [58] , but it is also inhibited by rapamycin-induced autophagy [60] . for all this, and despite existing differences between studies that are almost certainly due to the use of distinct experimental systems, the modulation of autophagy usually affects the replication of coronaviruses, and therefore it becomes a promising therapeutic target in the search for anticoronavirals. tables 4 and 5 compile the reported effects of autophagy inducers or inhibitors, respectively, on the infection of different coronaviruses in cell cultures. half of them are already fda-approved drugs for other diseases/disorders, and several have already shown inhibitory activity against sars-cov-2, i.e., ivermectin, (hydroxy-) chloroquine and nitazoxanide. as can be observed, even classic modulators, such as rapamycin, 3-methyladenine (3-ma) or chloroquine, usually exert an effect on coronavirus replication. in general, among all the autophagy modulators tested, independently of being autophagy inducers or inhibitors, the outcome is usually antiviral activity. this fact may reflect not only the precise viral control over the autophagy pathway, but also the difficulty of maintaining such a balance and the detrimental effect on viral replication if there is any dysregulation in this back-and-forth game. [58] and sars-cov [79] . as shown in table 4 , autophagy inducers generally antagonize coronavirus replication. among the autophagy inhibitors (table 5) , chloroquine (the most tested one) shows broad-spectrum anticoronaviral activity, which is probably because of its multimodal effects. briefly, chloroquine, apart from disorganizing the golgi, induces lysosomal alkalinization, which prevents amphisome/autophagosome-lysosome fusion and blocks the vesicle trafficking system [53] [54] [55] 93] , which potentially affects the replication cycle of coronavirus systemically, including their entry, which is mediated by ph-dependent endocytosis and requires a low ph for the s protein to trigger its membrane fusion activity [94, 95] . nitazoxanide is another late-stage autophagy blocker [96] that shows high anti-sars-cov-2 activity in cell cultures (ic 50 : 2.12 âµm) [97] , although it should be considered that its main metabolite, tizoxanide, induces autophagy by inhibiting the pi3k-akt-mtor pathway [98] . at this moment, the scientific community is focusing efforts in searching, by different approaches, for effective drugs against this pathogen and continuously revealing autophagy modulators [99] [100] [101] . as shown here, drugs that target autophagy, as well as those involved in regulating the endocytic pathway [110] could be added to the arsenal of compounds against coronavirus infections (for an extensive list see zumla et al. (2016) [111] ). as a consequence, the discovery of new autophagy regulatory drugs may be a source of new antivirals that is worth testing for this purpose. in this sense, we propose that the alternative autophagy routes are still scarcely explored in this field and can provide unexpected positive outcomes in the fight against viruses, and particularly coronaviruses. in a follow-up prospective effort, we think that interference with rab9 activity, a key element in these pathways, might be a promising approach. in this sense, the targeting of gdi/rabgdi (gdp dissociation inhibitor), which forms a complex with rab9 in the cytosol and mediates its activity in the endosome-trans golgi network, and specific "gdi-displacement factors" such as rabac1/yip3 (rab acceptor 1) are also candidates worth testing for this purpose [112, 113] . to conclude, we observed that most of the reviewed works tested the anticoronaviral effect of each autophagy modulator individually in order to accurately unravel the mechanisms involved. thus, having shown that autophagy and coronavirus replication cycles converge in several different stages, treatment strategies including the combination of autophagy-modulating agents might result in synergistic effects that are worth studying. in this vein, among present combinatory treatments, a frequent one is (hydroxy-) chloroquine together with azithromycin [114, 115] , a macrolide antibiotic with extensively reported autophagy-blocking activity, as well as other family members [116, 117] . another important factor to consider is the scarce number of in vivo studies in this field [118] , which is certainly due to the required and necessary biosafety restrictions. however, these studies are essential to assess the true potential of these drugs for clinical implementation because the outcome within the complex biosystem can be very different from that of in-cell culture tests. in this sense, despite the inhibitory effects observed in vitro, (hydroxy-) chloroquine treatments, either alone or in combination with azithromycin, has shown no benefits against sars-cov-2 infection in clinical trials [119, 120] . besides, the in vivo context allows the identification of not only possible side effects, but also paradoxical issues such as the fact that the virulence of coronaviruses may be different even if showing similar replication levels [8] . finally, it is important to mention that autophagy also plays a significant role in adaptive immune responses [24] [25] [26] , and in vivo tests are essential for determining the possible implications in this sense when using autophagic modulators in experimental treatments, as they could be either detrimental or beneficial in the long term. coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 aids, avian flu, sars, mers, ebola, zika broad-spectrum 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kinase signaling pathways coronavirus nsp6 restricts autophagosome expansion coronavirus replication complex formation utilizes components of cellular autophagy autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion bafilomycin a1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, h-4-ii-e cells does bafilomycin a1 block the fusion of autophagosomes with lysosomes? taylor & francis: abingdon an overview of their replication and pathogenesis autophagy negatively regulates transmissible gastroenteritis virus replication porcine epidemic diarrhea virus induces autophagy to benefit its replication coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication rapamycin-induced 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blocking the pak1/akt axis in breast cancer the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro niclosamide-induced wnt signaling inhibition in colorectal cancer is mediated by autophagy ro 31-8220 and ro 31-7549 show improved selectivity for protein kinase c over staurosporine in macrophages protein kinase c inhibits autophagy and phosphorylates lc3 autophagy regulates selumetinib (azd6244) induced-apoptosis in colorectal cancer cells chemical genetics approach to restoring p27kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells bay 43-9006 exhibits broad spectrum oral antitumor activity and targets the raf/mek/erk pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis activation of phosphatidylinositol 3-kinase/akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells protective autophagy elicited by rafâ�� mekâ�� erk inhibition suggests a treatment strategy for ras-driven cancers perturbation of intracellular k+ homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes the hairpin-type tail-anchored snare syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus nitazoxanide, an antiprotozoal drug, inhibits late-stage autophagy and promotes ing1-induced cell cycle arrest in glioblastoma remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro tizoxanide induces autophagy by inhibiting pi3k/akt/mtor pathway in raw264. 7 macrophage cells network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 sars cov-2: recent reports on antiviral therapies based on 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isolated rat hepatocytes the anti-hypertensive drug reserpine induces neuronal cell death through inhibition of autophagic flux targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 coronaviruses-drug discovery and therapeutic options targeting rab gtpases to distinct membrane compartments rab gtpases as coordinators of vesicle traffic in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 shows synergistic effect hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated chop induction in myeloma cells antiviral activity of chloroquine against human coronavirus oc43 infection in newborn mice observational study of hydroxychloroquine in hospitalized patients with covid-19 outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19 we thank diego sanz for graphical support. the authors declare no conflict of interest. key: cord-316234-vtjsfi2c authors: sultankulova, kulyaisan t.; kozhabergenov, nurlan s.; strochkov, vitaliy m.; burashev, yerbol d.; shorayeva, kamshat a.; chervyakova, olga v.; rametov, nurkuisa m.; sandybayev, nurlan t.; sansyzbay, abylay r.; orynbayev, mukhit b. title: new oligonucleotide microarray for rapid diagnosis of avian viral diseases date: 2017-04-05 journal: virol j doi: 10.1186/s12985-017-0738-0 sha: doc_id: 316234 cord_uid: vtjsfi2c background: we developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (aiv), newcastle disease virus (ndv), infection bronchitis virus (ibv), and infectious bursal disease virus (ibdv) in singleand mixed-virus infections. the objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. methods and results: the paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cdna on the microarray and its specificity tested with use of aiv, ndv, ibv, ibdv strains as well as biomaterials from poultry. sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 aiv, ndv, ibv and ibdv strains, different in their origin, epidemiological and biological characteristics (ribsp microbial collection). this microarray demonstrates high diagnostic sensitivity (99.16% within 95% ci limits 97.36–100%) and specificity (100%). specificity of the developed technique was confirmed by direct sequencing of np and m (aiv), vp2 (ibdv), s1 (ibv), np (ndv) gene fragments. conclusion: diagnostic effectiveness of the developed dna microarray is 99.18% and therefore it can be used in mass survey for specific detection of aiv, ndv, ibv and ibdv circulating in the region in the course of epidemiological surveillance. rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic methods is proposed. intensive poultry farming leads to higher risk of infectious disease emergence causing great economical losses. boundary spanning between clinical manifestations of different agents is peculiar to the course of many infections nowadays. more and more infectious diseases progress in association with different microorganisms and it effects significantly the clinical manifestation and differential diagnosis of the disease. currently, the viral infections such as avian influenza, newcastle disease, infectious bronchitis, and infectious bursal disease, etc., are a potential threat to poultry farming in the republic of kazakhstan. monitoring these economically significant avian diseases is the question of the day for poultry industry. avian influenza virus belongs to the orthomyxoviridae family, influenza a virus genus. from the beginning of year 2016 the disease outbreaks were recorded in 30 countries [1] . different aiv strains can cause 10 to 100% mortality among poultry. the agent of the newcastle disease is an rnacontaining virus, a member of the paramyxoviridae family, rubulavirus genus. in 2016 13 countries reported newcastle disease cases to the oie [1] . in poultry industrial farms, all infected birds need to be sacrificed due to threat of dissemination of the infection across countries [2] . the agent of the infectious bursal disease is rnacontaining virus of avibirnavirus genus in birnaviridae family. in outbreaks of the infectious bursal disease practically the entire population is affected and the lethality rate can approach 90% [3] , the reconvalescent birds become susceptible to the majority of infectious diseases of viral and bacterial etiology [4] . the causative agent of infectious bronchitis is an rna-containing coronavirus avia of coronavirus genus in coronaviridae family [5] . economical losses due to infectious bronchitis is composed of reduced egg and meat productivity, compulsory slaughter of sick birds, high death rate in young population. when the infection circulates in the farm for the first time the lethality rate can reach 70% [6] . currently, standard immunological methods [7] or methods based on polymerase chain reaction (pcr) [8, 9] are widely used to identify the above mentioned viruses. unfortunately, they can detect only one agent in a specimen. there are also multiplex rt-pcr assays that make possible simultaneous detection of more than one infectious agent by using multiple primer pairs. advantage of the multiplex rt-pcr is in combination of sensitivity and quickness of pcr alongside with elimination of need to test clinical specimens for each agent separately [10, 11] . avian viruses can cause diseases independently, in alliance with each other or in association with bacterial agents [12] . thereby, rapid and sensitive methods of detection are required that are able to differentiate viral infections for surveillance of newly emerging avian viruses as well as for disease control. application of dna microarray technology that makes possible multivariate analysis of genetic material is a highly promising way for simultaneous detection of several agents (aiv, ndv, ibv and ibdv) in one specimen. the paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescentlabelled viral cdna on the microarray and its specificity tested with use of aiv, ndv, ibv, ibdv strains as well as biomaterials from poultry. the objective of this study is to develop an oligonucleotide microarray for rapid diagnosis of avian influenza, newcastle disease, infectious bronchitis, and infectious bursal disease that will be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. four aiv and 4 ndv strains, 2 ibv and 5 ibdv strains from the ribsp (me&s rk/sc) microbial collection were used in the study (see table 1 for the list of these strains). one-hundred and twenty-two samples (44 cloacal swabs from sick birds and 78 tissue samples from dead ones) were delivered by veterinarians from different regions of kazakhstan to rge ribsp in the routine epidemiological surveillance for diagnosing sickness and death of birds (table 2) . rna was extracted from virus-containing material with trizol ("invitrogen", usa) according to the manufacturer's instruction. for selection of oligonucleotide primers and probes as microarray components the representative sample from international data base of ncbi (national center for biotechnological information) genbank (https:// www.ncbi.nlm.nih.gov/) containing genomes of aiv, ndv, ibv and ibdv was used. all full-sized encoding sequences of virus nucleotides were aligned by using clustal w algorithm in mega 6.0 software by method of progressive multiplex alignment. oligonucleotide probes with optimal physicochemical characteristics were selected with use of oligowiz 2.1 and picky 2.20 programs. specificity of the chosen oligonucleotide primers and probes was tested with use of blast (basic local alignment search tool) program that could compare the existing sequence with sequences in database in ncbi blast-analysis (ncbi, https://blast.ncbi.nlm.nih.gov/ blast.cgi). oligonucleotide primers and probes were synthesized in dna/rna synthesizer h-16 (k&a laborgeraete, germany) according to the manufacturer's instruction. pcr-amplification was carried out in multi-prime format. rt-pcr was performed with use of super script iii onestep rt-pcr system with platinum taq (invitrogen, usa) according to the manufacturer's instruction (in 50 μl of reaction mixture: super script iii rt/platinum taq mix-1 μl; 2x reaction mix-up to 1x; primers 20 pmol-1 μl each; rna-5 μl each; depc-treated water up to 50 μl). mixture of primers complementary to np and m (aiv), vp2 (ibdv), s1 (ibv), np (ndv) gene regions was used for specimen amplification. fluorescent labeling of specimens was carried out by direct embedding of cy5-dctp ("amersham", usa) in the process of rt-pcr, the reaction mixture was supplemented in this case with 2 μl of 1 mm cy5-dctp. oligonucleotide probes were diluted 1:1 with twofold buffer for oligonucleotide printing ("arrayit corporation", usa), in concentration 50 pmol applied on glass slides without support ("sigma", usa) by a method of contact printing with use of nanoprint lm60 spot-printer ("arrayit corporation", usa). slide contained 16 arrays, where oligonucleotide probes complementary to certain aiv, ndv, ibv, ibdv genome loci were identically immobilized as separate spots 300 μm in diameter. to 1.0 μl of pcr-mixture containing cy5-cdna hybridization solution was added, the total volume was brought with h 2 o up to 50.0 μl and heated in the solidstate thermostat at 99°c for 2 min, then cooled in ice for 2 min and at once applied onto the microarray. in parallel the oligonucleotide probes on the microarray were denatured by boiling of the slide in h 2 o for 1 min followed by incubation in 96% ethanol (−20°c) for 1 min. after that the slide was dried by centrifugation at 300 g for 2 min. hybridization was performed with use of a frame for 16 subarrays fast® frame ("whatman", usa) for 2 h at 37°c and stirring at 250 rpm. after hybridization the slide was rinsed in 3 × ssc buffer for 2 min and in 1 × ssc buffer for 2 min to remove unbound molecules of the sample and hybridization buffer. after that the frame was removed and the slide was rinsed with water for 2 min. it was dried by centrifugation at 300 g for 2 min. microarray scanning was carried out by use of innosca-n710al scanner ("innopsys", france) at 5 μm resolution and wavelengths 532 nm and 633 nm. the resulted data were processed with the help of mapix ver. 5.5.0 software ("innopsys", france) and the matrix corresponding to the probe layout on microarray was applied on the obtained pixel image. afterwards the applied array was used to detect probes by intensity of fluorescence with quantitative output with the help of the program module. median fluorescence values of probes minus background signals were considered as effective data. the bigdye terminator cycle sequencing kit was used according to the manufacturer's instructions. the sequencing run was carried out using the 16-capillary abi prism 3130 xl genetic analyser, (applied biosystems). the specificity of the assay was theoretically assessed by evaluating the primers and probes for relevant homologies using the blast tool (https://blast.ncbi.nlm.nih.gov/ blast.cgi). real-time rt-pcr was performed with use of light cycler 2.0 manufactured by roche company to detect aiv [13] , ndv [14] , ibv [15] and ibdv [16] . in assessment of laboratory tests effectiveness trueposi[17] . ninety-five percent confidence intervals (95% ci) were calculated according to wilson [17] . oligonucleotide microarray for rapid detection of aiv, ndv, ibv, ibdv in the result of analysis of nucleotide sequences from ncbi "influenza virus sequence database"genes encoding m and np proteins were chosen for aiv identification. nucleotide sequences of gene encoding np protein were selected for ndv. segment a of vp2 protein was used for ibdv. nucleotide sequences of gene encoding s1 protein were chosen for ibv identification. oligonucleotide primers and probes were produced in the course of standard automatic synthesis in dna/ rna synthesizer h-16 (k&a laborgeraete, germany), their sequences are shown in tables 3 and 4 . the selected oligonucleotide probes were used to develop a microarray for rapid diagnosis of aiv, ndv, ibv, and ibdv. probes were applied on the support by the method of contact printing in nano print lm60 (arrayit corp., usa). the study has shown that amplification products hybridize on the microarray only in case of obtaining sufficient number of single-chain fluorescently-labeled fragments of np and m (aiv), np (ndv), vp2 (ibdv), and s1 (ibv). the electrophoregram has shown pcr products-305 bp (aiv), 411 bp (ndv), 421 bp (ibv), 593 bp (ibdv) (fig. 1) . hybridization on the microarray of the obtained fluorescently-labeled fragments of np and m2 (aiv), vp2 (ibdv), s1 (ibv), np (ndv) genes of the viruses under study resulted in formation on glass slide of stable hybridization complexes with high binding energy and oligonucleotides the sequences of which were complementary to sequences of hybridized np and m2 of aiv, vp2 of ibdv, s1 of ibv, np of ndv gene fragments. for interpretation of the results the microarray layout is shown on fig. 2 . the first two horizontal rows contain universal oligonucleotide probes to np and m2 genes of aiv, next are probes for detection of vp2 gene of ibdv and s1 gene of ibv. last row contains probes for detection of np gene of ndv. in the microarray layout there are 16 identical subarrays arranged into 2 columns and 8 rows containing oligonucleotide probes that are complementary to antisense strand of aiv, ndv, ibv and ibdv genes. the dna-chip was scanned with innoscan710al ("innopsys", france) by cy5 channel activation. the results were interpreted with use of mapix ver. 5.5.0 software. the signal exceeding the background value was adopted as a positive result. the finding of the study considered reliable if in the course of scanning by cy5 channel bright fluorescent spots were observed. in the assay of samples the value of specific fluorescence reliably exceeded the value of the background signal (p < 0.05) (figs. 3 and 4) . as figs. 3 and 4 show m2 and np genes of aiv, vp2 gene of ibdv, s1 gene of ibv and np gene of ndv were reliably detected in all specimens. for testing specificity of the method direct sequencing of the pcr-products in abi prism 3130 xl genetic analyser, applied biosystems was carried out. the synthesized nucleotide sequences were analyzed using blast program. these nucleotide sequences that are the fragments of np and m2 (aiv), vp2 (ibdv), s1 (ibv), np (ndv) genes were compared with the data of the genbank database. computer analysis has shown the amplified specific pcr-products of aiv, ndv, ibv, ibdv to be parts of their genomes. so, homology of compared sequences confirms specificity of the developed method. the results of the performed experiments prove again that the used fragments of tested genes are highly specific for aiv, ndv, ibv, ibdv. the limit of detection of the assay method is 10 2 copies of rna. testing of the microarray with use of viruses from the ribsp microbial collection different strains of aiv, ndv, ibv and ibdv were used to test the oligonucleotide microarray. testing was carried out in comparison with real-time rt-pcr (table 5) . fifteen different strains of aiv, ndv, ibv and ibdv, diverse in their origin, epidemiological and biological characteristic, were identified correctly with use of dna microarray. diagnostic results of testing dna microarray with use of virus strains from the ribsp microbial collection were comparable to the results of the realtime rt-pcr. sensitivity of the microarray was comparable to the sensitivity of real-time rt-pcr. in large-scale epidemiological studies possibility to analyze concurrently one specimen on many diagnostic probes is extremely important for agent identification. it allows minimizing the time of analysis from several days to several hours. the microarray efficacy in rapid diagnosis of avian viral diseases was evaluated versus virus isolation in embryonated eggs and real-time qpcr for aiv, ndv, ibv and ibdv with use of 122 samples-44 cloacal swabs and 78 tissue samples from dead birds (table 6) . true disease status is determined by the most accurate diagnostic method possible that is called gold standard. in diagnosis of influenza and other avian infections it is virus isolation in chicken embryos followed by identification in hemagglutination inhibition test [18] , the whole procedure taking from 2 to 5 days. in our study aimed at evaluation of the microarray and real-time rt-pcr effectiveness we used the test of virus isolation in chicken embryos as gold standard. among 44 cloacal swabs analyzed with use of microarray, real-time rt-pcr and virus isolation in embryonated eggs 15.90% of specimens were positive for aiv, 63.64% for ndv. none of cloacal swabs was shown to be ibv and ibdv positive. aiv was detected by the microarray in 14.10% of 78 tissue samples from dead birds, ndv-in 84.62%, ibv-in ndv was detected in 90.00% of samples from great cormorants (phalacrocoracidae family), 100% from dalmatian pelican (pelecanidae family) as well as in 100% of samples from broiler chickens of poultry factory "allel agro" (almaty region) and in 100% of samples from dead domestic chickens in small households of korday district (zhambyl region). ibv and ibdv were detected respectively in 8.57 and 5.71% of samples from dead domestic chickens in small households of korday district (zhambyl region). so, 118 samples of 122 were positive for avian infections being diagnosed with use of the dna microarray and virus isolation in embryonated eggs, 117 samples displayed positive results in real-time rt-pcr. diagnostic results of the dna microarray testing with use of experimental specimens from sick and dead birds were comparable to the results of virus isolation in embryonated eggs and real-time rt-pcr. advantage of the dna microarray is simultaneous assay of samples for presence of 4 infections-avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease of birds, while virus isolation in embryonated eggs and real-time rt-pcr allow detecting only one agent in a sample. the results of aiv, ndv, ibv and ibdv detection in clinical specimens by different methods are shown in sensitivity and specificity characteristics of the developed dna microarray and of the real-time rt-pcr as well as positive and negative prognostic values at 95% confidence interval are shown in table 7 . while virus isolation in embryonated eggs was used as a standard method in our studies the dna microarray demonstrated diagnostic sensitivity equal to 99.16% within 95% confidence limits 97.36-100%) and diagnostic specificity equal to 100%. high microarray sensitivity is comparable to the diagnostic sensitivity of the realtime rt-pcr equal to 99.15% within 95% ci limits 97.30-100%. diagnostic specificity of the dna microarray and real-time rt-pcr is 100%. positive predictive values for the dna microarray and real-time rt-pcr are 100%. negative predictive values are 75 and 80% respectively. currently, most methods of aiv, ndv, ibv, ibdv and other avian viral agents detection are adapted to specific detection of one agent in a sample. multiplex rt-pcr is successfully used for detection of aiv and its subtypes [19, 20] and for diagnosing double infections such as combination of ndv and aiv [21] . also methods with use of fig. 3 results of scanning labeled cdnas of aiv, ndv, ibv and ibdv strains on microarray. 1 -"a/duck/alberta/35/76" (h1n1) (aiv); 2 -"a/duck/ germany/215" (h2n3) (aiv); 3 -"a/duck/california/72" (h3n8) (aiv); 4 -"a/duck/czechoslovakia/56" (h4n6) (aiv); 5 -"vinterfild" (ibdv); 6 -"bg" (ibdv); 7 -"201" (ibdv); 8 -"koktal" (ibdv); 9 -"52/70" (ibdv); 10 -"h-120" (ibv); 11 -"10-95" (ibv); 12 -"63/00" (ndv); 13 -"52/98" (ndv); 14 -"bor-74 vgnki" (ndv); 15 -"columba livia/kz/eko/15/2014" (ndv) multiplex real-time rt-pcr for aiv, ndv and ibv subtypes differentiation have been developed [22] [23] [24] . at present development of a test based on microarray technology for simultaneous detection of aiv, ndv, ibv and ibdv in one sample is important for poultry industry in the republic of kazakhstan. use of microarray improves quality and shortens the analysis duration in molecular diagnosis of infectious diseases and therefore is employed as an independent method in screening for several genes of large numbers of pathology samples [25] [26] [27] . there are biochips for influenza diagnosis that allow screening not only for ha and na, but for m and np genes of influenza a virus [25, 28] . in identification of ndv molecular methods with use of oligonucleotides specific to conservative regions of np-gene of ndv were used [29] . recently vp2 fig. 4 intensity profiles of hybridization signals of labeled cdnas of strains "a/duck/alberta/35/76" (h1n1) (aiv); "a/duck/germany/215" (h2n3) (aiv); "a/duck/california/72" (h3n8) (aiv); "a/duck/czechoslovakia/56" (h4n6) (aiv); "vinterfild"(ibdv); "bg" (ibdv); "201" (ibdv); "koktal" (ibdv); "52-70" (ibdv); "h-120" (ibv); "10-95" (ibv); "63/00" (ndv); "52/98" (ndv); "bor-74 vgnki" (ndv); "columba livia /kz/eko/15/2014" (ndv) gene region of ibdv is successfully used in synthesis of oligonucleotide primers and probes from highly conservative regions for molecular diagnosis [30] [31] [32] [33] . molecular methods for ibv diagnosis are oriented at using more conservative sequences located in s1 and s2 genes of ibv [34, 35] . in the proposed microarray probes were developed on the basis of conservative regions of gene fragments encoding np and m (aiv), np (ndv), vp2 (ibdv), s1 (ibv) array proteins from genbank database. all viral gene fragments demonstrated high rate of conservatism and therefore the test is universal for detecting aiv, ndv, ibv and ibdv strains. so, high homology of nucleotide sequences of gene regions encoding aiv, ndv, ibv and ibdv array proteins compared to genbank data confirms specificity of the developed microarray for rapid diagnosis of avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease. total analysis duration without time required for the viral rna extraction is 5-6 h, and 16 specimens can be simultaneously assayed. duration of the assay with use of the proposed microarray is not longer than in other molecular methods and simultaneous testing of samples for aiv, ndv, ibv and ibdv provides its advantage over other methods. various methods have been developed for the diagnosis of bird infection, such as virus isolation in cell culture, embryonated chicken eggs, or young specificpathogen-free (spf) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (elisa), or immunohistochemistry. all these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. these methods lack the ability to detect low levels of antigens in tissues [36] [37] [38] [39] [40] . in the present study field samples (122 in total) were used to test effectiveness and reliability of the microarray. nevertheless, positive result of using molecular and biological methods, being very important in emergency cases, should always be confirmed by the method of virus isolation. the results of the study show that diagnostic sensitivity (99.16%) and diagnostic specificity (100%) of the dna microarray are comparable with the same of the real-time rt-pcr (99.15 and 100%, respectively). diagnostic effectiveness as percentage ratio of true results to the total number of obtained results for the developed dna microarray and real-time rt-pcr was 99.18%. analysis of the obtained data shows that the microarray test for rapid diagnosis of avian infections demonstrates the effectiveness comparable to that of the molecular method real-time rt-pcr and is more rapid and less resource-consuming owing to its ability to detect simultaneously aiv, ndv, ibv ibdv positive samples in the course of one experiment. universality of the test makes it suitable for wide use in veterinary laboratories for prompt detection of avian infections. the developed microarray for rapid diagnosis of avian viral diseases can be used in mass analysis in the system of routine epidemiological surveillance owing to its ability to test one sample for simultaneous detection of aiv, ndv, ibv and ibdv in cases of single and mixed viral infections. at the same time duration of the analysis decreases many times versus classical methods and the proposed scheme of specimen preparation allows conducting assays immediately in small veterinary laboratories thus avoiding transportation of thermolabile rna. the study described in the article "new oligonucleotide microarray for rapid diagnosis of avian viral diseases" was conducted at the research institute for biological safety problems (ribsp), republic of kazakhstan. patent application called "method for rapid diagnosis of avian viral diseases (avian influenza, newcastle disease, infectious bursal disease, infectious bronchitis) on the basis of microarray technology" was registered at the national patent office under the number 2016/0784.1). in adherence to the virology journal guidelines the ribsp will make freely available any materials and information described in the publication that are reasonably requested by others for the purpose of academic, noncommercial research. this does not alter the authors' adherence to all the virology journal policies on sharing data and materials. consent for publication not applicable. no personal data were collected in the context of this study. re: scientific interest in newcastle disease virus is reviving molecular and structural bases for the antigenicity of vp2 of infectious bursal disease virus laboratory manual for isolation and identification of avian pathogens infectious bronchitis virus in jordanian chickens: seroprevalence and detection coronavirus avian infectious bronchitis virus review of rapid diagnostic tests for influenza rapid detection and simultaneous subtype differentiation of influenza a viruses by real time pcr detection and subtyping (h5 and h7) of avian type a influenza virus by reverse transcription-pcr and pcr-elisa rapid multiplex reverse transcription-pcr typing of influenza a and b virus, and subtyping of influenza a virus into h1 a multiplex reverse transcription-polymerase chain reaction assay for newcastle disease virus and avian pneumovirus (colorado strain) avian influenza virus detection and quantitation by real-time rt-pcr rapid detection and differentiation of newcastle disease 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classical, very virulent and variant strains infectious bursal disease viruses: molecular differentiation of antigenic subtypes among serotype i viruses molecular identification of infectious bursal disease virus strains detection of infectious bursal disease viruses in commercially reared chickens using the reverse transcriptase/polymerase chain reaction-restriction endonuclease assay phylogeny of antigenic variants of avian coronavirus ibv the coronavirus surface glycoprotein detection of infectious bursal disease virus in different lymphoid organs by single-step reverse transcription polymerase chain reaction and microplate hybridization assay point-counterpoint: is the era of viral culture over in the clinical microbiology laboratory comparison of a rapid antigen test with nucleic acid testing during cocirculation of pandemic influenza a/h1n1 2009 and seasonal influenza a/h3n2 comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection not applicable. the present work was supported by grant research project "development and testing of microarray for rapid diagnosis of avian viral diseases", no. 0920/gf 4 (ministry of education and science, republic of kazakhstan). the data-sets analyzed during the current study was available from the corresponding author on reasonable request.authors' contributions ks, nk and vs conceived and designed the experiments. yb, ks, och and nr performed the experiments. ks, nk and vs analyzed the data. ks, ns, as and mo contributed to the work with data analysis and interpretation of results. ks wrote the paper. all authors read and approved the final manuscript. key: cord-310536-u30cufg7 authors: finger, paula fonseca; pepe, michele soares; dummer, luana alves; magalhães, carolina georg; de castro, clarissa caetano; de oliveira hübner, silvia; leite, fábio pereira leivas; ritterbusch, giseli aparecida; esteves, paulo augusto; conceição, fabricio rochedo title: combined use of elisa and western blot with recombinant n protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis date: 2018-12-12 journal: virol j doi: 10.1186/s12985-018-1096-2 sha: doc_id: 310536 cord_uid: u30cufg7 background: the avian infectious bronchitis virus (ibv) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. this study examined the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein (n) of ibv. the coding sequence for n was amplified by rt-pcr and expressed in escherichia coli. a soluble recombinant n protein (rn) of approximately 50 kda was obtained. a total of 389 sera were tested against the rn in elisa and the results were compared with those of the commercial idexx ibv ab test. elisa-rn achieved a 90.34% sensitivity and 90.16% specificity. wb confirmed all false negative sera in elisa-rn or idexx test as truly positive. the current study indicate that the combined use of rn in elisa and wb is a powerful tool for the immunodiagnosis of avian infectious bronchitis. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl21(de3) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp 1 ml columns pre-packed with pre-charged ni sepharose in the äktaprime automated liquid chromatography system (ge healthcare). a total of 389 serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rn antigen (50, 100 and 200 ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version 6.0 for windows (graphpad software, usa). results: the expected cdna fragment of approximately 1240 bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 kda. out of 244 sera that scored positive in the commercial elisa idexx ibv ab test, 220 were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of 90.16%. out of 145 sera that scored negative in the idexx ibv ab test, 131 also scored negative in the elisa-rn, indicating a specificity of 90.34%. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl21(de3) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp 1 ml columns pre-packed with pre-charged ni sepharose in the äktaprime automated liquid chromatography system (ge healthcare). a total of 389 serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rn antigen (50, 100 and 200 ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version 6.0 for windows (graphpad software, usa). (continued on next page) (continued from previous page) results: the expected cdna fragment of approximately 1240 bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 kda. out of 244 sera that scored positive in the commercial elisa idexx ibv ab test, 220 were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of 90.16%. out of 145 sera that scored negative in the idexx ibv ab test, 131 also scored negative in the elisa-rn, indicating a specificity of 90.34%. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. keywords: ibv, nucleoprotein, recombinant antigen, diagnosis, indirect elisa background avian infectious bronchitis (ib) is caused by a virus in the coronaviridae family, genera gammacoronavirus. it is a highly contagious disease with a short incubation period [1] . the avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (ibv). the ibv is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms [2, 3] . infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. secondary bacterial infections will also contribute to economic losses. carcass condemnation due to the development of airsacculitis [4, 5] negatively impacts commercial sales of bird meat and eggs. brazil was once the world's largest exporter of poultry and currently the world's third largest producer of bird meat [6] . the consequences of ibv are a significant threat to brazil's poultry industry. the ibv genome consists of a non-segmented positivesense single-stranded rna that is approximately 27.6 kb in length. it encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (n), the spike protein (s), the envelope protein (e), and the matrix protein (m). the nucleocapsid protein, or n protein, consists of 409 amino acids. it has a molecular mass of approximately 50 kda and directly binds with the viral genome to form the virion nucleocapsid [6, 7] . its structure is highly conserved, with different strains of ibv sharing a high degree of identity (94-99%) [8] . the n protein is also known for its immunogenicity, inducing specific antibody and cytotoxic t-cells mediated responses [9, 10] . there is significant interest in the use of the ibv n protein as an important target for diagnosis since it possesses the antigenic characteristics required for the development of serological assays that can be applied to detect or quantify antibodies against the ibv [11] . the laboratory diagnosis of ib is dependent on direct and indirect techniques. the direct techniques are employed for viral isolation and genomic or phenotypic identification of the virus, while the indirect methods are used to detect specific antibodies [12] . in addition to being applied for serodiagnosis, serological techniques can also be employed to evaluate the immune responses stimulated by vaccines. commercial elisa kits are typically used to indirectly diagnose ibv. these kits, however, are expensive when large number of samples require screening and they are not acessible for applications with the scale of the brazilian poultry industry [13] [14] [15] . elisa techniques currently available are designed to detect polyclonal antibodies that target the whole virion. the use of nucleoprotein as the antigen for diagnosis and evaluation of vaccine immune responses is an interesting target to explore since this protein plays a important role in ibv virus replication and the induction of a specific immune response in infected birds [16, 17] . the use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and, thereby, reduce or eliminate background reactions. the use of recombinant antigens also represents a viable method of reducing immunoassay development costs. easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis [18] . the aim of the current study was to evaluate the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein of ibv. a previously characterized brazilian viral sample of ibv strain massachusetts 41 (m41-cnpsa -embrapa -concórdia, sc, brazil) was propagated after 9 days of incubation in the chorioallantoic cavity of specific pathogen free (spf) embryonated chicken eggs. the allantoic fluid was then collected and stored at − 70°c. viral rna extraction was carried out with trizol® ls reagent (invitrogen™, eua), according to the manufacturer's instructions. extracted rna from ibv strain m41 was used for cdna synthesis with random oligonucleotides. reverse transcription (rt) was carried out using superscript® one-step rt-pcr system (invitrogen, usa). the resulting cdna samples were used to for pcr amplification of the whole orf of the n protein gene. primers based on the ibv m41 n protein gene sequence available at gen-bank (accession number m28566) were designed to align between 102 and 120 and 1312-1331 bp of the gene and include cleavage sites for restriction enzymes. there was a restriction site for xhoi in the forward primer (5′ -ccgctcgagatggcaagcggtaaggcaa -3′) and a restriction site for kpni in the reverse primer (5′ -ggggtacctcaaagttcattctctccta -3′). the pcr reaction was performed with approximately 25 ng of the extracted cdna, 3.5 mm mgcl, 0.2 mm dntps, 2 units of taq dna polymerase, 1x reaction buffer, 1 pmol of each primer, and 5 m n,n,n-trimethylglycine (betaine) under the following conditions: 1 cycle of 95°c for 7 min, 1 cycle of 70°c for 1 min, then 45 cycles of 94°c for 1 min, 50°c for 1 min, and 72°c for 4 min, and a final extension of 72°c for 10 min. the pcr amplification product was confirmed on a 1% agarose gel and purified using gfx pcr dna and gel band purification kit (ge healthcare, chicago, usa), according to the manufacturer's instructions. the pcr product was cloned into pae vectors by a t4 dna ligase (invitrogen) binding reaction after cleavage with restriction enzymes kpni and xhoi. the constructed recombinant pae/n expression vector was used to transform e. coli bl21(de3) star competent cells (invitrogen). the resulting recombinant clones were cultivated in 10 ml of lb broth medium with 100 μg/ml of ampicillin (37°c, 16 h, 250 rpm). the whole culture volume was transferred to flasks containing 200 ml of lb and incubated at 37°c with agitation (200 rpm) until the optical density (o.d.) at 600 nm reached 0.8. the expression of the recombinant n protein (rn) was induced by adding isopropyl-β-d-thiogalactopyranoside (iptg, 0.5 mm final concentration) to the culture and incubating for 3 h at 37°c with agitation (200 rpm). the cells were harvested at 10,000 x g for 10 min at 4°c and the culture pellet containing the rn protein was subjected to a solubilization procedure in äkta wash buffer (0.234% nah 2 po 4 , 2.92% of nacl, 0.068% imidazole, ph 8.0, supplemented with 20 μg/ml lysozyme). the resuspended cells were submitted to seven sonication cycles of 20 s at 60 hz and centrifuged again. the rn was purified by affinity chromatography using histrap hp 1 ml columns pre-packed with pre-charged ni sepharose in the äktaprime automated liquid chromatography system (ge healthcare). the protein concentration was determined with qubit™ protein assay kits (thermo-fisher scientific, usa) according to the manufacture's instructions. the sds-page was carried out on 12% polyacrylamide gel. the gels were either stained with coomassie blue r-250 (bio-rad, california, usa) or electroblotted onto hybond-ecl 0.45 μm nitrocellulose membranes (ge healthcare) using the bio-rad mini trans-blot cell (bio-rad) for western blot. briefly, the membrane was blocked with 5% non-fat milk in phosphate buffer saline containing 0.05% tween-20 (pbs-t) (137 mm nacl, 2.7 mm kcl, 100 mm na 2 hpo 4 , 2 mm kh 2 po 4 , ph 7.4), incubated with mouse monoclonal antibody (mab) anti-6xhis (sigma, usa) diluted to 1:10000 in pbs-t, and incubated with polyclonal antibody anti-mouse igg conjugated to hrp (sigma). all incubation steps were performed at 37°c for 1 h under slight agitation followed by three washes with pbs-t. the immunoblot was developed using 3,3′-diaminobenzidine (sigma). a total of 389 chicken serum samples (n = 389) were used to develop and evaluate the elisa-rn. the serum samples were kindly provided by mercolab laboratories (garibaldi, brazil), a laboratory accredited by the mapa (ministry of agriculture, livestock and food supply) that uses the commercial elisa kit ibv ab test (idexx) for characterization. serum samples from chickens with a positive diagnosis for newcastle disease were used for the evaluation of the elisa-rn specificity. the samples used in this study originated from birds with a known history of vaccination, including vaccination against the newcastle disease virus, and were categorized into three groups: commercial egg-layers, broiler breeders, and meat-producing chickens. this information was further used to improve the evaluation of the elisa-rn test. were tested. positive and negative sera for ibv were used as controls. the 96-well microtiter plates (nunc max-isorp®, thermo fisher, usa) were coated with 100 ng/ well of rn diluted in 0.05 m carbonate-bicarbonate buffer (ph 9.6) and incubated overnight at 4°c. the plates were incubated at 37°c for 1 h with a blocking solution (5% non fat dry milk in pbs-t). serum samples diluted 1:200 in pbs-t were added in duplicate and the plates were again incubated for 1 h at 37°c. after this period, the secondary antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) diluted 1:10,000 in pbs-t was added and plates were once more incubated at 37°c for 1.5 h. after each incubation, the plates were washed three times with pbs-t. in the final step, plates were washed five times and 100 μl/well of peroxidase substrate o-phenylenediamine dihydrochloride (sigma aldrich) was added. the reaction was interrupted with 2 n h 2 so 4 and the results were read as o.d. using a spectrophotometer at 492 nm. to determine the cutoff value, negative sera were used, totalizing 20 sera, considering the mean of sera added of two standard deviation. the roc analysis was used to simulate the influence of different cutoff values on the sensitivity and specificity of the test. the results were compared with those obtained with the commercial kit. serum that scored as negative in the commercial kit but as positive in the elisa-rn were submitted to western blot analyses using the rn protein as antigen. in order to evaluate the repeatability of the elisa-rn, three separate batches of recombinant n protein were produced and purified following the methodology described above with distinct purification times, to demonstrate the reproducibility of the recombinant protein expression procedure. serum samples were selected for testing against each batch of antigen and the averages, and standard deviations, of the o.d. at 492 nm were calculated. of the samples used for this test, one was strongly reactive positive, four were moderately reactive positive, and four were negative serum samples. to evaluate the specificity of the test, negative serum samples to ibv and positive serum samples to newcastle disease were tested in the elisa-rn. the western blot was performed almost as described above. the rn was deposited in all wells of the sds-page gel and, after transfer to the nitrocellulose membrane, it was cut to obtain 14 strips. each strip was incubated with a 1:200 serum dilution after blocking with 5% non-fat milk. the antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) (1:10000) was added to all membranes and the reaction was revealed using 3,3′-diaminobenzidine (sigma). all incubation steps were performed at 37°c for 1 h under slight agitation and were followed by three washes with pbs-t. the results of the elisa-rn compared to the commercial kit results, the receiver-operating characteristics (roc) curves, the area under the curve (auc) and the confidence intervals were obtained through the software graphpad prism version 6.0 for windows (graphpad software). the expected cdna fragment of approximately 1240 bp was successfully amplified by pcr using primers designed to obtain the coding region of the n protein. successful recombinant pae/n vector construction was confirmed through cleavage with the same restriction enzymes used for the plasmid construction (date not shown). the recombinant n protein (rn) was expressed as a soluble protein, dispensing refolding steps, and, after purification, a yield of 10 mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 kda and 50 kda (fig. 1a) . the same bands were observed in the western blot and confirmed the presence of bands that corresponded to the protein of interest (fig. 1b) . other studies have also described the cloning of the 1200 bp corresponding to the n protein gene from ibv [18, 19] . they reported that the recombinant protein presented as two distinct molecular masses, one 50 kda and other 45 kda. the 45 kda mass is the truncated form of the n protein [18, 19] . this corroborates with the results observed through sds-page and western blot in the current study (fig. 1) . the results of the elisa-rn were compared with the results obtained with the ibv ab test (idexx). receiver-operating characteristics (roc) with 95% confidence intervals were used to analyse these results (fig. 2) . out of 244 sera that scored positive in the ibv ab test, 220 also scored positive in the elisa-rn test. the sensitivity of the elisa-rn test was 90.16%. out of the 145 sera that scored negative in the idexx test, 131 also scored negative in the elisa-rn, indicating a specificity of 90.34%. the area under the curve (auc) was 0.9588 (p < 0.001 and cutoff 0.5415). three different protein batches were used as coating antigens for testing the elisa-rn and the results from all three were similar (p > 0.05) and, thus, indicate antigen stability (fig. 3) . all newcastle disease positive sera tested negative in the developed elisa-rn, providing evidence of test specificity (data not shown). all sera that tested negative in the commercial test and positive in the elisa-rn were submitted for western blot analyses using the rn protein as an antigen. all serum samples reacted with the two bands of rn, confirming the results obtained in the developed elisa (fig. 4) . sera that tested negative in the elisa-rn and previous studies that focused on the development of more efficient diagnostic techniques were not limited to avian infectious bronchitis detection, but also aimed to control and monitor chickens vaccination [12, 20] . the nucleoprotein from ibv is widely considered the choice protein for the development of immunoassays for antibody detection since this protein plays an important role in inducing an antibody response in ibv infected or vaccinated animals [10, 13] . other studies have demonstrated that the nucleocapsid protein is highly conserved among ibv isolates (91-96.5% similarity), immunogenic, and abundantly expressed during infection [8, 10, 21] . these features make this protein an interesting candidate for use in diagnostic techniques, such as elisa and wb [4] . the elisa is a powerful tool because it provides a safe and an easy way to evaluate the ibv vaccine efficiency, and perform serological diagnosis as well as epidemiological surveillance [18] . in contrast to the elisa presented in this study, the elisa developed by lugovskaya et al. [15] used two fragments of the recombinant n protein expressed in e. coli as an antigen, achieved a specificity of 87.36%, and a sensitivity of 93.81%. these results are comparable with the commercial test that is currently used for routine ibv diagnosis that has a specificity of 88.97% and a sensitivity of 92.86% [15] . the elisa-rn developed in this study presented similar specificity and sensitivity results (90.16 and 90.34%, respectively), and thus highlights that our study uses a western blot technique to complement the results obtained through elisa and further support the ibv diagnosis. the western blot technique may prove useful for epidemiological studies, for monitoring specific pathogen free farms, and in vaccine potency tests. the association of elisa and western blot can be seen as a tool to be considered to increase sensitivity for serodiagnosis purposes [22] . since positive sera react with two bands in the western blot, the technique is even more specific. the recombinant n protein produced in this study was expressed in the soluble fraction. our rn was easily recoverable from the culture without using denaturing agents. in contrast to previous studies where the n protein was expressed in its insoluble form [10, 14] , our procedure avoids the necessity to refold the recombinant protein. the production of rn was repeatable since the same elisa-rn test results, and about the same yield, were obtained when different production and purification times were used. besides the use of the n protein for the diagnosis of ibv, an elisa that was developed using the s protein was also described and compared to a commercial test [23] , and achieved a specificity and sensitivity of 89.83 and 92.38%, respectively. however, the n protein offers specific advantages when used for the purpose of diagnosis during viral infection since it is produced in larger quantities than the s protein (the n proteins is produced at a ratio of 6:1 relative to the s protein [24] and plays an important role in the virus's replications and assembly process [25] . additionally, the s protein has hypervariable regions that cause mutations in its sequence and therefore offers low efficiency as a protein for diagnosis [1] . the results from the elisa test developed using the rn protein indicate that the test could be effectively applied for ibv diagnosis [17] . the yield of rn per litre of lb broth culture would coat approximately 1000 96-well microtiter plates allowing for the diagnostic analysis of approximately 45,000 serum samples in duplicate. also noteworthy is the production cost reduction from using rn in a soluble form, since this eliminates costs associated with the refolding step while it also preserves important conformational epitopes. the currently available commercial test on the other hand, employs the whole ibv as an antigen which requires viral propagation and the implementation of robust laboratory biosafety standards [23] . it is worth mentioning that, by comparing the developed elisa-rn and wb with the idexx ibv ab test, the analysis conducted in the current study indicated that it was possible that some serum identified as negative for anti-ibv antibodies on the commercial test be positive when tested using the elisa-rn and by wb. this could be of concern as false negative results are undesirable, especially if a lot of birds are misdiagnosed. thus, the elisa-rn could be applied together with the wb developed in this study for the routine detection of ibv in diagnostic laboratories. false negative results that contribute to the spread of the disease can be avoided, or at least decreased, when the two tests are applied together. the indirect elisa developed here with rn as an antigen allowed for the detection of anti-ibv antibodies in chicken serum at high specificity and sensitivity. the association between elisa and western blot techniques developed with a subunit of ibv (rn) were able to detect antibodies that were not detected with the commercial elisa test suggesting greater sensitivity in the developed elisa-rn. in addition, the elisa-rn with the advantages of easy preparation and improved safety could be a promising alternative to the whole live virus elisa. relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of crossprotection in vivo infectious bronchitis virus: immunopathogenesis of infection in the chicken efficacy of infectious bronchitis virus vaccines against heterologous challenge coronavirus avian infectious bronchitis virus bronquite infecciosa das galinhas: conhecimentos atuais, cepas e vacinas no brasil polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses comparisons of the structural proteins of avian infectious bronchitis virus as determined by western blot analysis specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in brazil recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry high-level protein expression following single and dual gene cloning of infectious bronchitis virus n and s genes using baculovirus systems detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens selective replication of coronavirus genomes that express nucleocapsid protein elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus diversidade genética de amostras brasileiras do vírus da bronquite infecciosa determinada pelo sequenciamento de nucleotídeos dos genes n e s1 development of a recombinant nucleoprotein-based enzyme-linked immunosorbent assay for quantification of antibodies against porcine reproductive and respiratory syndrome virus combined use of western blot / elisa to improve the serological diagnosis of human tuberculosis development of a multiepitope antigen of s protein-based elisa for antibodies detection against infectious bronchitis virus the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3′ genomic rna coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides we thank mercolab laboratory (garibaldi, rs, brazil) for giving the sera that were analyzed in this study. not applicable. all data generated or analyzed during this study are included in this article. all authors read and approved the final manuscript. this article does not contain any studies with human participants or animals performed by the author. the authors authorize the publication. no known competing interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-316153-wet0go35 authors: jia, w.; karaca, k.; parrish, c. r.; naqi, s. a. title: a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains date: 1995 journal: arch virol doi: 10.1007/bf01309861 sha: doc_id: 316153 cord_uid: wet0go35 an antigenic variant of avian infectious bronchitis virus (ibv), a coronavirus, was isolated and characterized. this strain, cu-t2, possesses a number of unusual features, which have not been previously observed in ibv. the s1 glycoprotein of cu-t2 carries virus-neutralizing and serotype-specific epitopes of two ibv serotypes, arkansas (ark) and massachusetts (mass). sequence analysis revealed that the virus, originally an ark serotype, has acquired the mass-specific epitope by mutation(s). this provides evidence that point mutations may lead to generation of ibv antigenic variants in the field. it was further observed that two independent recombination events involving three different ibv strains had occurred in the s2 glycoprotein gene and n protein gene of cu-t2, indicating that genomic rna recombination in ibv may occur in multiple genes in nature. it was especially significant that a sequence of holland 52 (a vaccine strain) had replaced half of the n gene of cu-t2. this proves that recombination among vaccine strains is contributing to the generation of ibv variants in the field. based on these observations it is predicted that every ibv field isolate could have unique genetic nature. therefore, several recently reported diagnostic and serotyping methods of ibv which are based on dot-blot hybridization, restriction fragment length polymorphism (rflp), and polymerase chain reaction (pcr), may not reveal the true antigenic and/or genetic nature of ibv isolates, and may in fact yield misleading information. results in heavy economic losses to the commercial poultry industry, worldwide [24] . ibv has a single-stranded, positive sense rna genome. it is 27.6 kilobases long, encodes three major structural proteins, and is organized as 5'-pol-s-gene3-m-gene5-n-3' (fig. 1) [9, 35, 36] . the three structural proteins of ibv are the spike glycoprotein (s protein), the membrane glycoprotein (m protein) and the nucleocapsid protein (n protein). the s protein of ibv is cleaved post-translationally into n-terminal s1 and c-terminal $2 proteins [13] . the s1 protein carries antigenic epitopes which induce virus neutralizing (vn) antibody, and also determine the virus serotype [14, 15] . the n protein may induce cellular immune responses against ibv [6] . ibv isolates have been grouped into serotypes based on vn test [16, 18] . although more than twenty distinct ibv serotypes have been reported in the usa [17, 32] , most isolates causing disease in the field belong to massachusetts (mass), connecticut (conn), or arkansas (ark) serotypes [24] . live vaccines containing strains of those three serotypes have been routinely used in the field. however, more frequently than ever, outbreaks of the disease are being observed in vaccinated flocks. these vaccine failures, however, are not always due to infection by distinct serotypes, but could be caused by antigenic variants of ibv emerging from wild-type or vaccine viruses by point mutation(s) or genomic rna-rna recombination [12, 25] . although point mutation(s) are believed to contribute to the generation of new antigenic variants of ibv, no direct evidence for that has been presented. rna-rna recombination has been shown to occur at a high frequency both in vivo and in vitro in mouse hepatitis virus (mhv), a coronavirus i3, 22, 23, [27] [28] [29] 31 ], yet, no isolation of recombinant mhv from natural disease in mice has been reported. in the case of ibv, one japanese and three european isolates have been suggested to be possible recombinants [11, 25] . however, the origin of ~-~'~unknown-oriqin sequence[-------]hol152-1ike sequence ~ hvr fig. 1 . schematic characterization of the cu-t2 genome. pol rna polymerase gene. s s protein gene; $1 s1 protein gene; $2 $2 protein gene. n n protein gene. u tr 3' end untranslated region. hvr hypervariabte region the recombination fragments in those isolates was unknown. more recently, two american field isolates were found to contain fragments of mass-like sequences in the s1 gene, which were 94% to 95% homologous to ibv strain mass41 [38] . all the recombinations suggested in ibv so far have been in the s gene, and none of those reports have addressed whether the recombinations had altered the antigenic characteristics of the viruses. here we describe an ibv serotype and recombination variant, cu-t2, which reacted with two monoclonal antibodies (mabs) specific to the vn epitopes of ibv serotypes mass and ark, respectively. we present evidence that the cu-t2 was originally an ark serotype which subsequently acquired a mass-specific epitope on the s1 protein through mutation(s). we also show that cu-t2 had undergone two independent genomic rna recombination events within the $2 and n protein gene (n gene), respectively, involving three different ibv strains. furthermore, we provide evidence that cu-t2 had acquired a sequence of holland 52 (ho1152), a vaccine strain of mass serotype used in the field. implications of such genetic and antigenic changes in diagnosis and serotyping of ibv are discussed. four previously characterized ibv strains, ark99, hol152, corm46, and mass41 [19, 20] were obtained from our own laboratory stocks. cu-t2 was isolated from an adult commercial chicken flock in new york state, which was experiencing reduced egg production and poor egg shell quality associated with ibv infection. virus isolation from the flock was performed through the use of "sentinel" chickens. briefly, ten specificpathogen-free (spf) chickens were placed among the affected birds in separate cages for ten days. subsequently, the "sentinel" chickens were euthanized and the tracheas were collected for passage in spf chicken embryos. cu-t2 strain was recovered from the allantoic fluid of inoculated embryos, and was subsequently propagated and plaque purified in chicken kidney cell (ckc) culture monolayers [16] . mabs 1318, 1588, and 940 specific to sl-associated serotype-specific epitopes of ibv prototype ark, mass, and conn, respectively, have been described elsewhere [21] . the protocols for both antigen-capture and competitive-binding elisas have also been reported previously [21, 34] . the elisas were performed using whole virions, or purified s1 protein of cu-t2 according to the procedures used previously [21] . viruses were propagated in chicken embryos and purified by sucrose gradient as described previously [37] . viral genomic rnas were extracted with phenol-chloroform and used for cdna synthesis and rna sequencing. cdna synthesis, cionin9 and sequencin 9 for cdna synthesis, an uni-zap ii kit (stratagene, la jolla, ca) was used in conjunction with a synthetic primer complementary to a sequence located downstream of the s genes of 262 w. jia et al. several mass serotype strains [5] (5'-gaactagtctcgaggaaggacgtgggact-ttg-y). the cdna library was screened by hybridization with a 5' end-radiolabeled probe prepared from a styi fragment derived from a cdna clone (pbsm41m) of s gene of mass41 obtained from solvay animal health, inc., mendota heights, mn. phagemids containing ibv genes were prepared by in vivo excision in escherichia coli (e. coli) using a protocol furnished by stratagene (la jolla, ca). dna sequencing was carried out by the dideoxy method, using a sequenase 2.0 dna sequencing kit (united states biological (usb), cleveland, oh). cdna clones park99-8pl, pbark99-8p2, pbt2-t223, pbt2-t229, and pbt2-t237 were used for sequencing. both strands of each cdna clone were sequenced. sequence data were obtained from cdna clones, except for the first 1100 bases of the s gene, the last 200 bases of the n gene, the 3' end non-coding region of cu-t2, and the partial gene3 of ark99 and ho1152, which were obtained from direct sequencing of ibv genomic rna. direct sequencing of ibv genomic rna was also performed at least twice for those regions, using an rna sequencing kit (usb, cleveland, oh). sequence alignments and analysis were performed using the genetics computer group (gcg) program. the nucleotide sequences reported here have been deposited with the genbank. the accession numbers are as follows: s protein gene of ibv ark99, l10384; s protein gene of ibv cu -t2, u04739; n protein gene of ibv cu-t2, u04805; 3' end non-coding region of ibv cu-t2, u04804. tables 1 and 2 summarize results of the three types of elisas performed with the ark, conn, and mass serotype-specific mabs. in both the indirect and antigen-capture elisa, the ark-specific and mass-specific mabs reacted with both the whole virions and the purified s1 protein of cu t2 strain (table 1) . in the competitive-binding elisa, unlabeled mass-and ark-specific mabs blocked reaction of the homologous labeled mabs, but not with the heterotogous mabs. in order to determine the genetic basis for the expression of the ark-and mass-specific epitopes on the $1 protein ofcu t2, the entire s gene of cu t2 was sequenced. the entire s gene of ark99 was also sequenced for comparison. all base positions indicated in this paper are calculated from the s gene start codon [4] . amino acid positions indicated are of the s protein precursor, including the signal peptide. the gene of ark99 was found to be 3480 bases long, with a capacity to code 1159 amino acids. the s1 and $2 genes were 1629 and 1851 bases long, respectively, and were predicted to encode 543 and 616 amino acids, respectively. comparison of the deduced amino acid sequences of the s proteins of ark99 and mass41 [5] , revealed an 88.2~o homology (1022/1159). however, the s1 protein showed only 78.1~o homology (424/543), compared to 97.1% (598/616) for the $2 (data not shown). amino acid variations in the s1 proteins of the two viruses were more than 7 times as frequent as for the $2 protein. alignment of the $1 gene of ark99 with that of mass and several european ibv isolates revealed that fifteen bases (5'-tggaagtgctacgcc-3') between bases 419 and 435 of the ark99 strain were unique to that virus (data not shown). a 95.3~ homology was observed between the first 1100 bases of the $2 gene of ark99 and mass41, while the last 751 bases of the two genes were identical (fig. 2) . the nucleotide sequences of s1 genes of cu-t2 and ark99 exhibited over 98~o homology° the fifteen-base insertion in the s1 gene of ark99 described above was also observed in the cu-t2 $1 gene, but its location was between bases t agggtcttaa tgactc-tt-atagaccttg aaa-a-t-tc aatactcaaa acttatatta agtggccttg gtatgtgtgg ttagccatag cttt-gccac 3330 3429 t c a a a gc tc (~ gt c q a tattatcttc atc-taata-tagg-tgggt tttcttcatg actgg-tgtt gtggttgttg ttgtggatgc tttggcatta tgcctctaat gagtaagtgt 3430 3529 t c t t 416 and 432, as compared to bases 419 and 435 for ark99. the difference in the position of the insertion was due to a three-base deletion (taa) between bases 356 and 360 of the s1 gene of cu-t2 compared to the s1 gene of ark99. a single noncoding substitution was noted within the fifteen-base insertion of cu-t2 (t versus c at position 428). a 96.7% homology was observed between the amino acid sequences of the s1 proteins of cu-t2 and ark99. seventeen amino acids were found to be different in the s1 protein of cu-t2 compared to those in ark99, and those differences were distributed throughout the gene (data not shown). a 96.6~o homology was observed in the first 1100 bases of the $2 genes of ark99 and cu-t2, but, the following 751 bases had only 87.63/o homology (fig. 2) . comparison with published sequences showed that the sequence of this 751-base region of cu t2 was more similar to a fragment in the 3' end of the $2 gene of a japanese isolate kb8523 [37] than to those of all other strains. although 76 nucleotide substitutions were found in that fragment of the cu-t2 and kb8523, 66 of those were synonymous substitutions (fig. 2) . the downstream crossover site of this fragment was about 12 to 13 bases downstream of the stop codon of the $2 gene of cu-t2. through a substitution of t by g in the taa stop codon, the $2 gene of cu-t2 was 27 bases longer than that of ark 99, but of the same length as the $2 gene of kb8523 (fig. 2) , indicating that a fragment of approximately 751 bases was incorporated into the $2 gene of cu-t2 by recombination. to further study the possible evolutionary origin of the cu-t2 strain, the first 268 bases of gene3, the n gene and the 3' non-coding region of cu-t2 were sequenced subsequently. the same regions of gene3 of ark99 and ho1152 were also sequenced for comparison. the first 268 bases of the gene3 of cu-t2 matched more closely with those of ark99 gene3 (97.6~o homology), than of mass41 [7] , kb8523 [37], or ho1152 (table 3) . the n gene of cu-t2 was t230 bases long, with a capacity to code 410 amino acids. around 436 bases at the 5' end and 150 bases at the 3' end of i i00 g a g g a c t a g t g atggc~gcg gt~ggc~c tggaaagaca gacgccccag cgccagtcat caaactagga ggaccaaa-c cacctaaagt tggttcttct ggaaatgcat 101 200 c t t c tt t a t t t cttggtttca agc~t#~ gcc~gaagc taaattcacc tccacct~g tttg~ggta gcggtgttcc tgat~tg~ ~tcttaaaa c~gccagca 201 300 g t c ac t c a t t a g c t g t a t acatgg-tac tggagacgcc ~gc-aggtt t~gccaggt aaaggcggaa gaaaaccagt cccagatgct tggtacttct attatactgg aacaggacca 301 400 t g c c gt c a t a gccgctgacc tg~ttgggg tgatagcc~ gatggtatag tgtgggttgc tgcaaagggt gctgatacta ~tctagatc t~ccagggt ac~gggacc 401 500 t t tt g a c gaa at t t t atc~cagc-gcttcatcag cagcatctag tagagcaccg tcgcgtgatg gctcgcgtgg -cgtag~g-gg-gctg~g atgatcttat tgctcgtgca 601 700 t a ~ ag t c c c t gc~j~agatca ttcaggatca gcag~g~g ggttctcgca ttactaa-gc t~ggc-gat gaaatggctc atcgccggta ttgt t c c a -gttacagca atgctcaacc tagtccctag cagccatgct tgtctttttg gaagtagagt gacgcccaaa cttc~ccag atgggctgca cttga-attt cc t t a a t q t ag t t g~tttacta ctgtggtttc acgtgatgat ccgcagtttg at~ttatgt gaaaatttgt gatcagtgtg tg c c a t a g c g g ta t t g~gcaggat gatgaagtag ataaagcatt gacctcagat gaggagagga acaatgcaca gctggaattt gatgatg~c cc~ggtgat t~-tggggg 1201 1230 ac t a gt g a tt a tt a c ac t a gattcagc-t-ggtgag~ tg~ctttga cut2 n gene were very similar to the comparable regions of ark99. however, two crossover sites were observed around bases 436 and 1090 (fig. 3) . between those two sites only five bases were different (99.2% homology) between the cu-t2 and the ho1152 strains, and of those, only two were non-synonymous (fig. 3) . within the same region, 42 bases were different between cu-t2 and ark99 (93.6% homology), and eight of those changes were non-synonymous (fig. 3 ). there were many more differences in that region between cu-t2 and other ibv strains, including mass 41, gray, kb8523 and beaudette (data not shown). the 3' end untranslated region (utr) of the ibv genome was previously shown to contain an approximately 190-base hypervariable region (hvr) [39] (fig. 1) . that region of the cu-t2 sequence had a high homology with ark 99 (96.4~), but lesser homology with kb8523 (89.3%), ho1152 (77.3%) and mass41 (52.4~) (data not shown). the results show that cu-t2 is a serotype and recombination variant which originated from an ark-like ibv. this is based on both the sequence data and mab studies of the virus. the nucleotide sequence homologies between the s 1 gene, most of $2 gene, gene3 and the 3' utr of cu-t2 and ark99 all attest to the origin of cu-t2 from an ark-like virus. the fifteen-base insert observed in the $1 genes of both ark99 and cu-t2, which was absent in the mass serotype strains, further strengthens this conclusion (fig. 1) . the seventeen amino acids in the s 1 protein of cu-t2 which were different from those in ark99 were distributed throughout the gene, indicating that these amino acid changes were not due to recombination. therefore, it may be concluded that cu-t2 acquired the mass-specific serotype epitope on the s1 protein as a result of point mutation(s). the results of competitive-binding elisas indicated that the ark-and mass-specific epitopes on cu-t2 are independent. these facts provide direct evidence that point mutation(s) can result in the generation of antigenic variants during a natural infection. the results show that an approximately 751 base-long fragment of unknown origin was incorporated into the 3' end of cu t2 $2 gene by recombination. interestingly, this recombinant fragment was similar to one previously observed in kb8523, a possible recombinant strain of ibv [25] . recombination fragments in other two european isolates (6/82 and d207) were also found within the same regions [25] , although they were of shorter length than those of cu-t2 and kb8523 strains. in an in vitro transfection experiment it was observed that rna recombination in mhv occurred preferentially at certain selected sites (hot spots) [1] . in ibv, a secondary structure most likely exists at the 5' end of gene3 [30] . we found that the downstream crossover sites of both cu-t2 and kb8523 were located around that secondary structure (data not shown). the clustering of recombinations in ibv suggests that a recombination 'hot spot' most likely exists around the 3' end of the s2 gene and the 5' end of the gene3, although the clustering may be a result of selection [2] . an additional recombination which resulted in the replacement of half (52.4~) of the n gene was also observed, indicating that genomic rna recombination of ibv may involve more than two strains and may occur in multiple genes during natural infection. more interestingly, that recombination fragment in the cu-t2 is almost identical to a sequence (99.2~o homology) in the n gene of ho1152 strain. since both ark99 and ho1152 strains have been used simultaneously as live vaccines in the usa [24] , this finding provides convincing evidence that recombination among vaccine strains is contributing to the emergence of variants in the field. the significance of recombination in the n gene must also be viewed in the light of a recent report that the n protein may induce cellular immune responses against ibv [6] . the sequence of s1 portion of ark99 s gene obtained from a cloned pcr product has been published more recently [38] . when the s1 portion of the entire s gene sequence of ark99 obtained in this study was compared with that sequence, nine bases were found to be different, which would result in four amino acid changes in the s1 protein at positions 143, 335, 348 and 356. our data suggest that both inter-strain recombinations and mutation(s) are contributing to the generation of ibv variants in the field. we have shown here that genomic recombination between ibv strains may lead to replacements of large rna fragments in multiple genes. the replacement, however, may not result in change of antigenic nature of ibv. in contrast, mutation(s) on the s1 protein alone may generate new vn epitope(s). the data imply that every field isolate of ibv could be unique in rna sequence. therefore, recently reported diagnostic and serotyping methods of ibv, such as dot-blot hybridization [33] , restriction fragment length polymorphism (rflp) and polymerase chain reaction (pcr) [26, 40] , may not reflect the true antigenic and/or genetic nature of the virus. indeed, an exclusive use of such assays may yield misleading information on the antigenic and/or genetic characteristics of the virus. a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus random nature of coronavirus rna recombination in the absence of selection pressure establishing a genetic recombination map for murine coronavirus strain a59 complementation groups cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6/82 with that of ibv beaudette induction of anti-viral immune responses by immunization with recombination-dna encoded avian coronavirus nucleocapsid protein sequencing of coronavirus ibv genomic rna: three open reading frames in the 5' 'unique' region of mrna d sequence of the nucleocapsid genes from two strains of avian infectious bronchitis virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the 1960s in the infectious bronchitis virus: evidence for recombination within the massachusetts serotype location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: partial amino terminal sequencing of spike polypeptide $2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor polypeptide of ibv strains beaudette and m41 coronavirus ibv: virus retaining spike glycopolypeptide $2 but not s1 is unable to induce virus-neutralizing or haemagglutination inhibiting antibody, or induce chicken tracheal protection coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells serotyping of avian infectious bronchitis viruses by the virus-neutralization test variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens serological comparisons of strains of infectious bronchitis virus using plaque purified isolates the neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures immunologic differences in strains of infectious bronchitis production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes in vivo rna-rna recombination of coronavirus in mouse brain rna recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus a59 and fusion-negative mouse hepatitis virus 2 avian infectious bronchitis in: calnek bw sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis rna recombination in animal and plant viruses recombination between nonsegmented rna genomes ofmurine coronaviruses rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus high-frequency rna recombination of murine coronaviruses an overview of infectious bronchitis virus serotypes dot-blot hybridization using digoxigenin-labeled cdna probe complementary to the s1 gene of avian infectious bronchitis virus permits discrimination between virus strains a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes rapid detection and identification of avian infectious bronchitis virus we thank ms. beverley bauman, and ms. alice adriguetto for their technical assistance, and dr. bruce calnek and dr. randall renshaw for critical reading of the manuscript. the clone of mass41 s gene was kindly provided by dr. georges thiry, sotvay animal health, inc. this work was partially supported by a grant from sotvay animal health, inc.recombination among ibv strains key: cord-313676-6rebpe57 authors: de la torre, david i.; nuñez, luis f.; astolfi-ferreira, claudete s.; piantino ferreira, antonio j. title: enteric virus diversity examined by molecular methods in brazilian poultry flocks date: 2018-03-29 journal: vet sci doi: 10.3390/vetsci5020038 sha: doc_id: 313676 cord_uid: 6rebpe57 enteric viruses play an important role in the brazilian poultry industry due to the economic impact of resulting low yields of broilers, layers, and breeders. the most common enteric viruses affecting commercial flocks in brazil include fowl adenovirus of group i (fadv-i), chicken parvovirus (chpv), chicken astrovirus (castv), avian nephritis virus (anv), infectious bronchitis virus (ibv), avian reovirus (areo), and avian rotavirus (artv). the aim of this study was to identify single and multiple infections using data obtained from 270 samples from eleven brazilian states, corresponding to the period between 2010 and 2017. this was accompanied by an analysis of the relationship between the age of birds, clinical signs, and geographical distribution, using polymerase chain reaction (pcr) and reverse transcription-pcr (rt-pcr) techniques. twenty-five profiles of virus combinations were detected. single infections were encountered in 86.3% of samples, and multiple infections were present in the remaining 13.7%. both single and multiple infections affected all kinds of commercial chickens with digestive problems, stunting syndrome, decreases in egg and meat production, increased mortality, and respiratory signs. fadv-i, chpv, castv, anv, and artv were mostly detected in young broilers, in contrast with ibv, which was detected in hens from one to greater than 51 weeks of age. these results exhibit the complexity of enteric diseases and the still poorly understood role of each pathogen as a unique etiological agent. enteric viruses are the etiological agents for a series of health disturbances for commercial chickens around the world. they cause severe economic losses for the poultry industry because they negatively affect productive parameters, causing growth retardation, low feed consumption, high mortality, poor egg and meat production, and runting-stunting syndrome (rss) [1] [2] [3] [4] . these kinds of infections affect mostly young birds, but it is common to find viral infections in birds of all ages, including broilers, layers, and breeders [5] . the main enteric viruses reported to cause enteric diseases are found in single and multiple infections and include the fowl adenovirus of group i (fadv-i); chicken parvovirus (chpv); two viruses from the astroviridae family: chicken astrovirus (castv) and avian nephritis virus (anv); two viruses from the reoviridae family: avian reovirus (areo) and avian rotavirus (artv); and a member of the coronaviridae family, infectious bronchitis virus (ibv) [4, [6] [7] [8] [9] . several laboratory analytical methods have been used to detect enteric viruses in organic tissues from sick and healthy birds. conventional polymerase chain reaction (pcr) and reverse the tissues were mechanically macerated and suspended in phosphate buffered saline (pbs), 0.1 m, ph 7.4, in a 1:1 proportion in 1500 µl microtubes. the mixture was vortexed, subjected to three freeze-thaw cycles, and subsequently clarified at 12,000× g for 20 min at 4 • c. an aliquot of each supernatant was used for dna and rna extraction, using the organic phenol-chloroform method, according to procedures described in [15, 16] , respectively. the concentration and quality of nucleic acids were evaluated in a nanodrop 2000 (thermo fisher scientific, wilmington, de, usa). final dna and rna preparations were stored at −20 • c. the samples screened for viruses with rna genomes (castv, anv, ibv, areo, and artv), were submitted to a reverse transcriptase (rt) reaction to produce complementary dna (cdna), which was then used in the corresponding pcr. rt was conducted with the extracted rna at a concentration of 3-5 µg/µl, which was denatured at 95 • c for 5 min and added to a mix containing 250 ng of random primers, 500 µg/ml of oligo (dt) 12 , and sterile distilled water, to reach 20 µl in final volume. the reaction was performed with the following temperature conditions: an initial incubation at 25 • c for 10 min, a second incubation at 37 • c for 50 min, and inactivation of the reaction at 70 • c for 15 min. rt products were stored at -20 • c. the primers used for detection of fadv-i were those described in [10] , as shown in table 1 , and amplify an 897 bp segment corresponding to the variable region loop 1 of the gene that encodes the structural protein, hexon. through analysis of the nucleotide and amino acid sequences, it is possible to characterize the 12 different serotypes of fadv-i (fadv-1-fadv-8a, fadv-8b-fadv-11) [10] . dna amplification was carried out in 200 µl microtubes by adding 2.5 µl of extracted dna with a concentration of 1 µg/µl to a mix containing 1x pcr magnesium free (−mg) buffer (invitrogen), 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer, (hexon a and hexon b), 1 u of platinum tm taq dna polymerase, 1.5 mm of mgcl 2 (invitrogen), and sterile distilled water, to reach 22.5 µl. the following temperature conditions were used for the pcr reactions: a cycle of 94 • c for 5 min, 35 cycles at 94 • c for 60 s, 52 • c for 45 s, and 72 • c for 1 min, followed by a final extension at 72 • c for 10 min. the primers used for detection of chpv were those described in [17] (table 1) and amplify a 561 bp segment corresponding to a well-conserved region of the nonstructural gene (ns) usually used for detection of chicken and turkey parvovirus [17] . dna amplification was carried out in 200 µl microtubes by adding 2 µl of extracted dna with a concentration of 1 µg/µl into a mix containing 1x pcr magnesium free (−mg) buffer, 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer (pvf1 and pvr1), 1.25 u of platinum tm taq dna polymerase, 2 mm of mgcl 2 , and enough sterile distilled water to reach 23 µl. the following temperature conditions were used for the pcr reaction: a cycle of 94 • c for 3 min, 35 cycles at 94 • c for 30 s, 55 • c for 30 s, 72 • c for 1 min, followed by a final extension at 72 • c for 10 min. pcr was conducted with the cdna produced by the rt reaction, using the primers described in [12] (table 1) . this was used for the diagnosis of castv and anv, which results in the amplification of 362 bp and 473 bp segments, respectively, both of which are located in the rna polymerase gene (orf 1b) [12] . dna amplification was done in 200 µl microtubes by adding 2.5 µl of cdna to a mix containing 1x pcr magnesium free (−mg) buffer, 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer (cas pol 1f and cas pol 1r for castv; anv pol 1f and anv pol 1r for anv), 1 u of platinum tm taq dna polymerase, 1.5 mm of mgcl 2 , and enough sterile distilled water to reach 22.5 µl. the following temperature conditions were used for the pcr reaction: a cycle at 95 • c for 5 min, 35 cycles at 94 • c for 30 s, 50 • c for 30 s, 72 • c for 1 min, and a final extension at 72 • c for 10 min. pcr was conducted with the cdna produced by the rt reaction, using the primers for detection of all known coronaviruses, described in [11] (table 1) , which amplifies a 266 bp segment located in a highly conserved region of the 3 -untranslated region (3 -utr) in the first pcr reaction (utr-11 and utr-41) and a 179 bp segment in a second, hemi-nested reaction (utr-31 and utr-41) [11] . amplification of the first and second pcr reactions was performed in 200 µl microtubes by adding 2.5 µl of cdna in the first pcr reaction and 1 µl of the pcr product for the second reaction. the mix for the two pcr reactions contained 1x pcr magnesium free (−mg) buffer, 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer (utr-11, utr-31 and utr-41), 1.25 u of platinum tm taq dna polymerase, 2 mm of mgcl 2 , and enough sterile distilled water to reach 22.5 µl for the first pcr reaction and 24 µl for the second pcr hemi-nested reaction. the following temperature conditions were used for both pcr reactions: a cycle at 94 • c for 3 min, 35 cycles at 94 • c for 1 min, 48 • c for 90 s, 72 • c for 90 s, and a final extension at 72 • c for 10 min. pcr was conducted with the cdna produced by the rt reaction, using the primers described in [12] (table 1) , which amplify a 630 bp segment located in segment 10 (s10) of the viral genome, encoding the non-structural protein, nsp4 [12] . dna amplifications were conducted in 200 µl microtubes by adding 2.5 µl of cdna to a mix containing 1x pcr magnesium free (−mg) buffer, 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer (nsp4-f30 and nsp4-r660), 1 u of platinum tm taq dna polymerase, 1.5 mm of mgcl 2 , and enough sterile distilled water to reach 22.5 µl. the following temperature conditions were used for the pcr reaction: a cycle at 95 • c for 5 min, 35 cycles at 94 • c for 30 s, 50 • c for 30 s, 72 • c for 1 min, and a final extension at 72 • c for 10 min. pcr was conducted with the cdna produced by the rt reaction, using the primers described in [13] (table 1) , which amplify a 1120 bp fragment located in segment 4 (s4) of the viral genome, encoding the structural protein 4 (vp4) [13] . dna amplifications were conducted in 200 µl microtubes by adding 5 µl of cdna to a mix containing 1x pcr magnesium free (−mg) buffer, 1.25 mm of each deoxynucleotide triphosphate, 0.5 µm of each primer (s4-f13 and s4-r1133), 1.25 u of platinum tm taq dna polymerase, 2 mm de mgcl 2 , and enough sterile distilled water to reach 20 µl. the following temperature conditions were used for the pcr reaction: a cycle at 94 • c for 5 min, 35 cycles of 94 • c for 30 s, 50 • c for 1 min, 72 • c for 1 min, and a final extension at 72 • c for 10 min. rt-pcr and pcr products were analyzed by electrophoretic diffusion in 1.5% ultrapure tm agarose (invitrogen) gels submerged in 0.5x tris-borate-ethylenediamine tetraacetic acid (edta). the size of dna fragments was estimated by comparison with the 100 bp dna ladder (invitrogen). a uv trans-illumination camera was used to visualize the dna bands. some samples were chosen randomly for dna sequencing to validate the rt-pcr-and pcr-specific reactions: fadv-i (n = 3), chpv (n = 3), castv (n = 3), anv (n = 4), areo (n = 1), and artv (n = 3). the nucleotide sequences for each sample were submitted to genbank with the exception of the fadv-i and chpv sequences, which had already been submitted in previous studies. ibv sequences were not analyzed phylogenetically due to the high diversity of coronaviruses in different animal species. pcr products from each sample were purified using the gpx™ pcr dna and gel band purification kit (ge healthcare, piscataway, nj, usa), following the manufacturer's instructions. purified products were sequenced in the forward and reverse directions using the bigdye ® terminator cycle sequencing kit v3.1 (invitrogen) based on the manufacturer's instructions. descriptive statistics were used to represent the variability of the positive samples, using the single and multiple viral infections, type of birds (broilers, layers and breeders), age of birds (days for broilers and weeks for layers and breeders), clinical signs (respiratory signs, digestive signs, stunting-mortality-drop production, and no clinical signs), and geographic distribution of the samples. minitab 18 (minitab ® statistical software, v18.1, minitab inc., state college, pa, usa) was used to develop the variance analysis of frequencies of single and multiple infections for each virus, and clinical signs pertaining to each type of bird. the alfa value for significance level was α = 0.05. table 2 . multiple infections occurred with 25 profile combinations of viruses (table 3) . anv, castv, ibv, chpv, and artv were present in combination with each other, resulting in between two and five virus combinations. fadv-i was present in combination with chpv, anv, and ibv. areo showed a single infection. the variance analysis showed no significant difference between the frequencies of single and multiple viruses (p = 0.36). 1 + 22 8.1% 2 + + 1 0.4% 3 + + 2 0.7% 4 + + + 2 0.7% 5 + + + 1 0.4% 6 + 19 7% 7 + + 3 1.1% 8 + + 1 0.4% 9 + + 1 0.4% 10 + + + 7 2.6% 11 + + + 3 1.1% 12 + + + 1 0.4% 13 + + + + 2 0.7% 14 + + + + 1 0.4% 15 + + + + 1 0.4% 16 + + + + + 1 0.4% 17 + 15 5.6% 18 + + 6 2.2% 19 + + + 1 0.4% 20 + 164 60.7% 21 + + 1 0.4% 22 + + 2 0.7% 23 + 8 3% 24 + 4 1.5% 25 + 1 0.4% the samples received for viral diagnostics were from broilers, layers, and breeders of all ages. according to the age of birds, the samples corresponding to layer and breeder hens were divided into 5 groups, with intervals of 10 weeks of age, from week 1 to week 50, and two more groups, one for birds older than 51 weeks, and a group for which the ages were not reported (n/r; table 4 ). the samples corresponding to broilers were divided into 6 groups with intervals of 7 days of age, from day 1 to day 42, and two more groups, one for birds older than 43 days, and an n/r age group (table 5 ). 11 .7% for the 43-day group, 7.4% for the 1-7-day group, 6.7% for the 22-28-day group, 4.9% for the 29-35-day group, and 6.7% for the n/r age group (table 5) . single viral infections were found mostly in broilers, with 129/233 (55.4%), followed by 49/233 (21%) in layers and 34/233 (14.6%) in breeders. multiple viral infections were found with more frequency in broilers, with 14/17 (82.4%) with two viruses, 15/15 (100%) with three viruses, and 4/4 (100%) with four viruses. the only sample with five viral infections also belonged to the broilers group. a total of 3/17 (17.6%) samples with multiple viral infections (3 viruses) were found in samples from breeders ( table 6 ). the organs corresponding to positive results for fadv, chpv, castv, and artv included the liver, intestines, and pancreas. areo was present in one sample of intestines and ibv was present in all the five organs used for this study (table 7) . fadv-i was found in samples from breeders (12/28) and broilers (14/28). chpv was found in samples from broilers (20/22) . castv was found in samples from breeders (10/27) and broilers (17/27) . anv was found in samples from broilers (41/42). ibv was found in samples from layers (49/196) , breeders (16/196) , and broilers (116/196). areo was found in one sample, but the origin was not reported. artv was found in samples from breeders (2/17) and broilers (15/17) . all data describing the positive samples according to the age of layers, breeders, and broilers affected by the enteric viruses in this study are shown in table 8 . * poor production was defined as culling, stunting, and mortality. positive samples were divided into five groups according to clinical signs and type of birds. respiratory signs were reported in 39/270 samples, digestive signs in 9/270, stunting-mortality-decreased production signs in 69/270, no clinical signs in 5/270, and the remaining 148/270 samples did not include reports regarding clinical signs. the most common disease symptoms reported were stunting syndrome, mortality, and a decrease in egg and meat production, which occurred in 69/270 (25.6%) of the positive samples. broilers showed the highest values of stunting-mortality-decrease in production, with 55/69 (79.7%), followed by breeders with 12/69 (17.4%) and layers with 2/69 (2.9%). an important factor to be considered is that respiratory pathogens causing respiratory problems were found in digestive organs, for example ibv, with 12/39 (30.8%) positive samples in broilers, 23/39 (59%) in layers, and 3/39 (7.7%) in breeders. digestive diseases were not commonly reported as having unique clinical signs, and only 9 positive samples were obtained from broilers. all frequencies of clinical signs are described in table 6 . the variance analysis showed no significant difference between clinical signs for each type of bird (p = 0.45). samples originated from 12 brazilian states from the north-east, east, and south-east regions. table 9 . the following accession numbers correspond to the sequences used in the phylogenetic analysis: fadv-i and castv sequences were already submitted to the genbank database in previous studies [18] . fowl adenovirus sequences were characterized according to their position in the phylogenetic tree ( figure 1 ). isolate 424-4 was clustered with the reference sequence of strain tr59 with a bootstrap value of 100 in the common ancestral line, suggesting that this isolate should be classified into the fadv-8a serotype. isolate 420-12 was clustered with reference sequences of strain 764, and some isolates originated from china and peru, with a bootstrap value of 100 in the common ancestor of the 6 sequences, suggesting that this isolate should be classified as fadv-8b serotype. isolate 471-14 clustered with the reference sequence of strain atcc, suggesting that this isolate should be classified as fadv-11 serotype. isolates 475-5, 656-3, 691-6, and 752-3, clustered with reference sequences of avian nephritis virus from the united states, korea, australia, china, and japan, and a second group of isolates (541-2, 541-12, and 541-16) were clustered with reference sequences of chicken astrovirus from india and the united states, showing high bootstrap values for each group of both types of astroviruses detected in this study ( figure 2 ). the three sequences that were positive for artv were analyzed with reference sequences from the united states, peru, south korea, ireland, germany, and nigeria, all of them clustering with a high bootstrap value with the common ancestor. reference sequences for groups d, f, and g rotavirus were used as external groups to show the differentiation of the positive samples from avian rotavirus a (figure 3) . isolate 806-1 clustered with reference sequences of areo from hungary. the branch lengths and low bootstrap values from the common ancestor for reference sequences from hungary, the united states, and china, all show the magnitude of the genetic change among different isolates from these countries (figure 4 ). the three isolates corresponding to chpv were analyzed phylogenetically with strains from different countries from america, europe, and asia ( figure 5) . isolate 786-1 clustered with an isolate of chpv from peru, whereas isolates 752-3 and 691-1 clustered in a different group, together with brazilian isolates, but remained in the main group with all isolates of chicken parvovirus. goose parvovirus was used in this tree as an external group. the phylogenetic trees were inferred using the neighbor-joining method [19] . the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are joined to the branches [20] . the tree was drawn to scale, with branch lengths in the same units as the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the tamura-nei method [21] and are in units of the number of base substitutions per site. many etiological agents, such as bacteria, fungi, parasites, mycotoxins, and viruses, are related to enteric diseases. enteric viruses have been considered the main causative etiological agents of rss and acute enteric disturbances that negatively affect the yield of commercial chickens [1, 2, 4, 22, 23] . the most common viruses associated with the enteric diseases of chickens and turkeys include fadv-i, chpv, castv, anv, ibv, areo, and artv [7, 9] . clinical signs reported for each sample used in this study were related to digestive problems (diarrhea and feed intake), respiratory signs, stunting, high mortality, and a decrease in egg and meat production, which is consistent with results for clinical signs of enteric viruses obtained in other studies around the world [3, 4, 7, [24] [25] [26] [27] . molecular techniques such as pcr and nucleotide sequencing were employed due to their wide use in the diagnosis of enteric viruses [9, 22, 28] . the association of enteric virus with the age of broilers, breeders, and layers (tables 4 and 5) showed that molecular diagnosis of these viruses can be performed at different stages of production, which can be useful in the control of vertical infections. for example, in our results, chpv, anv, and ibv were present in broilers in the first week of age (table 8) , constituting possible vertical transmission. a high number of enteric viruses were found in birds from all ages and in different stages of production, both in broilers and hens (tables 4 and 5) . enteric viral infections mostly affect young birds, probably due to their immature intestine epithelium, which is the first organ affected during a viral infection. this results in poor feed absorption with the consequent general symptoms [5] . fadv-i was detected in young and old birds (tables 4 and 5) , corroborating results obtained previously [29] , where the virus was found in several samples from broilers, layers, and domestic chickens of all ages, even though the clinical signs were more severe in younger birds [30] . chpv infections affect young birds in their first four weeks [23, 27] , which is supported by our findings since the virus was detected in 20 infected samples from broilers between 1 and 28 days old (table 8 ). areo was found in 15/333 detections, corresponding to broilers in the growth phase (table 8) , especially between 8 to 28 days old, showing that rotavirus infections mainly affect young birds, promoting rss and enteric lesions [1] . both astroviruses, castv and anv, were found in samples from young broilers, despite having nine positive samples from breeders older than 51 weeks old (table 8) . astrovirus infections usually affect young birds [24] , although these viruses can be found in older birds in single and multiple infections with other enteric viruses [8] . ibv affects birds of virtually all ages [31] as demonstrated by our results, where samples of broilers, layers, and breeders from one week and greater than 51 weeks (table 8) presented positive results for coronavirus in all digestive organs tested, despite the fact that some clinical signs reported for these samples were respiratory signs. enteric viruses such as fadv-i, chpv, castv, anv, ibv, and artv were present in the liver, intestines, and pancreas. caecal tonsils and cloacal swabs were used exclusively for diagnosis of ibv (table 7 ). viral particles of fadv-i were found in the liver, intestines, and pancreas. the organs where this virus can be detected are the liver, intestines, caecal tonsils, proventriculus, bursa of fabricius, thymus, spleen, lungs and kidneys [32] [33] [34] [35] , especially in the first week of infection. based on the pancreatic tissue samples that were positive for fadv-i (5/28) (table 7) , we confirmed the importance of this organ for diagnosis of diseases caused by fowl adenovirus and even in cases of pancreatitis [36] . chpv was detected in the liver, intestines, and pancreas, which are areas commonly used for chpv detection due its pathogenicity as an enteric virus, considering the variety of organs where this virus can be found, including the brain, duodenal loop, and even on cloacal swabs [27, 37, 38] . astroviruses, such as castv and anv, are commonly detected in the digestive organs and fecal samples of sick and healthy birds [39] [40] [41] , supporting our findings of astrovirus in the liver, intestines, and pancreas (table 7) . ibv can be detected in the intestinal content, trachea, lung, liver, bursa of fabricius, pancreas, thymus, kidney, proventriculus, and spleen of affected birds [13, 42] , according to the dynamic distribution of the virus, resulting in a wide range of useful organs for molecular detection of this virus, which in our case helped to determine the presence of the pathogen in liver, intestine, pancreas, cloacal swabs, and caecal tonsils (table 7) . in our study, we found three different serotypes of fadv-i, without ruling out the possibility of multiple fadv infections in the same sample because multiple strains of different serotypes can affect to the same animal [29] . multiple infections between fadv-i and other enteric viruses were found in our study, especially with chpv, anv, and ibv, supporting previous results showing that fadv-i is often present in co-infections with other enteric viruses including castv, areo, and artv [7] . chpv was present in co-infections with one to four viruses, including ibv, anv, fadv-i, and castv, which has also been reported in metagenomic [3] and epidemiological surveys [38] with broilers showing rss. the most common virus found in this study belongs to the family coronaviridae, which indicates the presence of infectious bronchitis virus in the processed samples. a high percentage of positive samples (60.7%) ( table 3 .) indicated the presence of single ibv infections, probably due to the absence of co-infections with other enteric viruses or because only ibv analysis was requested. ibv co-infections with multiple enteric viruses were demonstrated in 32 samples (table 2 ), making it difficult to define the exact role of ibv in enteric diseases [43] , especially in multiple viral infections. all of the enteric viruses detected during 2010 and 2017 are widely distributed around the world. fadv-i, found in this study, is a common virus, widely distributed in brazil and well-reported throughout the world [9, 13, [44] [45] [46] [47] [48] , demonstrating its wide distribution among commercial birds. the phylogenetic tree showed that chpv has a well-conserved genome in non-structural genic regions, as evidenced by the branch sizes in comparison with reference sequences from genbank, further demonstrating the distribution of chpv around the world [14, 49, 50] . astroviruses affecting commercial flocks are also widely distributed worldwide [28, 39, 51] . phylogenetic analysis of astroviruses clustered anv and castv in two different groups, and each of them was sub-grouped with the nucleotide sequences of the rna polymerase gene from isolates originating in north-america and asia, providing a resource to determine the global distribution of these viruses. the artv analyzed in this study showed a close phylogenetic relationship with isolates from the united states, peru, south korea, ireland, germany, nigeria, and brazil, particularly in the length of the branches in the phylogenetic tree, which may confirm the complete distribution of the virus around the world [22, 24, [52] [53] [54] . coronaviruses such as ibv are some of the most widely distributed enteric viruses in brazil and have attracted special interest from the poultry industry due the economic losses caused by its impact on the health of birds [55] [56] [57] . the geographical distribution of the enteric viruses analyzed in this study was reported previously in some regions from the brazilian territory. according to this study, fadv-i was disseminated in the states of paraiba, pernambuco, são paulo, and santa catarina, in addition to previously reported isolates of fadv-i and chpv from the state of rio grande do sul [58] . castv was found in samples from minas gerais, and the other member of the family, astroviridae (anv), was found in samples from ceara, paraiba, pernambuco, bahia, minas gerais, and são paulo, increasing the previously reported distribution of astroviruses in the states of rio grande do sul, parana, and mato grosso [59, 60] . chpv was distributed in the states of ceara, paraiba, pernambuco, bahia, minas gerais, and são paulo, supporting the results of [38] , who reported the presence of this virus in an outbreak in são paulo, and expanding upon the report of [27] , who worked with isolates of chpv from poultry farms in the state of rio grande do sul. the artv results suggest that this virus was disseminated in the states of pernambuco, minas gerais, and são paulo, consistent with other previous reports that describe the presence of artv in the states of minas gerais, parana, rio grande do sul, and goias [61, 62] . ibv is the most disseminated virus among poultry flocks in brazil [63] . this virus requires more specific analysis for the characterization of the different genotypes that affect commercial chickens in brazilian states and other countries in south america, to determine the epidemiological dynamics and pathogenesis of the virus within the poultry industry. these results may contribute to 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grants from the fundação de amparo à pesquisa do estado de são paulo (fapesp-grant #2013/08560-5 and 2015/09348-5), conselho nacional de desenvolvimento científico e tecnológico (cnpq-grant #453920/2014-4), and by the coordenação de aperfeiçoamento de pessoal de nível superior, brasília-df, brazil (capes-grant #1706271). the first author also thanks the "secretaría de educación superior, ciencia, tecnología e innovación-senescyt" for economic support through the universities of excellence 2014 scholarship program in ecuador. the authors would like to thank the poultry companies in brazil that generously sent the samples included in this study and for the diagnosis of enteric viruses. the authors declare that they have no conflict of interest. key: cord-308591-cs8os2f5 authors: tawakol, maram m.; nabil, nehal m.; samy, ahmed title: evaluation of bacteriophage efficacy in reducing the impact of single and mixed infections with escherichia coli and infectious bronchitis in chickens date: 2019-11-29 journal: infect ecol epidemiol doi: 10.1080/20008686.2019.1686822 sha: doc_id: 308591 cord_uid: cs8os2f5 infectious bronchitis virus (ibv) represents a major threat to poultry production worldwide particularly when complicated with bacterial infection. in the present study samples were collected from forty broiler farms with respiratory manifestations to characterize ibv and e. coli. bacteriophages were isolated and enriched from sampled farms to study its efficacy to control single and mixed infections with e. coli and ibv in vivo. twelve out of forty farms were positive for ibv. phylogenetic analysis of partial spike protein revealed that all positive cases clustered within the gi-23 genotype. eight out of forty farms were positive for e. coli serogroups o26, o78, o86, o114, o119, with o125 found on three farms. bacteriophage treatment delayed the onset and reduced the severity of clinical signs, and prevented the mortality associated with single and mixed infection with ibv and e. coli. furthermore, in mixed infections, bacteriophage treatment significantly reduced e. coli as well as ibv shedding. groups treated with bacteriophages showed a significant reduction of e. coli shedding that gradually decreased over time, in contrast to higher and gradually increasing shedding without bacteriophage treatment. in conclusion, bacteriophage treatment significantly reduced the pathogenicity and shedding of ibvand e. coli in mixed infections. avian respiratory tract infections are associated with massive economic losses particularly under poor intensive rearing conditions in winter seasons. several pathogens, adverse environmental conditions and poor managemental factors are involved [1] [2] [3] . pathogens include avian influenza virus (aiv), newcastle disease virus (ndv), infectious bronchitis virus (ibv), avian pneumovirus (apv), mycoplasma gallisepticum (mg), and avian pathogenic e. coli (apec). avian respiratory tract infections can be caused by single pathogens or in combination with each other in form of preceding, concurrent or secondary infections [3] [4] [5] . change in the ecosystem of the respiratory microbiome [6] , mechanical damage of the epithelium, loss of ciliated cells and the impairment of innate immune responses are a consequence of primary respiratory infections and associated with enhanced disease and economical losses during superinfections [7] . in egypt, respiratory diseases outbreaks caused significant economic losses in commercial poultry production during recent years [8] . laboratory investigations revealed that ibv represents the most common pathogen detected during these outbreaks [8] [9] [10] . ib is a highly contagious disease affecting the respiratory, renal and reproductive tract of chickens. the disease caused by single-stranded, enveloped rna virus belongs to the genus of gamma coronavirus [11] . the spike (s) glycoprotein represents the major structural and immunogenic domain, and plays a pivotal role in tissue tropism and carry the receptor-binding site [12] . the s glycoprotein is cleaved post transitionary into s1 (globular head) and s2 (stalk) domain [13] . the s1 domain possesses three hypervariable regions (hvr 1, 2 and 3) responsible for the high genetic and antigenic variability of ibv [14] . despite massive vaccination strategies used in egypt, ibv extensively circulates in all over egypt in vaccinated and non-vaccinated chickens with continuous viral evolution [9, 15, 16] . complications between apec and ibv infections are widely observed under field conditions and are reproduced under experimental conditions resulting in enhanced severe colibacillosis [7] and enhanced pathogenicity post ibv infection. under field conditions, commercial broiler farms with inadequate biosecurity measures and with the presence of colibacillosis in early age bird are associated with more severe ibv infections in late age birds (>20 days old). despite the wide spread use of antibiotics and vaccination against ibv, complications as a consequence of mixed infections of avian bacterial pathogens including apec and ibv became a general phenomenon associated with significant economic losses. in the present study samples were collected from broiler farms with birds with respiratory manifestations. all samples were tested for the presence of ibv and apec. recovered ibv isolates were genetically characterized by partial spike protein sequencing. recovered apec were characterized by serotyping and antibiotic profiling. bacteriophages were isolated from water samples collected from the same farms and enriched against apec o78. in vivo study was conducted to evaluate the efficacy of bacteriophages in reducing the pathogenicity of single and mixed infections with apec and ibv. one hundred recently succumbed chickens suspected of being ibv infected were collected from forty broiler farms. the study was conducted between late 2017 and early 2018 and samples collected in northern egypt (dakahila and damietta governorates). lung, trachea, kidney, and liver samples were aseptically collected. on the same day of sampling, half of the organs were cultured for bacteriological investigation in buffered peptone water at 37ºc for 24 h. for ibv screening, lung and trachea samples were homogenized with an equal mass of phosphate buffer saline and then centrifuged at 7,000 × g for 5 min. supernatant was collected and stored at −80ºc until further investigation. viral rna was extracted using the qiaamp viral rna mini kit (qiagen) in accordance with the manufacturer instructions. one-step rt-pcr was performed for ibv screening as previously described [17] . genotyping with forward and reverse primers for amplification of hvr1, amplicon purification and sequencing was performed as previously described [17] . obtained sequences in this study aligned with sequences represent all infectious bronchitis virus genotypes proposed by [18] . all samples used in the phylogenetic analysis were downloaded from genbank (http://www.ncbi.nlm.nih.gov). the sequences were aligned using multiple alignment mafft version 7 (https://mafft.cbrc.jp/alignment/server/). the tree was constructed with the mega 6 software [19] using the nucleotide substitution of the hasegawa-kishino-yano model with the gamma-distributed rate (with four rate categories) with bootstrap value based on 1000 replicates [20] . then, the tree was viewed and edited using the figtree v1.4.2 software (http://tree.bio.ed.ac. uk/software/figtree/). for the bacteriological investigation, e.coli isolation and confirmation was performed in accordance with [21] . confirmed, positive e.coli strains were subjected to serotyping using all available o (o1 to o181) antisera in accordance with [22] , cross-reacting antigens were used to ensure the removal of cross-reactivity. all confirmed strains were tested against antibiotics commonly used in egyptian farms by disc diffusion, with testing procedures and interpretations of the results performed in accordance with reference laboratory protocols. one of the field isolates e.coli (serotype o78) was tested for purity using api 20e (biomérieux, inc.) and counted in colony-forming units (cfu). over the surveillance period, sewage water samples were collected from poultry farms and used for the isolation of bacteriophages specific to apec o78. samples were centrifuged at 10,000 rpm for 10 min and then filtered through a 0.22 μm filter (millipore). bacteriophage purification and enrichment was performed in accordance with [23] . briefly, filtered samples were mixed with luria bertani (lb) broth (sigma), early-log grown apec o78 added and samples incubated overnight at 37ºc with shaking set to 120 rpm/min. then, samples were centrifuged at 10,000 rpm for 10 min and filtered through a 0.22 μm filter. the presence of bacteriophages was initially tested by the spot test method based on the double layer plaque technique in accordance with [24] . for this, 100 µl of apec o78 were cultured on lb agar for 8 h, then 10 µl of the prepared bacteriophage suspension spotted onto it, and plates incubated at 37ºc overnight. the appearance of a clear zone in the plate indicated the presence of the lytic phage. plaque assay was used for the titration of bacteriophages as previously described [25] . in vivo evaluation of bacteriophage efficacy in the reduction of e.coli and/or ib pathogenicity animal experimental designs and procedures were reviewed and approved by rlqp in accordance with guidelines of the ministry of agriculture and land reclamation and ministry of environment of egypt. pure e.coli o78 isolate recovered in the present study were titrated by colony-forming units (cfu). birds were challenged with 10 8 cfu diluted in 100 µl sterile pbs. the ibv strain ibv-eg/1212b-2012 (accession number: jq839287) belonging to egy/var ii [15] was used for the intratracheal challenge with 10 6 eid 50 in 100 µl per bird. previously described primers and probes were used to create a standard curve for absolute quantification of ibv shedding [26] . a total of 70 healthy one-day old chicks were purchased from a local poultry hatchery and housed in isolators with ad libitum access to feed and water. one day later, chicks were monitored for the presence of ibv or apec o78 as previously described and then randomly divided into seven groups. groups 1, 2, and 3 were treated with bacteriophages by intratracheal inoculation with 10 8 pfu at 1, 5, 8 and 13 days; whilst groups 4, 5, 6 and 7 were not treated with bacteriophages. groups 1 and 4 were challenged intratracheally at day 2 with 10 8 cfu apec o78. groups 2 and 5 were challenged intratracheally with 10 6 eid 50 of ibv. groups 3 and 6 were infected with both pathogens in a 3 days interval. group 7 was kept as a negative control and received pbs. the experimental design is shown in figure 1 . three birds per time point were humanely culled at 4, 9 and 15 days postinfection (dpi). the carcasses were weighed and dissected and trachea and lung samples collected aseptically for the detection and titration of apec o78, bacteriophages and ibv as previously described. clinical signs scoring and sampling all birds were observed twice daily and clinical signs recorded in accordance with [27] . briefly, no signs were recorded as 0, mild signs included mild gasping, coughing or depression and were recorded as 1, and severe gasping, coughing or depression with ruffled feathers was considered as severe signs and recorded as 2. birds with severe signs unable to move were recorded as 3 and humanely culled and samples collected. birds which unexpectedly succumbed to disease were also recorded as 3 and samples collected. statistical significances of viral and bacterial shedding between different groups were evaluated using student's t-test in microsoft excel based on the mean of shedding quantities in three tested birds sampled from each group at each time point. history, clinical findings, and prevalence of apec and ibv in the examined flocks forty broiler flocks, aged between 30 and 40 days and suffering from respiratory affections, from the dakahlia and damietta governorate were examined. unwell birds showed respiratory manifestations visible as nasal discharge, sneezing, gasping and swollen head. in all farms examined unwell birds were isolated and subjected to intense antibiotic treatments. however, clear differences in activity and feed intake remained. screening of collected samples focused on the detection of ibv and e.coli. eight farms were positive for e.coli (20% of all farms examined), five farms from the dakahlia and three farms from the damietta governate. serotyping of the isolated strains revealed the presence of the o26, o78, o86, o114, o119 serogroups, with the o125 serogroup detected on three farms. disc diffusion tests showed the following resistance rates: 100% to penicillin, 75% to gentamycin and streptomycin, 62.5% to ciprofloxacin, 50% to cefotaxime and 25% to doxycycline. 75% of all isolated strains were multidrug-resistant (mdr). twelve farms were positive for ibv (30% of all farms examined) determined by real-time pcr, with ten farms from the dakahlia and two farms from the damietta governate. postmortem examination of the recently succumbed birds revealed the presence of caseous plug at the tracheal bifurcation, caseous trachitis, and fibrinous pericarditis, perihepatitis and air sacculitis. for virus isolation, specific pathogen free embryonated chicken eggs were inoculated. total figure 1 . schematic outline of the experimental design rna was extracted from the allantoic fluid and the partial s1 gene of each isolate sequenced. results obtained revealed that the tested samples clustered within the egyptian variant group of gi-23 genotype subgroups ( figure 2 clinical signs observed including gasping, coughing or depression started to appear from three days postchallenge with apec or a mixed apec and ibv infection. bacteriophage treatment delayed the onset of the clinical signs to 6 days post-challenge (dpc) and in addition markedly reduced their severity in both groups (figure 3) . regarding ibv infection, clinical signs were observed from four-days postchallenge, with bacteriophage treatment leading to a reduction of their severity, but not delaying their onset (figure 3) . bacteriophage treatment was not associated with mortality in single apec or mixed apec and ibv infected groups. in contrast, birds challenged with apec alone and mixed apec and ibv infection without bacteriophage treatment showed a 16% and 29% mortality rate at 8 and 7 days post-infection respectively (figure 4 ). bacteriophage treatment in combination with single ibv infection did not reduce the mortality of 26% (figure 4 ). [18] with samples from the present study indicated by black circles. the tree was constructed using the nucleotide substitution of the hasegawa-kishino-yano model with the gammadistributed rate (with four rate categories) with bootstrap value based on 1000 replicates bacteriophage treatment significantly reduced apec shedding after single apec or mixed apec and ibv challenge, with a gradual decrease of bacterial loads in lung tissues over time. in contrast, a nontreated and challenged group showed a significantly higher apec load with a gradual increase over time especially at 9 and 15 dpc ( figure 5 ). interestingly, bacteriophage treatment significantly reduced ibv shedding in the mixed infected group but not in the ibv alone infected group comparing to the mixed infected group without bacteriophage treatment. the bacteriophage treated group infected with ibv showed relatively comparable results to the infected non-treated group. groups with single ibv infection and mixed apec and ibv infection with bacteriophage treatment showed a reduction, but not statistically significant, of ibv comparing to single ibv infection without bacteriophage treatment, with the reduction only becoming statistically significant at 15 dpc ( figure 6 ). infectious bronchitis in particular complicated by mixed viral and/or bacterial infections is associated with catastrophic economic losses for the global poultry industry, and a significant threat for food security. under field conditions complications caused by mixed ibv and apec infections are linked with with accentuating respiratory signs in addition to airsacculitis, pericarditis and possible perihepatitis [28] . under experimentally controlled conditions, mixed ibv and apec infections can induce more pronounced clinical lesions in the air sacs that persist for a longer time [29] . in the present study samples were collected from birds with respiratory manifestations from a total of forty farms, and subjected to characterization based on currently circulating ibv and apec strains. in addition, the efficacy of bacteriophage treatment in reducing apec replication in the avian respiratory tract was studied in vivo, as well as their effect in reducing ibv pathogenicity after mixed apec and ibv infections. out of forty farms, ibv was detected on 12 farms (30% of all farms examined). the high incidence of ibv in egyptian poultry flocks suffering from respiratory manifestations (39-75.6%) has been recorded in several previous studies [8, 10, 30, 31] . the range in the incidence rate is apparently due to the differences in the age of birds, with a higher incidence in 20-30 days old birds [8] . based on partial spike protein gene sequencing, all positive ibv samples were clustered within the egyptian variant ibv that belongs to the gi-23 genotype [18] . all recent molecular characterization of ibv circulating in egypt revealed the circulation of two distinct genotypes, the gi-1 vicinal strains genotype and the gi-23 local variant genotype . average percentage of mortality in different groups (birds per group at each time point). the highest and earliest mortality was associated with mixed infections without bacteriophage treatment. in contrast, no deaths occurred in bacteriophage treated groups with mixed infection. figure 5 . the e.coli bacterial counts. samples were collected at 4, 9 and 15 dpc and are represented by the mean log10 cfu/ml of samples collected and tested from three birds. ttests were performed. * p < 0.05, * * p < 0.01, ***p < 0.001 or ns for non-significant. the asterisks over columns represent a significant difference comparing to apec the infected group. asterisks over lines represent a significant difference between the mixed infection groups with and without bacteriophage treatments. significantly higher counts with gradually increased bacterial shedding are observed in nontreated groups. figure 3 . clinical signs scoring in different groups. no signs were recorded as 0, mild signs included mild gasping, coughing or depression and were recorded as 1, and severe gasping, coughing or depression with ruffled feathers was considered as severe signs and recorded as 2. birds with severe signs unable to move were recorded as 3 and humanely culled and samples collected. birds which unexpectedly succumbed to disease were also recorded as 3 and samples collected. each dot represents the mean of the clinical signs scoring of all birds in the group at each time point. 34 ]. despite wide spread vaccination with classical and/or variant strain based vaccines in egypt, ibv outbreaks were frequently reported in vaccinated and non-vaccinated flocks of farms [16] , indicating poor protection against the prevalent local variant strain [35] . in chickens, diseases associated with apec cause significant economic losses due to mortality, decreased feed conversion rates, carcass contaminations and the high cost of its control and prevention. these losses in addition to the high prevalence of e.coli in poultry farms' environment necessitate the use of antibiotics. however, the constant unwise use of antimicrobials in egypt led to the high prevalence of multidrug-resistant strains that threaten human health and are associated with economic losses in the commercial poultry sector [36, 37] . in the present study, 20% of the collected samples were positive for e.coli. 75% of them showed resistance to three or more antibiotics which were defined as multidrug resistant. this highlights the importance for the development of alternative therapeutic approaches preventing colibacillosis. bacteriophage therapy has been proven as an effective tool in the treatment of colibacillosis which initiated in the respiratory tract of chicken [38] [39] [40] . the ability to generate bacteriophages against selected bacterial species, serotypes or strains, that do not affect the commensal bacterial flora [41] , make them safe in comparison to antibiotics and provide an alternative approach for eliminating pathogens. in the present study, the efficacy of bacteriophage therapy in single and mixed infections with egyptian ibv variant and/or apec o78 has been evaluated. results obtained revealed a significant higher persistence of apec and ibv shedding together with higher morbidity and mortality in the mixed infection group comparing to single infected groups. this finding agrees with several previous studies [29, 42, 43] . mechanisms behind the escalation of pathogenicity after mixed ibv and apec infections are not yet fully understood. however, alteration of the immune response is most likely the cause not mechanical altering of the mucociliary barrier [29] . bacteriophage treatment significantly reduced the severity of single apec o78 challenge as well as a mixed infection with apec and ibv. in brief, a high dose of bacteriophages administered intratracheally reduced the morbidity, prevented mortalities and significantly decreased apec shedding compared to non-treated apec challenge. local application of bacteriophages at the site of apec infection that is relatively inaccessible via the circulatory system, i.e. intra air sac administration, enables bacteriophages to eliminate the disease [44] . this explains the success of intrathoracic air sac injections in the reduction or prevention of mortality in several studies [38, 44] . in the present study with a higher bacteriophage dose and intra-tracheal inoculation, apec was significantly reduced in the bacteriophage treated lung tissue. in the case of mixed infections, bacteriophage treatment did not only reduce bacterial counts but also significantly reduced ibv detected in the lung, and associated with reduction in morbidity and mortality in the mixed infection group but not the single ibv infected group. these results show the efficient inhibition of apec replication associated with a reduction of ibv severity during mixed infections. in conclusion, the intra-tracheal administration of a high dose of bacteriophages limited apec replication in the respiratory tract, and subsequently diminished the consequences of single and mixed infections with apec and ibv. bacteriophage treatment represents a promising tool for the prophylactic control of colibacillosis, and minimizes the consequences of mixed apec and ibv infections. figure 6 . ibv titers. the ibv titers of samples collected at 4, 9 and 15 dpc are represented by the mean log10 eid50/ml of samples collected and tested from three birds. t-tests were performed. * p < 0.05, ** p < 0.01, and ns for non-significant. the asterisks over columns represent a significant difference compared to the ibv infected group. asterisks over lines represent a significant difference between mixed infection groups with and without bacteriophage treatments. significantly lower viral titers were observed in the mixed infection group treated with bacteriophages. to thank the broilers farm's owners for their cooperation 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and characterization of bacteriophages for avian pathogenic e. coli strains isolation and characterization of bacteriophages infecting salmonella spp bacteriophages isolated from chicken meat and the horizontal transfer of antimicrobial resistance genes development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens pathogenicity of infectious bronchitis virus isolates from ontario chickens avian infectious bronchitis virus course of infection and immune responses in the respiratory tract of ibv infected broilers after superinfection with e. coli incidence of avian nephritis and infectious bronchitis viruses in broilers in egypt molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 isolation and identification of egypt/beni-seuf/01 a novel genotype of infectious bronchitis virus emergence of a novel genotype of avian infectious bronchitis virus 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inoculated with infectious bronchitis virus and/or e. coli the experimental infection of chickens with mixtures of infectious bronchitis virus and escherichia coli evaluation of the influence of bacteriophage titer on the treatment of colibacillosis in broiler chickens1 the authors would like to thank collogues in reference laboratory for veterinary quality control on poultry production for their technical help. also, we would like key: cord-337128-yyz7z0xj authors: abdel-moneim, ahmed s; zlotowski, priscila; veits, jutta; keil, günther m; teifke, jens p title: immunohistochemistry for detection of avian infectious bronchitis virus strain m41 in the proventriculus and nervous system of experimentally infected chicken embryos date: 2009-02-05 journal: virol j doi: 10.1186/1743-422x-6-15 sha: doc_id: 337128 cord_uid: yyz7z0xj background: infectious bronchitis virus primarily induces a disease of the respiratory system, different ibv strains may show variable tissue tropisms and also affect the oviduct and the kidneys. proventriculitis was also associated with some new ibv strains. aim of this study was to investigate by immunohistochemistry (ihc) the tissue tropism of avian infectious bronchitis virus (ibv) strain m41 in experimentally infected chicken embryos. results: to this end chicken embryos were inoculated in the allantoic sac with 10(3 )eid(50 )of ibv m41 at 10 days of age. at 48, 72, and 120 h postinoculation (pi), embryos and chorioallantoic membranes (cam) were sampled, fixed, and paraffin-wax embedded. allantoic fluid was also collected and titrated in chicken embryo kidney cells (cek). the sensitivity of ihc in detecting ibv antigens in the cam of inoculated eggs matched the virus reisolation and detection in cek. using ihc, antigens of ibv were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. these results were consistent with virus isolation and denote the wide tissue tropism of ibv m41 in the chicken embryo. most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with ibv strain m41. conclusion: ihc can be an additional useful tool for diagnosis of ibv infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with ibv strains of different virulence. moreover, these results underline that embryonic tissues in addition to cam could be also used as possible source to generate ibv antigens for diagnostic purposes. infectious bronchitis virus is the prototype species of the family coronaviridae in the order nidovirales. more than 25 genotypes are distributed worldwide. ibv causes an acute highly contagious viral respiratory disease of chickens which is characterized by respiratory rales, coughing and sneezing [1] . some ibv strains replicate in the gastrointestinal tract, oviduct, and kidney, and due to their nephropathogenic properties they have the potential to cause severe losses with up to 44% mortality [1, 2] . in other cases, infection of the proventriculus leads to 75% to 100% mortality in young birds [3] . most isolates of ibv replicate well in the developing chicken embryo following inoculation of the allantoic cavity, and high titers of virus can be isolated from the allantoic fluid [4] . replication of ibv strains m41 and beaudette in vitro is restricted to primary chicken cells and depends on the expression of 2,3linked sialic acids on the cell surface. thus, these molecules are supposed to serve as receptor determinants for primary attachment of ibv to host cells [5, 6] . the conventional diagnosis of the ibv is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. since two or three blind passages are often required for successful primary isolation of ibv, this procedure could be tedious and time consuming. alternatively, ibv may be isolated by inoculation in chicken tracheal organ cultures. this method is sensitive [7] but highly laborious. furthermore, ibv may be detected directly in tissues of infected birds by means of immunohistochemistry [8, 9] or in situ hybridization [10] . the reverse transcription-polymerase chain reaction (rt-pcr) has proved useful in the detection of several rna viruses [11] . aim of this study was first to evaluate the suitability of ihc for detection of ibv antigen in paraffinwax embedded cam and second to analyse the viral antigen distribution in different embryonic tissues between 48 and 120 h after experimental infection. inoculation of 10 3 eid 50 ibv m41 in spf ece resulted in death of embryos at 24 h (3 embryos), 41 h (2 embryos) and single embryonic death at 65, 87 and 120 h after ibv inoculation. allantoic fluid and cam were harvested at the times given in table 1 which also showed that high virus titers were obtained 24-87 h pi that decreased sharply to 10 2 tcid 50 at 120 h pi (table 1) . immunostaining of the cam was positive in all inoculated eggs from which infectious virus was also recovered. only one embryo showed non specific death at 24 h pi as it showed negative results in both immunostaining and virus recovery assays. ihc detected infected cam that possesses virus titers of only 10 2 or 10 3 tcid 50 in the corresponding allantoic fluid (table 1) . ibv antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos (table 1, figure 1 ). moderate number of positive cells (++) were detected in the mucosa, smooth muscle fibres and vasculature of gizzard at 41, 48, 65 and 72 h pi. few (+) to moderate (++) number of positive cells were detected in the proventriculus mucosa as well as its smooth muscle fibres in embryos dead at 41, 87 and 120 h pi ( figure 1a ). positive immunostaining was also detected in macrophages of both spleen (41, 48, 65, 72, 87 h pi) ( figure 1c ) and liver (kupffer cells) (41, 48, 65, 87 h pi). ibv antigens were also present in neurons of both spinal cord (65 and 72 h pi) and brain (65 and 120 h pi) ( figure 1b) , heart (41, 48, 72 h pi) ( figure 1d) classic methods for ibv diagnosis include serological tests for analysis of antibody titers against ibv [12] and virus isolation in embryonated chicken eggs since ibv grows well in the developing chicken embryo. these methods are inherently slow and time consuming. currently, detection and serotype analysis of ibv is performed by rt-pcr and restriction fragment length polymorphism analysis [13] or by sequencing rt-pcr products of s1 gene [14] . to establish ihc on paraffin-wax embedded tissues using a polyclonal rabbit anti-ibv serum, embryonated eggs were inoculated with 10 3 eid 50 ibv m41. cam and embryos were collected till 120 h pi, since the presence of ibv antigen in inoculated eggs by an antigen detection method is preferably performed 2 to 3 days after inoculation [15] . this also confirmed in the current study where ibv titers declined sharply at 120 h pi. it is well known that maximum ibv virus titers reached 1 to 2 days postinoculation [16] [17] [18] but interestingly, allantoic fluid showed high virus titers 24-87 h pi. immunostaining of the cam was positive in all inoculated eggs from which infectious virus was recovered. it is worth to note that also samples of infected cam with respective allantoic fluid virus titers of only 10 2 or 10 3 tcid 50 were obtained, stained positively with the ibv antiserum which equals approximately 10 4 or 10 5 eid 50 [19] . this indicates a relatively high sensitivity of the immunohistochemistry applied in this study. using a monoclonal antibody for immunostaining, the detection limit for ibv antigen was 10 6.2 eid 50 as described by [20] . these differences in sensitivity may be due to larger number of antigenic epitopes recognized by the polyclonal serum. this finding prompted us to apply ihc for screening of antigen distribution in different tissues in the ibv inoculated embryos. antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos. however, isolation or detection of virus by virological methods may indicate only that virus is present in the tissue due to viraemia and thus does not necessarily prove productive replication in the respective organs. hence, tissue tropism can not be determined by virus isolation or detection only [21] but requires morphologically based techniques. although chong & apostolov [22] , failed to detect virus by immunofluorescence in the intestine and cecal tonsils of chickens experimentally infected with m41 of ibv, lucio & fabricant [23] found that m41 can infect a variety of tissues and that some isolates may be recovered frequently from the digestive tract. ibv infec-tion of the proventriculus was firstly recorded in china [3] then detected with unam-97 ibv mexican variant that produced decrease in the proventricular gland papillary branching and electrodense material scattered in proventriculus with a structure consistent with coronaviruses [24] . to the best of our knowledge, this is the first time that the prototype ibv strain m41 was also detected within the muscle layer of the proventriculus. because ibv causes an upper respiratory tract disease, viral antigens in nasal mucosa, trachea and lung were expected. ibv m41 viral antigen was found in the renal tubules and glomeruli. this observation is consistent with the finding that ibv m41 has also been isolated and/or detected in kidneys of naturally or experimentally inoculated chickens immunohistochemical detection of ibv antigens in chicken embryos after experimental infection with ibv m41 figure 1 immunohistochemical detection of ibv antigens in chicken embryos after experimental infection with ibv m41. (a) proventriculus, focally extensive, there is strong immunolabelling of smooth muscle cells and scattered cells within the vasculature (arrows). bar = 100 μm (b) brain, within the cytoplasm of numerous neurons there is strong staining of ibv antigen. bar = 40 μm (c) spleen, scattered through the parenchyma there are numerous singular polygonal cells with red intracytoplasmic staining for ibv antigen, interpreted to be macrophages. bar = 40 μm (d) myocardium, capillary endothelium stains strongly positive for ibv antigen. bar = 100 μm. [23, 25, 26] . as earlier described for other ibv genotypes, antigens of m41 were present not only in renal tubules, but also in the glomerular tuft epithelium [27] . the detection of ibv antigen in the spleen, is discussed controversially in the literature. in some reports, ibv antigens or mrna were not found within the spleen [28] [29] [30] , in others, splenic infection was observed [27, 31, 32] . studies to determine virus distribution in embryonic tissues were conducted previously [29, 30] indicating that ibv antigen or nucleic acid was present in trachea, bursa, kidney, intestine, lung, heart, esophagus, mesentery, shell gland, and air sac, but not in spleen or thymus. the different virus distribution between the present paper and other papers may be due to the difference of embryo age at inoculation; 10 days old embryos (present paper) and 17 or 18 days old embryos [29, 30] . it is probable that more immature tissues are more susceptible for ibv. the susceptibility for ibv infection is associated with the expression of 2,3linked sialic acid which is used by the virus for primary attachment to the cell surface [5, 6] . for tight binding and subsequent fusion with the cellular membrane interaction with a second receptor appears to be required [5] . the initial target of avian influenza viruses and ibv in chicken is the respiratory epithelium. presence of 2,3-linked sialic acid is the prerequisite for avian influenza viruses to initiate respiratory infection. this molecule may also be used by ibv for infection of the respiratory tract. ibv, like avian influenza viruses, infects many non-respiratory tissues, including alimentary tract, oviduct and kidney [33, 34] . the broad distribution of 2,3-linked sialic acid in different organs and species contradicts the view that this type of sugar is a major determinant of the narrow host tropism of ibv. in this study ibv antigen was found in musculature of both gizzard and proventriculus of inoculated embryos which is consistent with the known presence of 2,3-linked sialic acid receptors in the intestinal tract of chicken [35] . thus it raises the question whether ibv infection of the proventriculus is a classic feature of ibv and occurs in association with an old ibv strain. a recently isolated m41 strain [26] resulted in an increased proventriculus index 7-28 days after experimental infection of 1-day-old chickens [mohamed aa: studies on infectious proventricultis in broiler chicken. master thesis, in progress]. for our knowledge, ibv was neither isolated from nor detected in the brain of young or adult chickens [32] . our finding that ibv antigens are present in the nervous system of embryonic chicken, may be explained by the presence of polysialylated n-cam (neural cell adhesion molecules) in chicken embryos which might mediate virus entry into the neurons. the abundance of polysialylated n-cam declines gradually during the embryonic development and the synthesis dramatically decreases right before birth [36, 37] . our results show that the classic ibv strain m41 exhibits a wide tissue tropism including the nervous system and the proventriculus in chicken embryos and demonstrate that ihc as described here is a very sensitive tool for detection of productive virus replication in situ and therefore allows further studies to improve the understanding of the pathogenesis of the ibv infection. the ibv strain m41 used in the current study was kindly provided by m. hess, intervet, boxmeer, nl. chicken embryo kidney cells (cek) were prepared from specific pathogen free (spf) embryonated chicken eggs (ece) as described elsewhere [38] . briefly, kidneys from 18-dayold spf ece were isolated, washed with hanks' balanced salts solution, minced and disaggregated in trypsin solution. after centrifugation the cells were resuspended in dulbeccos' modified eagle's medium (dmem) supplemented with 10% fetal calf serum (fcs) and grown at 37°c in a 5% co 2 incubator to confluent monolayers. twelve ten days old spf ece were inoculated in the allantoic sac with 200 μl of ibv strain m41(10 3 eid 50 ). inoculated ece were candled twice daily. at 48, 72, or 120 h dead and/or living whole embryos as well as allantoic fluid and cam were collected. embryos died at any time after inoculation, were also sampled accordingly. the allantoic fluid of inoculated eggs was harvested and clarified by centrifugation for 10 min at 925 g. the obtained supernatants were used for titration in cek. the presence of ibv antigens was investigated in cam and embryos using the avidin-biotin complex (abc) method for immunohistochemistry [39] . to this end, sampled cam and individual whole embryos, containing all organs were fixed for 4 h in carnoy solution (absolute ethyl alcohol:chloroform:acetic acid; 6:3:1) followed by dehydration in absolute ethanol, and paraffin waxembedding. sections were cut at 2 μm, and mounted on electrostatically charged glass slides. tissue sections (cam and whole embryos cut in transversal or sagittal orientation) were dewaxed in xylene, rehydrated, treated with 3% hydrogen peroxide and then subjected to antigen retrieval by microwaving in 10 mm citric buffer (ph 6.0) for 10 min. treated slides were cooled at room temperature for 20 min. non specific background staining was blocked by incubating the sections for 20 min. with goat serum. the sections were then incubated for 1 h with 1:3000 rabbit anti-ibv polyclonal serum (prepared previously in the laboratory of g. keil, fli, using purified ibv m41) in tris buffered saline (tbs), 1% bovine serum albumin (bsa), in each tissue 10 randomly selected areas of each compartment were evaluated at high power by light microscopy. the judgments were made semiquantitatively via side-byside comparisons of one section to another and the purpose was to evaluate antigen distribution in relation to labelling intensity in different embryonic tissues. 100 μl of tenfold serial dilutions in mem were added to 10 5 cek cells per well. after 48 h of incubation, cells were fixed with acetone-methanol, and virus titers expressed as tissue culture infective dose fifty (tcid 50 ) were determined by indirect immunofluorescence using a polyclonal serum raised in rabbits against purified ibv m41 virions. ibv infected cek cells were fixed with acetone-methanol for 15 min. after washing, cells were incubated with the polyclonal rabbit anti-ibv serum (1:2000) for 1 h and subsequently with fitc-conjugated goat anti-rabbit antibodies (sigma) (1:2000) for 1 h. both primary and secondary antibodies were diluted in pbs. plates were rinsed three times with pbs after each step. infectious bronchitis coronaviruses in poultry and other birds characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens infectious bronchitis sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of the tracheal epithelium by 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bronchitis virus in egypt in vitro characterization and pathogenesis of egypt/beni-suef/01; a novel genotype of infectious bronchitis virus early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe the control of avian infectious bronchitis/nephrosis in australia establishment of persistent avian infectious bronchitis virus infection in antibodyfree and antibody-positive chickens recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china the quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza a viruses to human type receptors properties and developmental regulation of polysialyltransferase activity in the chicken embryo brain developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, pst and stx cell culture methods iontophoretic application of unconjugated cholera toxin b subunit (ctb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons we thank katrin giesow and gabriele czerwinski for excellent technical assistance. the authors declare that they have no competing interests. asa performed virus inoculation in ece and cell culture, ifa, contributed to ihc, analyzed the data, and drafted the manuscript. pz contributed to ihc and histopathological examination, jv performed virus isolation and ifa. gmk and jpt initiated the project, provide continued directions and critically reviewed the manuscript. jpt also performed and supervised histopathology and ihc. all authors read and approved the final manuscript. key: cord-296611-ma32oz4o authors: ma, tianxin; xu, liwen; ren, mengting; shen, jie; han, zongxi; sun, junfeng; zhao, yan; liu, shengwang title: novel genotype of infectious bronchitis virus isolated in china date: 2019-01-29 journal: vet microbiol doi: 10.1016/j.vetmic.2019.01.020 sha: doc_id: 296611 cord_uid: ma32oz4o recombination events are known to contribute to the emergence of novel infectious bronchitis virus (ibv) genotypes. in this study, we carried out detailed phylogenetic analysis and sequence comparisons based on 74 complete nucleotide sequences of the ibv s1 gene, including strain i0636/16 and 73 representative sequences from each genotype and lineage. the results showed that strain i0636/16 represented a novel genotype, designated as lineage 1 within genotype vii (gvii-1). further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage 18 within genotype i (gi-18)-like virus with an as-yet-unidentified sequence, likely derived from another ibv strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. to the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/ch/lgx/111119. these results emphasize the importance of limiting exposure to novel ibvs that may serve as a source of genetic material for emerging viruses, as well as the importance of ibv surveillance in chicken flocks. avian infectious bronchitis, caused by infectious bronchitis virus (ibv), was first described in north dakota, usa in the 1930s (schalk and hawn, 1931) . it is a highly contagious viral respiratory disease that is considered as one of the most important causes of heavy economic losses to the poultry industry worldwide (cavanagh and gelb, 2008) . all ibv strains replicate primarily in the respiratory tract and cause respiratory diseases in birds. however, some virus strains are also able to replicate in many other epithelial surfaces, including kidneys and oviducts, and infection with these strains may thus result in nephritis, decreased egg production and quality, and significant mortality. numerous different ibv strains have been reported to date, with pathologies ranging from mild respiratory symptoms to severe kidney and oviduct diseases (cavanagh, 2007) . ibv is an avian coronavirus with a single-stranded, positive-sense, rna genome of approximately 27 kb. the 3′ end of the genome encodes four structural proteins, including spike glycoprotein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, and four non-structural accessory proteins, 3a, 3b, 5a, and 5b. the 5′ end of the genome encodes two polyproteins (1a and 1ab) that contain proteins necessary for rna replication (boursnell et al., 1987) . similar to other coronaviruses, genetic diversity in ibv is created by both recombination events (kottier et al., 1995) and by mutations, including substitutions, deletions, and insertions (shi et al., 2000) , that occur during replication of the viral genome. the high mutation rates are attributed to the limit of replication fidelity although it has been shown that coronaviruses possess proofreading enzyme (3′-to-5′ exoribonuclease) which is essential for replication fidelity (denison et al., 2011) , while recombination events are thought to result from a unique templateswitching copy-choice mechanism during rna replication (simon-loriere and holmes, 2011) . the spike protein comprises about 1145 amino acids and undergoes post-translational cleavage to form s1 and s2 subunits (cavanagh, 1983) . the s1 subunit contains virus-neutralizing epitopes and carries serotype-specific determinants. furthermore, the s1 gene has demonstrated the highest variability in the whole viral genome (niesters et al., 1986) , with mutations and recombination events in the s1 gene considered as critically important for the emergence of new virus genotypes, serotypes, and variants. many different ibv types have been https://doi.org/10.1016/j.vetmic.2019.01.020 received 14 october 2018; received in revised form 9 january 2019; accepted 27 january 2019 found worldwide, and new variants continue to emerge in different parts of the world. a new classification based on analysis of the whole s1 gene has recently been proposed (valastro et al., 2016) , including grouping and naming 32 lineages, comprising six genotypes (gi-1-gi-27, gii-gvi), and a number of inter-lineage recombinants. some of these lineages are distributed in several continents, countries, or regions, such as gi-1 (formerly massachusetts; mass), 4/91, or cr88) , , gi-16 (ck/ch/ldl/97i (ldl/97i) or q1), gi-21 (italy 02), and gi-23 (var2) (de wit et al., 2011; valastro et al., 2016) , while others are geographically confined to specific regions. among the widely distributed ibv lineages, gi-1, gi-13, gi-16, and gi-19 are currently circulating in china (de wit et al., 2011; valastro et al., 2016) , while strains assigned to some of these lineages, such as gi-16 and gi-19, were also found to originate in china. in addition, ibv variants of indigenous lineages were also found to be circulating in chicken flocks in china. new lineages, gi-7 , gi-28, and gi-29 (chen et al., 2017; jiang et al., 2017) , were recently found in chickens suffering from respiratory and urinary health problems. these results indicated that novel ibv lineages have been emerging continuously in china in recent years. an epidemiological study conducted in china from january 2016 to december 2017 based on the s1 gene sequence isolated and characterized a new variant, designated i0636/ 16, in guangxi province in 2016 (xu et al., 2018) . despite the fact that most heterogeneity of ibvs occurs in the s1 gene, sequence analysis of this gene alone was not sufficient to characterize this novel strain. we therefore carried out a detailed investigation of the s1 gene and the complete genomic sequence of the i0636/16 strain to clarify its genetic characteristics and gain further insights into the origin of the strain. we also investigated the antigenicity and pathogenicity of the ibv strain i0636/16 in the present study. fertile white leghorn specific pathogen-free (spf) eggs and chickens were purchased from harbin veterinary research institute, chinese academy of agricultural sciences. ethical approval to carry out this study was obtained from the ethical and animal welfare committee of heilongjiang province, china (license no. hsy-iacuc-2018-186). we used the virus γcov/ck/china/i0636/16, referred to as i0636/ 16, in this study. this virus was previously isolated in spf chicken eggs from a disease outbreak associated with respiratory disease in a layer flock. the virus underwent six passages in spf eggs and was tentatively classified as a variant (xu et al., 2018) . a further eight ibv strains (h120 (gi-1) (chen et al., 2015) , 4/91 (gi-13) , ldl/ 092022 (ldl/092022) (gi-19) (liu et al., 2013) , ldl/97i (gi-16) (liu et al., 2007) , ck/ch/lsc/99i (lsc/99i) (gi-22) (liu et al., 2006) , ck/ ch/lgx/111119 (lgx/111119) (gi-28) , i0111/14 (gi-29) , and i0725/17 (gvi-1) (xu et al., 2018) ), representative of different ibv lineages circulating in china in recent years, were used in cross virus-neutralization (vn) tests. viral stock was prepared by inoculating each of the viruses into 9-day-old spf embryonated chicken eggs by the allantoic cavity route, followed by incubation for 48-72 h and chilling at 4°c overnight. allantoic fluid was harvested from the inoculated eggs and clarified by low-speed centrifugation at 1500 × g for 10 min. cleared supernatant was stored in aliquots of 0.3 ml at −70°c until further processing. virus titration was performed using 9-day-old spf chicken eggs, as described previously (liu et al., 2009 ). the eggs were examined for ibv lesions including curling and dwarfing up to 7 days post-inoculation. viral titers were calculated according to reed and muench (1938) and expressed as the 50% egg infective dose per milliliter (eid 50 /ml). 2.3. rna extraction, reverse transcription-polymerase chain reaction, sequencing, and sequence determination viral rna was extracted from the allantoic fluid using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's protocol. the genomic sequence was amplified by reverse transcriptionpolymerase chain reaction (rt-pcr) using a primescript™ one-step rt-pcr kit ver. 2 (takara bio inc., shiga, japan) according to the manufacturer's instructions. the strategy and primers used for amplifying, cloning, and sequencing the complete genome of strain i0636/16 have been described previously (liu et al., 2013) . the primers were previously designed for ibv complete genome sequencing (liu et al., 2013) , and if the primers failed to work due to sequence differences, new primers were designed based on the newly determined sequences flanking those genome regions. the 3′/5′ ends of the genome were determined using a 3′/5′ race kit (takara bio inc.) according to the manufacturer's protocol. the pcr products were cloned into the pmd18-t vector (takara bio inc.) according to the manufacturer's instructions. each genome fragment was sequenced three times. chromatograms were analyzed using the program chromas (http:// technelysium.com.au/wp/chromas/) and sequences were aligned using bioedit (http://www.mbio.ncsu.edu/bioedit.htm). open reading frame (orf) predictions were carried out using the orf-finder program (https://www.ncbi.nim.nih.gov/orffinder/), and orfs were compared with the beaudette strain (genbank accession number: nc_001451). the complete genomic sequence of i0636/16 has been deposited in the genbank database, with the accession number mh924835. the full s1 gene sequence generated in this study was subjected to blast searches using the national center for biotechnology information database, and then analyzed phylogenetically using a dataset consisting of 73 sequences (supplemental table 1 ), including representative sequences for each genotype and lineage, as recently recommended (valastro et al., 2016; chen et al., 2017; jiang et al., 2017) , and the gx-nn130021 strain, which was closely related to i0636/16 by blast analysis. the s1 gene sequences of the ibv strain i0636/16 and the selected viruses were aligned using clustalw, and phylogenetic analysis was carried out using mega version 6.0 software (http://www. megasoftware.net/) using the maximum likelihood method with the tamura-nei substitution model and 1000 bootstrap replicates to assess the robustness of the branches. only one ibv representative was selected from each lineage based on the phylogenetic trees to calculate the percentage identities between strain i0636/16 and the representative strains at both the nucleotide and amino acid levels. recombination events and the probable parental genotypes of strain i0636/16 were analyzed by selecting and downloading the complete genomes of 121 ibv strains (supplemental table 2 ) from genbank (www.ncbi.nlm.nih.gov/genbank/), representing sequences of ibv strains from all continents and each lineage if possible, three turkey coronavirus (tcov) strains, including north american and european strains, and the only available strain of gfcov. the complete genomic sequences of the ibv strain i0636/16 and the other selected viruses were aligned using clustalw, and phylogenetic trees were constructed using the above-mentioned method to reduce the database size, by collapsing the sequences into clusters with different sequence identities. sequence representative(s), including the complete genome sequences of ibv strains ck/ch/gd/gz14 and gx-yl9, which showed close sequence identity to the i0636/16 strain, were examined using simplot version 3.5.1 (http://sray.med.som.jhmi.edu/scropftware/simplot/) t. ma, et al. veterinary microbiology 230 (2019) 178-186 to identify likely recombination breakpoints (lole et al., 1999) .the ibv strain gx-yl9 was used as a query virus. the window width and step size were set to 500 bp and 30 bp, respectively. to confirm these recombination breakpoints, three phylogenetic trees were constructed on the basis of the results of similarity plot (simplot) analysis for the nucleotide fragments 1-20,265 (5′ untranslated region (utr) to 3′ end of gene 1), 20,266-24,151 (3′ end of gene 1 to 5′ end of gene 3), and 24,152-27,629 (5′ end of gene 3 to 3′ utr), from nine ibv strains, including i0636/16, five strains (lgx/ 111119, ck/ch/gd/gz14, γcov/ck/china/i0114/14 (i0114/14), gx-yl9, and gx-yl5) showing close relationships with i0636/16 based on the complete genomic sequence analysis, and three mass strains (beaudette, h120, and m41) as an outgroup. in addition, the nucleotide identities between i0636/16 and the eight ibv strains were calculated. finally, blastn (http://blast.ncbi.nlm.nih.gove/blast.cgi) analysis using the fragment (20,266-24,151) of the recombined region was conducted using the genbank database. antisera against the viruses h120, 4/91, ldl/091022, ldl/97i, lsc/99i, lgx/111119, and i0111/14 chen et al., 2017; jiang et al., 2017) were used in this study. antisera against strains i0725/17 and i0636/16 were produced in this study following a standard protocol . briefly, 1-month-old spf chickens were inoculated intratracheally with approximately 10 5 eid 50 per bird, followed by an intravenous injection of the same dose 3 weeks later. blood samples were collected after a further 3 weeks, and serum was harvested, pooled, and inactivated for 30 min at 56°c before being used in vn tests. we investigated the antigenic relationships between i0636/16 and the other reference strains by reciprocal β vn tests, using a fixed concentration of virus and serial dilutions of serum (ducatez et al., 2009) . two-fold serial dilutions of each antiserum were mixed with an equal volume of virus dilution containing 100 eid 50 in 0.1 ml and incubated for 1 h at 37°c. each serum-virus mixture was then inoculated into embryonated spf chicken eggs via the allantoic sac route. the eggs were evaluated for non-specific mortality 24 h after inoculation and for the presence of specific lesions at 1 week after inoculation. end points corresponded to the serum dilutions that neutralized 50% of the virus. end-point titers were calculated by the method of reed and muench (1938) . vn end-point titers were used to calculate the percentage of antigenic relatedness (r) according to the method of archetti and horsfall (1950) . isolates with r values between 50% and 100% were considered to be antigenically related (gelb et al., 1997) . forty-five 1-day-old spf chickens were separated in three groups of 15 birds each and housed in negative pressure isolators with ad libitum food and water. birds in groups 1 and 2 were inoculated via the ocular and nasal routes with 10 5 eid 50 /per bird of strain i0636/16 and lgx/ 111119 in 100 μl volume, respectively, while birds in group 3 served as a negative control. oropharyngeal and cloacal swabs and blood were collected from all birds in all groups at 4, 8, 12, 16, 20, 24, 28 and 32 days post-inoculation. the oropharyngeal and cloacal swabs were used to recover virus using 9-day-old spf chicken eggs via the allantoic cavity route (liu et al., 2013) and the allantoic fluids were used for rt-pcr to detect the presence of challenge viruses , to measure the duration of excretion of the strains i0636/16 and lgx/ 111119 during the course of the experiment. the sera were used to detect specific antibodies against ibv using a commercial enzymelinked immunosorbent assay (idexx corporation, westbrook, me, usa) according to the manufacturer's instructions. morbidity and mortality were recorded daily. five birds from each group were selected randomly and killed humanely at 5 days post-inoculation. trachea, lung, proventriculus, cecal tonsil, and kidney tissues were collected from these birds and used for virus titration in 9day-old spf chicken eggs, as described previously . trachea and kidney were considered as the two targeted organs for ibv strains. lung is the representative of respiratory tract of chickens. proventriculus and cecal tonsil were representatives of upper and lower digestive tracts which were often used for ibv isolation (alvarado et al., 2006; yu et al., 2001) . data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version 5 (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). based on blast analysis using the complete nucleotide sequence of the s1 gene, the i0636/16 strain was found to be closely genetically related to gx-nn130021, which was isolated in guangxi province in 2013 . a phylogenetic tree comparing 74 reference strains representing the well-established genotypes and lineages, strain gx-nn130021 and the current strain i0636/16 are illustrated in fig. 1 . the ibv strain i0636/1, which was isolated in guangxi province in 2016, was closely related to the reference strain gx-nn130021. strains i0636/16 and gx-nn130021 were clearly separated from other ibv reference strains, including the six well-established genotypes and 29 lineages. homology analysis of the ibv strains from the different lineages revealed similarities between isolate i0636/16 and the established lineages of 59.0%-71.7% and 51.3%-69.6% at the nucleotide and deduced amino acid levels, respectively. the deduced amino acid substitutions were scattered across the s1 subunit of the spike proteins in i0636/16, compared with the other lineages (data not shown). however, strains i0636/16 and gx-nn130021 shared 97% and 96.9% nucleotide and amino acid identities, respectively, with each other. alignment of the s1 deduced amino acid sequences identified 18 substitutions between i0636/16 and gx-nn130021, of which only two were located in the hypervariable region (hvr) (supplemental fig. 1a) . a consensus sequence of strain i0636/16 was obtained and the fulllength genome consisted of 27,629 nucleotides excluding the 3′ poly a tails. orf analysis predicted ten orfs, resulting in a typical ibv genome organization of 5′utr-1a-1b-s-3a-3b-3c(e)-m-5a-5b-n-3′utr. in addition, there was also an orf with the potential to code for a protein of 94 amino acids between gene m and 5a of strain i0636/16 which was identified previously in some ibv strains (bentley et al., 2013) . phylogenetic analysis of 121 complete genomes was conducted to investigate the relationships between the obtained strain i0636/16 and different avian coronaviruses downloaded from genbank. strain i0636/ 16 was grouped together with all the ibv strains used in this study, which had been isolated from different regions at various times but was grouped apart from coronaviruses isolated from other avian species (fig. 1b) . within ibv strains, i0636/16 showed close relationships with ibv strains lgx/111119 (93.5%), ck/ch/gd/gz14 (93.1%), i0114/14 (92.8%), ck/ch/2010/jt-1 (92.8%), gx-yl9 (92.4%), and gx-yl5 (92.1%), most of which were isolated from south china, particularly from guangxi province. we investigated the impact of probable recombination events on the origin of strain i0636/16 by conducting an analysis using simplot software ( fig. 2a) . two major recombination events were observed, one in the 3′ end of gene 1 (at approximately nucleotide 20,265) and another in the 5′ end of gene 3 (at approximately nucleotide 24,152). the phylogenetic trees (fig. 2b ) clearly supported the simplot results, with strain i0636/16 showing identities of > 96.6% with lgx/111119, ck/ch/gd/gz14, i0114/14, ck/ch/2010/jt-1, gx-yl9, and gx-yl5 for the genomic region 1-20,265, compared with an identity of < 86.9% with the mass-type strains. similar to this genomic region, i0636/16 showed an identity of 92.5% with lgx/111119 for the genomic region 24,152-27,640, compared with < 85.2% with the mass-type strains. these results suggested that the lgx/111119 (gi-28 lineage) virus, circulating in chicken flocks in china , was a potential parent of recombinant i0636/16. in contrast, in the second phylogenetic tree, the branch (genomic region 20,266-24,151) that included the spike gene separated the strain i0636/16 from all reference strain sequences, and shared no more than 75% nucleotide identity with any of the reference strains, making difficult to assess the origin of the fragment 24,152-27,629. blastn analysis using this fragment of i0623/16 showed that this strain was closely related to strain gx-nn130021 (97% nucleotide identity), but shared no more than 75% nucleotide identity with any other ibv strain in the genbank database. overall, these data suggest that gvii-1 viruses emerged as a result of replacement of the spike gene in strain lgx/111119-like viruses (gi-28) through recombination. the antigenic properties of i0636/16 were compared to those of reference strains by vn tests. the calculated antigenic relatedness values, r, of strain i0636/16 against homologous and heterologous strains are listed in fig. 3 . the r values confirmed the absence of relationships between i0636/16 and h120, 4/91, ldl/091022, ldl/97i, lsc/99i, lgx/111119, i0111/14, and i0725/17(all < 5%). the value of r for lgx/111119 was slightly higher but did not reach the 50% threshold for antigenic relatedness between strains, suggesting that the i0636/16 strain represented a novel serotype. respiratory signs started on day 4 in four birds post-challenge, followed by the development of symptoms in nearly all birds. the t. ma, et al. veterinary microbiology 230 (2019) 178-186 respiratory signs varied from mild respiratory distress to severe rales. generally, the respiratory signs of chickens challenged with strain lgx/ 111119 were more severe than those of chickens challenged with i0636/16. one bird died on day 9 after challenge with strain i0636/16. in contrast, three birds died on days 4, 8 and 16 after challenge with strain lgx/111119. macroscopic lesions in the dead birds were mainly confined to the kidneys, which showed pale, swollen, and mottled kidney parenchyma, and slightly distended tubules and urethras with uric acid crystals. petechiae were also observed in the trachea, pharynx, and larynx in most challenged birds. the clinical signs continued up to day 17 in one bird that showed mild respiratory distress. no clinical signs were observed in the control birds throughout the experiment. no birds seroconverted before day 8 post-challenge, 33.3% and 50% birds seroconverted on day 12 after challenge with i0636/16 and lgx/ 111119, respectively. nearly all birds seroconverted on day 16 after challenge with the two virus strains. as expected, no birds in the negative control group showed an antibody response against ibv. virus recovery from oropharyngeal swabs using 9-day-old spf chicken eggs challenged with i0636/16 revealed that 100% of birds were positive on day 4, 40% were positive on day 8, and 22.2% were positive on day 12 post-challenge. in contrast, using cloacal swabs, 100% were positive on days 4 and 8, and 44.4% were positive on day 12 post-challenge. no birds tested positive on day 16 or thereafter postchallenge. comparing with those of chickens challenged with i0636/ 16, prolonged virus shedding were observed from both the respiratory tract and cloaca of chickens challenged with the lgx/111119 strain (fig. 4a) . no birds in the negative control group tested positive for virus recovery at any timepoint. the titers of the challenge viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues were determined in five challenged chickens from each of the three groups, at 5 days post-inoculation using 9-day-old spf chicken embryos . infection with strain lgx/111119 resulted in significantly higher production of infectious viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues than those of corresponding tissues of chickens infected with strain i0636/16 (fig. 4b) . comparatively, infection with strain i0636/16 resulted in lower production of infectious virus in the trachea than those in the kidneys and cecal tonsils. no infectious viruses were detected in the lungs in the five chickens challenged with strain i0636/16. no viruses were detected in any tissues in the five control chickens. some studies have reported that genotyping based on sequencing the hvrs of the s1 gene or partial sequencing of the s1 gene is representative of the grouping based on the complete s1 gene (lee et al., 2003) ; however, others disagree with these findings (moreno et al., 2017; schikora et al., 2003) , and this discrepancy is considered to be due to the presence of recombination events involving the s1 hvr. in the present study, we used the complete s1 gene for ibv typing, as suggested previously (manswr et al., 2018; valastro et al., 2016) , and found no evidence of recombination events in the i0636/16 s1 gene sequence. we did identify a novel genotype consisted by ibv isolate i0636/1 and the reference strain gx-nn130021, which clustered together in the phylogeny and did not fall within established lineages. in agreement with this result, the two strains shared high nucleotide and amino acid identities (97% and 96.9%), compared with low identities with representative ibv strains from different lineages. based on these results, these two ibv strains should be grouped into a novel genotype, designated as gvii-1, although the designation of lineage/genotypes should be assigned to monophyletic groups of at least three viruses sampled from at least two different outbreaks (valastro et al., 2016) . generally, it is considered that the chance of having the same serotype is higher between strains with the same genotype (wang and huang, 2000) , though these data showed that this relationship was not very strong, given that a change in a small percentage of the amino acids in the s1 protein could result in a change of serotype (cavanagh et al., 1992) . the current virus cross-neutralization results showed that gvii-1 was not only a novel genotype, but also antigenically different from the currently tested genotype lineages. strain gx-nn130021 was isolated in guangxi province, china, in 2013 , suggesting that the novel gvii-1 had been circulating in china over a 5-year period. however, the source of this new virus introduction into the chicken population in china remains unknown. full-length genome analysis showed that the emergence of the i0636/16 strain involved a double crossover event that replaced the spike gene by that from an as-yet-unidentified ibv strain. however, the remaining parts of the genome originated from commonly known gi-28 ibv strains, suggesting that the i0636/16 strain was a mosaic, and that one of its putative parents was descended from gi-28 lineage viruses. the antigenic differences among ibv strains have been investigated, and previous work showed that s1 sequences had a stronger correlation with protective relatedness than with antigenic relatedness between strains (ladman et al., 2006) . the as-yet-unidentified parental virus of strain i0636/16, which shared the same spike gene with strain i0636/ t. ma, et al. veterinary microbiology 230 (2019) 178-186 16, might thus be antigenically different from vaccine strains used in china, such as h120 and 4/91; therefore vaccination with these live vaccines might not offer complete protection against infection with the parental virus of strain i0636/16. similarly, considering the high prevalence of the gi-28 lineage in south china where the novel gvii-1 viruses were isolated, it is reasonable to speculate that the gi-28 lineage may co-infect birds more frequently than other lineages, although the current commonly used vaccines cannot offer complete protection against gi-28 viruses . vaccination with these vaccines is therefore likely to induce non-sterilizing immunity, which may allow prolonged replication, shedding, and circulation of both the deduced parental strains of gvii-1 viruses. consequently, although vaccination with attenuated vaccines is carried out intensively in china, this procedure may produce an environment where co-infection with gi-28 and the as-yet-unidentified parental viruses can enhance the likelihood of recombination. theoretically, the short persistence and shedding, and limited numbers of i0636/16-like viruses isolated so far in china suggest that the probability of recombination between the as-yet-unidentified parental virus and gi-28 viruses may not be high, similar to the hypothesis suggesting that the shorter persistence and shedding of mass-based vaccines (bijlenga et al., 2004) and the low percentage of m41-like viruses (chen et al., 2015; moreno et al., 2017) lgx/111119 fig. 4 . infection of chickens with strains lgx/111119 and i0636/ 16. virus recovery from oropharyngeal (upper) and cloacal (lower) swabs from chickens challenged with ibv strains lgx/ 111119 and i0636/16 (a). virus recovery was performed by inoculating 9-day-old embryonated, specific pathogen-free eggs through the allantoic route with supernatant from the swabs. replication of strains lgx/111119 and i0636/16 in trachea, lung, kidney, cecal tonsil, and proventriculus in chickens (b). one-dayold spf layer chickens in groups 1 and 2 were inoculated via the ocular and nasal routes with 10 5 × eid 50 /per bird of strains lgx/ 111119 and i0636/16 in 0.1 ml, respectively, and trachea, lung, kidney, cecal tonsil, and proventriculus tissues were collected from five birds at 5 days post-challenge for virus titration in eggs. data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version 5 (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). t. ma, et al. veterinary microbiology 230 (2019) 178-186 the ibv genome undergoes mutations and recombination, and the exchange of a long region of the genome by recombination may allow viruses to rapidly explore ample areas of the sequence space, potentially leading to the emergence of novel strains with different features in terms of virulence and disease pathogenesis, antigenicity, and cell and tissue tropisms (cavanagh et al., 1992; kant et al., 1992; simon-loriere and holmes, 2011) . the genome of strain i0636/06 was closely related to gi-28 viruses, except in the spike gene. this phenomenon was similar to that of tcov, the genome of which was also closely related to ibv, except in the spike gene. replacement of the spike gene of tcov was responsible for the host shift from chickens to turkeys and a pathogenicity shift from upper-respiratory disease to enteric disease (jackwood et al., 2010) . even though the recombination events that replaced the spike gene did not result in a tissue tropism shift in the kidneys of chickens after challenge, the i0636/16 strain showed low affinity to the respiratory tract compared with the parental virus lgx/ 111119 . in contrast, the ability of i0636/16 to reach the cecal tonsils and subsequently to replicate in this tissue was slightly higher compared with that of respiratory tract. the replicase gene of avian coronavirus ibvs is known to be a determinant of pathogenicity (armesto et al., 2009) , and we were therefore unable to compare the pathogenicity of i0636/16 with one of its deduced parental virus until a source has been identified for the replicase gene because it is as-yetunidentified, though another deduced parental virus (lgx/111119) showed more severe pathogenicity than i0636/16 in spf chickens. however, we cannot conclude that this difference in pathogenicity was due to replacement of the spike gene until the source of the gvii-1 spike gene has been identified, and alternate explanations for the observed differences between i0636/16 and gi-28 spike should be considered. the frequency of successful inter-typic genetic changes may depend on the viability of the recombinant progeny able to establish in the host population, and on the increase in fitness of the recombinant offspring in the host population compared with their non-recombination parents (smits et al., 2003) . the recombinant i0636/16 was only detected sporadically and persisted for a short time, and thus probably represented strains that were not sufficiently competitive with respect to one of their progenitor strains, lgx/111119-like viruses . however, compared with the unidentified parental virus, i0636/ 16-like strains may be sufficiently competitive, and the parental virus may have been circulating at below cut-off levels for detection, due to its lower replicative capacity and lower percentage. we isolated the i0636/16 ibv strain and passaged it in spf chicken eggs . the number of passages of the virus in eggs was high (six passages), and the possibility that some genetic changes were introduced as a result of its propagation in the eggs cannot be ruled out. no complete genomic sequences of gvii-1 lineage viruses are currently available in the public database. the present results thus provide valuable information on the characteristics of the i0636/16 strain and contribute to a better understanding of the origin of gvii-1 viruses. further studies on these isolates will provide information on the emergence, evolution, and effectiveness of current vaccines against these isolates. detection of 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declare that they have no competing interests. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2019.01.020. key: cord-309469-2naxn580 authors: an, hongliu; cai, zhichao; yang, yuying; wang, zhaoxiong; liu, ding xiang; fang, shouguo title: identification and formation mechanism of a novel noncoding rna produced by avian infectious bronchitis virus date: 2019-01-05 journal: virology doi: 10.1016/j.virol.2018.12.019 sha: doc_id: 309469 cord_uid: 2naxn580 viral noncoding (nc) rnas have been shown to play important roles in viral life cycle. many viruses employ different mechanism to produce ncrnas. here, we report that coronavirus infectious bronchitis virus (ibv) produces a novel ncrna in virus-infected cells. this ncrna consists of 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-untranslated region of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome. using mutagenesis and reverse genetics, we reveal that this ncrna is a subgenomic rna generated by discontinuous transcription mechanism. viruses employ different mechanisms to produce a number of noncoding (nc) rnas excluding microrna. these ncrnas mainly include: (1) viral ncrnas transcribed by rna polymerase (pol) iii. for example, two virus-associated (va) rnas encoded by human adenovirus (reich et al., 1966; steitz et al., 2010) , eber1 and eber2 encoded by epstein-barr virus (skalsky and cullen, 2015) , mirna precursors encoded by murine γ-herpesviral trna-pre-mirna chimeras (bogerd et al., 2010; bowden et al., 1997; diebel et al., 2010) and retrovirus (kincaid et al., 2012) , and intragenic viral small ncrna encoded by human bocavirus 1 (wang et al., 2017) ; (2) viral ncrnas transcribed by rna polymerase ii. for example, polyadenylated nuclear (pan) rna encoded by kaposi's sarcoma-associated herpesvirus (sun et al., 1996; zhong and ganem, 1997) , the~2-kb latency-associated transcript (lat) expressed by herpes simplex virus (bloom, 2004 ), a con-served~5-kb intron expressed by human cytomegalovirus (hcmv) (kulesza and shenk, 2004) , and a 7.2-kb rna expressed by mouse cmv (kulesza and shenk, 2006) , u-rich rnas (hsurs) produced by herpesvirus saimiri (hvs), (albrecht and fleckenstein, 1992; ensser and fleckenstein, 2005) ; (3) subgenomic ncrnas from single-stranded rna viruses by incomplete degradation of genomic rna by the cellular 5-3′ exonuclease xrn1. for example, subgenomic rna (sfrna) produced by flaviviruses, such as dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), and zika virus [reviewed in (bidet and garcia-blanco, 2014; roby et al., 2014; pijlman et al., 2008; akiyama et al., 2016) , and subgenomic ncrna generated by some plant viruses such as barley yellow dwarf virus and red clover necrotic mosaic virus using similar mechanism (miller et al., 2016a) . viral ncrnas play important roles in viral life cycle. adenovirus va rnas are characterized for their role in counteracting the host antiviral defense through inhibition of protein kinase r (pkr) (mathews and shenk, 1991; wilson et al., 2014) . eber2 regulates the expression of a subset of ebv latent genes throung the interaction of eber2 and a cellular transcription factor paired box protein 5 (pax5) (arvey et al., 2012; lee et al., 2015) . degradation of mir-27 by mediated hsur 1 promotes activation and presumably proliferation of hvs-infected host t cells guo et al., 2014) . kshv pan rna is essential for virion production (borah et al., 2011) .β-herpesvirus hcmvencoded β2.7 prevents mitochondria-induced apoptosis, enabling steady atp production for viral processes and persistent infection (campbell et al., 2008; stern-ginossar et al., 2012) . sfrna produced by flaviviruses is required for cytopathicity and pathogenicity (pijlman et al., 2008) . it has been demonstrated to 1) interfere with cellular rna decay pathways by inhibiting xrn1 (moon et al., 2012) , 2) dampen the antiviral activity of type i interferon (schuessler et al., 2012) and 3) inhibit the rnai pathway in both the vertebrate and arthropod hosts, most likely by serving as a decoy substrate for dicer (schnettler et al., 2012) . inhibition of the host interferon response appears to be, at least in some flaviviruses, achieved by binding and inactivating cellular https://doi.org/10.1016/j.virol.2018.12.019 received 28 september 2018; received in revised form 19 december 2018; accepted 26 december 2018 regulators of translation of interferon-upregulated mrnas (bidet et al., 2014) . ncrnas encoded by some plant rna viruses can inhibit host translation and overwhelm host's rna interference system to favor virus infection (miller et al., 2016b) . avian infectious bronchitis virus (ibv) belongs to the genus gammacoronavirus within the order nidovirales. ibv is an enveloped positive-sense, single-stranded rna virus causing the acute highly contagious poultry disease infectious bronchitis (cavanagh, 2005) . like other coronaviruses, ibv can produce sgrnas via a discontinuous transcription mechanism to encode its structural proteins and specific accessory proteins (masters, 2006; sawicki et al., 2007) . briefly, ibv produces six mrna species in the infected cells, including its genomic mrna1, sgrna2 encoding spike (s) protein, sgrna3 encoding 3a, 3b and envelope (e) protein, sgrna4 encoding membrane (m) protein, sgrna5 encoding 5a and 5b, and sgrna6 encoding nucleocapsid (n) protein. recently, a low-abundance sgrna located between the sgrna4 and 5 has been identified (bentley et al., 2013) . in this study, we identify firstly an ncrna in the ibv-infected cells. moreover, we prove that this ncrna is derived mainly from 3′ utr of viral genome and is generated by discontinuous transcription process. vero and chicken embryo fibroblast df1 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml) and streptomycin (100 units/ml) (invitrogen). a recombinant ibv (ribv) (fang et al., 2007) generated from an infectious clone reference genome (ibv beaudette p65, genbank accession number dq001339.1) was used as the wild-type control. all ibv mutants were propagated in vero cells in fbs-free dmem. virus stocks were made through three repeated freezethaw cycles and kept at −80°c in 0.5-1 ml aliquots until use. constructs containing mutation or deletion were produced by using a quikchange site-directed mutagenesis kit (stratagene). the fulllength cdna was assembled as previously described (fang et al., 2007) by replacing the corresponding fragment with the mutant fragment. the mutations were verified by automated nucleotide sequencing. fulllength transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tx) according to the manufacturer's instructions, and electroporated into vero cells with one pulse at 450 v and 50 µf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing dmem and further cultured in dmem without fbs. the transfected cells were monitored daily for formation of cytopathic effect (cpe). recovered viruses were plaque purified and passaged on vero cells. vero cells in 6-well plates were infected with a dilution series of viruses for 1 h, washed twice with medium. cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% crystal violet. the number of plaques was counted and the virus titer was calculated as plaque-forming unit (pfu) per ml. ten-day-old embryonated, pathogen-free chicken eggs were inoculated with ibv as described previously (shen et al., 2009) . the allantoic fluid and different organs were harvested after the embryos were chilled at 4°c overnight. total rna was extracted from the homogenized tissues and used for rt-pcr analysis. total rna was isolated from ibv-infected cells using trizol reagent® (invitrogen) according to the manufacturer's instructions. the concentration of the total rna extracted was quantified using a nanodrop™ 1000 spectrophotometer (nanodrop technologies, inc., thermo fisher scientific, usa). reverse transcription (rt) was performed with oligo(dt)18 or specific primer using reverse transcriptase (promega) according to the manufacturer's instructions. for the detection of viral positive-stranded subgenomic (sg)rna, oligo(dt)18 was used for cdna synthesis; for negative-stranded sgrna, ibv-5′end-f (5′-1 acttaagatagatattaatatata) was used. ibv-5′end-f and ibv-3′end-r (5′-27608 tgctctaactctatactagc) were used for pcr. ibv-infected cells at different time point post-infection were washed with pbs and lysed with 2 ×sds loading buffer containing 100 mm dithiothreitol (dtt), boiled at 100°c for 5 min, and clarified. the proteins were separated by sds-page and transferred to a polyvinylidene difluoride (pvdf) membrane (stratagene). the membrane was blocked overnight at 4°c or for 2 h at room temperature in blocking buffer (5% fat-free milk powder in phosphate-buffered saline (pbs) buffer containing 0.1% tween 20 (pbst)) and then was incubated with diluted primary antibodies in blocking buffer for 2 h at room temperature. after the membrane was washed three times with pbst, it was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 h at room temperature. after the membrane was washed three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl kit; amersham biosciences) according to the manufacturer's instructions. the films were exposed and developed. quantitative real-time pcr (qpcr) was used to validate gene expression changes in infected cells. total rna (2 µg) was reversedly transcribed to cdna, and the resulting cdna was subjected to qpcr using power sybr green pcr master mix (applied biosystems). amplification and data collection were performed as manufacturer's instruction (applied biosystems 7500 real-time pcr system). the relative gene expression levels were measured using gapdh as an internal reference, and normalized to gene expression in mock-infected cells (relative expression = 1). all experiments were performed in triplicate. in the virus-infected cells, ibv produces six mrna species, including the genome-length mrna1 and five subgenomic mrnas via a discontinuous transcription mechanism. this mechanism is mediated by transcription-regulating sequences (trss) in the 3′ end of the leader (trs-l) and the preceding each mrna body (trs-b). trss comprise a conserved core sequence (cs) [cu(u/g)aacaa] in ibv. each mrna possesses a leader sequence of 64 nucleotides derived from the 5′-end of the genome. in general, six mrnas are readily detected by northern blotting in the infected cells. when probing the viral positive-or negative-stranded mrna6 by rt-pcr, an unexpected pcr product was concurrently amplified in the ibv-infected vero cells as well as in chicken embryos (fig. 1) . it is smaller than mrna6. subsequently, this product was cloned and sequenced. sequence analysis indicated that it consists of 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-utr (from nucleotides 27104-27608) of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome (fig. 2) . the results showed the generation of a novel sgrna in the ibvinfected cells. this sgrna may be overlooked in previous studies because of its smaller size and lower level of transcription. due to lack of start codon aug, this sgrna is designated as a noncoding rna (ncrna). insertion of an orf encoding egfp (carrying its own start codon) between 27149 and 27150 nt of ibv 3′-utr allowed the recovery of recombinant virus. rt-pcr analysis showed the presence of the egfp-containing sgrna and fluorescence confirmed egfp expression in virus-infected cells (fig. 3) , implying that the ncrna is an mrna. however, this virus was unstable and a deletion of 377 nucleotide acids (from 81 to 457 nt) of egep sequences was detected in passage 3 in vero cells (data not shown). further sequence comparison revealed that a sequence motif (uaaca), located in the junction between the 5′ end leader sequence and the 3′-utr of ncrna, is shared by 5′-terminus of 3′-utr and the core sequence (cuuaacaa) within ibv trs (fig. 2) . these results prompt us to speculate that this ncrna, like the other viral sgrna, may be generated by a discontinuous transcription mechanism via template switch mediated by a noncanonical core sequence (uaaca). to confirm this hypothesis, we analyzed the effect of several mutations on ncrna generation by mutagenesis and reverse genetics. as shown in fig. 4 , compared to wild-type ribv, single mutation u27104a and a27108u had no or minor effect on ncrna synthesis; a27105u resulted in a significant reduction in the ncrna generation, while mutation c27107g, a27106u and deletion of five nucleotides (δ27104-08) completely abolished the synthesis of both positive-stranded and negative-stranded ncrna (fig. 4) , suggesting that at least three fig. 1 . detection of a novel sgrna in virus-infected vero cells and chicken embryo by rt-pcr. total rna was extracted from the ribv-infected vero cells and chicken embryo. cdna was synthesized by reverse transcription using oligo (dt)18 as a primer. pcr was performed using primers ibv-5′end-f and ibv-3′end-r. the amplicons were analyzed on 1% agarose gel electrophoresis. fig. 2 . sequence of the junction between ibv 3′-utr and the leader at 5′-end of viral genome, indicating the formation of a novel sgrna mediated by 27104 uaaca 27108 . vero cells were infected with ribv at an moi of 1 pfu/cell. total rna extracted from the infected cells was used for reverse transcription using primer oligo(dt)18. pcr was carried out using primers ibv-5′end-f and ibv-3′end-r. pcr product was cloned and sequenced. nucleotides (a27105/a27106/c27107) are involved in the effective ncrna generation. the results confirmed previous report that ibv can synthesize sgrna via template switch mediated by a noncanonical core sequence (bentley et al., 2013) notably, the mutant virus carrying four mutations (a27100u/a27111u/g27113c/g27114c) was also unable to produce ncrna (fig. 4) , suggesting these nucleotides are required for ncrna production. because the sequence motif (a27100/a27111/ g27113/g27114) located downstream of the truncated cs (uaaca) also exists downstream of the cs (cuuaacaa) in the leader trs (table 1 , marked in bold), our result prove that the sequences downstream of the cs exert a stronger influence on coronavirus sgrna synthesis (sola et al., 2005) . taken together, the results confirm that the ncrna generation involves a discontinuous transcription process in which the 5′ leader sequence and 3′-utr are fused through the transcription-regulating sequences in the 3′ end of the leader and in the 5′ end of the 3′-utr. the mutant virus c27107g was selected for further experiments because it is not capable of producing ncrna and carries only one nucleotide change, compared to ribv. plaque assay on vero cells showed both viruses did not display major differences in plaque morphology and in virus growth properties (fig. 5a) . moreover, real-time rt-pcr and western blot were performed to analyze the expression of s and n genes at different time points post-infection, respectively. similarly, no significant differences were detected at both rna level and protein level (fig. 5b, c) . the results suggest that ncrna has little effects on viral replication and viral cytopathicity in vero cells. in this report, we have identified an ncrna in ibv-infected cells and revealed that this ncrna is generated via discontinuous transcription mechanism by reverse genetics and mutagenesis for the first time. although the ncrna has no or little effect on viral replication and pathogenesis in vero cells, roles in ibv pathogenesis in chicken and virus-host interplay are unknown, needing to further study. coronaviruses employ a discontinuous transcription mechanism to synthesize subgenomic mrnas through template switch taking place in the transcription-regulating sequences in the 3′ end of the leader (trs-l) and in the intergenic region preceding each mrna body (trs-b) during negative rna synthesis (baric and yount, 2000; zúñiga et al., 2004; masters, 2006; sawicki et al., 2007) . the five sgrnas (mrnas 2-6) of ibv, which are readily detected by northern blotting, possess the canonical core sequence (5′-cu(u/g)aacaa-3′) ( table 1 ). it has been reported that coronaviruses, such as severe acute respiratory syndrome coronavirus, mouse hepatitis virus and ibv, can use noncanonical cs to synthesize sgrna via discontinuous transcription mechanism (zhang and liu, 2000; hussain et al., 2005; bentley et al., 2013) . in this report, we have identified the existence of a novel sgrna derived mainly from the 3′ utr of ibv in the ibv-infected cells (fig. 1) . the synthesis of this sgrna is mediated by a truncated cs ( 27104 uaaca 27108 ) identical to nucleotides of 3-7 of ibv cs. among which at least three nucleotides (a27105/a27106/c27107) are involved in the effective sgrna generation (figs. 2 and 4) , providing more evidence for the use of noncanonical transcriptional signals in synthesis of coronavirus sgrnas. in addition, we verified that the sequence motif (a27100/a27111/g27113/g27114) located downstream of the truncated cs (uaaca) is necessary for ncrna generation (fig. 4) , reinforcing the importance of nucleotides immediately flanking cs in coronavirus sgrna synthesis (sola et al., 2005) . interestingly, when blast search in genbank, we find that the sequence motif (uaaca) is conserved at 5′ end of 3′ utr of ibv strain beaudette and its derivants, arkansas dp1, and turkey coronavirus but not for strain m41, h120, h52, a2, and several field isolates in china. therefore, whether these viruses can produce ncrna and how ncrna affects the viral pathogenecity remain to be determined. fig. 5 . effect of ncrna on plaque morphology and viral replication. a. plaque assay. vero cells in 6-well plates were infected with a dilution series of ribv and mutant virus ibv-c27107g for 1 h, respectively. after washing twice with medium, cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% crystal violet. b. quantitative analysis of sgrna synthesis of n and s. total rna (2 μg) extracted from the vero cells infected with ribv and ibv-c27107g at an moi of 0.5 pfu/cell at 8 and 20 h post-infection was used for reverse transcription using primer oligo(dt)18 respectively. amplification and data collection were performed as manufacturer's instruction (applied biosystems 7500 real-time pcr system). the relative gene expression levels were measured using gapdh as an internal reference, and normalized to gene expression in mock-infected cells (relative expression = 1). all experiments were performed in triplicate. c. western blotting. vero cells were infected with ribv and ibv-c27107g at an moi of 0.5 pfu/cell at the indicated time point. cell lysates from the indicated time point were prepared for western blotting using antibody against s or n protein. zika virus produces noncoding rnas using a multi-pseudoknot structure that confounds a cellular exonuclease nucleotide sequence of hsur 6 and hsur 7, two small rnas of herpesvirus saimiri an atlas of the epstein-barr virus transcriptome and epigenome reveals host-virus regulatory interactions subgenomic negative-strand rna function during mouse hepatitis virus infection identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses 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pathogenicity rna of low molecular weight in kb cells infected with adenovirus type 2 a contemporary view of coronavirus transcription noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development ebv noncoding rnas role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mrna synthesis noncoding rnps of viral origin decoding human cytomegalovirus polyadenylylated nuclear rna encoded by kaposi sarcoma-associated herpesvirus parvovirus expresses a small noncoding rna that plays an essential role in virus replication dissection of the adenoviral va rnai central domain structure reveals minimum requirements for rna mediated inhibition of pkr identification of a noncanonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear rna encoded by kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis this work was financially supported by grants from the national natural science foundation of china (no. 31572490) and the department of science and technology, hubei provincial people's government, china (no. 2013bhe020) key: cord-303794-fn3jkiil authors: hassan, kareem e.; ali, ahmed; shany, salama a.s.; el-kady, magdy f. title: experimental co-infection of infectious bronchitis and low pathogenic avian influenza h9n2 viruses in commercial broiler chickens date: 2017-06-30 journal: res vet sci doi: 10.1016/j.rvsc.2017.06.024 sha: doc_id: 303794 cord_uid: fn3jkiil in this study, commercial broilers were experimentally infected with single (classical ibv, variant ibv or aiv-h9n2) or mixed aiv-h9n2 with classical, variant or vaccine strains of ibv. birds were monitored for clinical and pathological outcomes and virus shedding for 10 days post infection (dpi). clinical signs were limited to the respiratory tract in all challenged groups and varied from mild to moderate mouth breathing to severe respiratory signs with snorting sound and extended head. mortalities were only recorded in mixed aiv-h9n2/variant ibv challenge group. aiv-h9n2 challenge caused tracheal petechial hemorrhage that progressed to tracheal congestion and caseation. in mixed aiv-h9n2/ibv vaccine challenge, severe tracheitis with bronchial cast formation was observed. in mixed aiv-h9n2/variant ibv challenge severe congestion of the tracheal mucosa and excessive exudates with a tendency to form tubular casts were observed. kidney ureate deposition was only observed in variant ibv challenge group. histopathologically, tracheal congestion, severe degeneration, and deciliation were noticed in all groups of mixed infection. interestingly, hemorrhage and atrophy were observed in thymus gland of birds challenged with single aiv-h9n2 or mixed aiv-h9n2/ibv. there was no difference in the tracheal shedding level of variant ibv between single and mixed infected groups while classical ibv shedding increased in mixed infection group. interestingly, the aiv-h9n2 showed constantly high shedding titers till 7dpi with variant or vaccine ibv co-infection. in conclusion, co-infection of ibv and aiv-h9n2 induced severe clinical outcome and high mortality. also, ibv co-infection increased the shedding of aiv-h9n2 in experimentally infected birds. respiratory diseases outbreaks in commercial broiler chicken flocks have increased recently in egypt causing severe economic losses in the broiler industry. avian influenza h9n2 and h5n1 subtypes, infectious bronchitis virus (ibv), and virulent newcastle disease virus (vndv) have been frequently isolated from different broiler chicken flocks (hassan et al., 2016) . these pathogens are of major significance and have a great economic impact because they are able to induce disease independently or in association with each other (roussan et al., 2008) . ibv is causing an acute highly contagious viral respiratory disease of chickens belonged to coronaviridae. secondary bacterial infection is the main cause of bird death due to some bacteria gain access to the blood circulation following damage of the respiratory tract caused by ibv (dwars et al., 2009) . some ibv strains are highly nephropathogenic, able to cause mortality up to 30% in young birds. regardless of regular vaccinations with massachusetts (mass) strains, ibv still has severe and diverse effects on the poultry industry in the country, causing mortality and high condemnations especially with other viral diseases being affecting poultry in egypt (abdel-moneim et al., 2006; hassan et al., 2016) . though the h9n2 are low pathogenic viruses, they vary considerably in pathogenicity in different host species. in the middle east, h9n2 outbreaks were reported to be associated with cast formation in the tracheal bifurcation (karimi-madab et al., 2010) . mixed infections of h9n2 aiv with other respiratory pathogens, particularly ibv, mycoplasma gallisepticum, staphylococcus aureus, avibacterium paragallinarum, escherichia coli, ornithobacterium rhinotracheale and/or immune suppressive agent can exacerbate h9n2 aiv infection resulting in severe clinical disease and variable mortality (kishida et al., 2004; nilli and asasi, 2003; perk et al., 2004) . ibv and aiv-h9n2n2 viruses were found to be widely spread in egyptian poultry with a great economic impact because they are able to induce disease independently or in association with each other (roussan et al., 2008) . the high prevalence of both aiv-h9n2 and ibv determined in field studies highlighted a potential role of ibv in exacerbating the manifestation of aiv-h9n2 infection in broiler chicken with high mortalities (haghighat-jahromi et al., 2008; hassan et al., 2016; seififi et al., 2010) . ibv could provoke ciliostasis in the host's airways (cook et al., 1976; hassan et al., 2016) and may, therefore, facilitate the opportunity for other related pathogens and aggravate their pathogenicity (haghighat-jahromi et al., 2008) . in this study, pathogenesis of both single and mixed aiv-h9n2 and ibv infections under experimental conditions was investigated in commercial broiler chickens. experimentally infected commercial broilers with classical and variant ibv and vaccine ibv strains in presence or absence of aiv-h9n2n2 infection were monitored for clinical outcomes, virus shedding, postmortem and histopathological lesions. an aiv-h9n2 subtype virus (a/chicken/egypt/bsu-bs-k7t/2012, acc. no. kf998212). the virus belongs to group b of g1-like lineage viruses. a variant ibv (ib/chicken/egypt/bsu-mn-kb44/2013, acc. no. kr010942), which is closely related to the recent egyptian variant ii ibv (e.g. eg/bsu-2/2011/acc. no. jx174185) (abdel-moneim et al., 2012) . the aiv-h9n2n2 and the variant ibv viruses were isolated in egypt from 26 day-old and 21 day-old broiler chickens in beni-suef and menia governorates respectively (hassan et al., 2016) . a classical locally isolated ibv strain (ib/chicken/egypt/mf/2012) was kindly provided by d. manal a. afifi, department of poultry diseases, cairo university, egypt. all viruses were propagated and titrated in 10-dayold specific pathogen free embryonated chicken eggs (spf-ece). commercial live attenuated 4/91 ibv vaccine (nobilis® ib 4/91, intervet international b.v., boxmeer-holland) was purchased and used to study the effect of variant ibv vaccines on the pathogenesis of aiv-h9n2 infection. for ibv hemagglutination inhibition (hi) test; purified, concentrated and phospholipase c type 1 enzyme treated variant (ibv-eg/ 11539f-201) and classical mass-like strain (ibv-eg/mevac01/201) strains with their positive control antisera were kindly provided by middle east for veterinary vaccines (me vac) co. research and development department. all experiments were conducted according to animal research ethics guidelines at the faculty of veterinary medicine, beni-suef university, egypt. one hundred and five-day-old commercial broilers were obtained from a private broiler breeder flock located at el-badrashin, giza, egypt. chicks were divided into 7 groups (15 chicks/ group) in separate isolated experimental. birds were reared till 20th day old provided with feed and water ad libitum and vaccinated against aiv-h5n1, newcastle disease virus (at 2 and 9 days old using bivalent mefluvac h5nd®, middle east for veterinary vaccines co., egypt) and ibdv (at 14 days old using bursa-vac®, intervet schering-plough animal health). experimental groups included; negative control group, variant ibv strain infected group, classical ibv strain infected group, aiv-h9n2 infected group, mixed aiv-h9n2/ibv variant strain infected group, mixed aiv-h9n2n2/ibv classical strain infected group, and mixed aiv-h9n2/ibv 4/91 vaccine strain infected group. blood samples were collected before challenge for hemagglutination inhibition test (hi) for aiv-h9n2n2 and agar precipitation test (agpt) for ibv. birds were challenged using intranasal and/or intra-tracheal inoculation of 10 6 eid 50 /0.5 ml of titrated viruses for aiv-h9n2n2 and ibv viruses, respectively. in the mixed aiv-h9n2n2/ibv 4/91 vaccine strain infected group, the ibv 4/91 vaccine was administered via eye drop according to the manufacturer instructions in terms of dose and route of administration. mixed infection groups were challenged simultaneously. serum samples were collected at 20-day old for aiv-h9n2 and ibv serology testing before the challenge. tracheal swabs were collected from experimentally infected chicks for virus's detection at 2, 5 and 7 dpi. at the same time points, 3 chicks from each group were euthanized and organs (trachea, thymus, and kidney) were collected for histopathological examination (bancroft and gamble, 2008) . swab samples were suspended in 2 ml sterile phosphate buffer saline (ph 7.2) and vortexed. sera were collected from the remaining chicks at 10 dpi for determination of the serum antibody titer against aiv-h9n2 using hi test (oie, 2014) . the ibv hi tests were conducted as previously described (king and hopkins, 1983) with minor modifications. briefly, both tests were done in u-shaped microtiter plates. serum samples were kaolintreated before testing (king and hopkins, 1983) . serial twofold dilutions of treated serum in pbs were done and to each serum dilution 25 μl of the diluted classical and variant ibv ha antigen (8 ha units/ 25 μl) was added separately. the plates were incubated at room temperature for 20 min then 25 μl of 0.5% chicken rbc's were added to each well, and the plate were mixed and incubated for 40 min at room temperature. the hi titer of a sample was calculated as the reciprocal of the last serum dilution with no ha. negative control serum and antisera against both classical and variant ibv antigen viruses were included in the test. the viral rna was extracted by bioflux® viral rna mini spin column kit (bioflux, china) in accordance with manufacturer's instructions. verso 1-step qrt-pcr kit (thermo scientific, usa) was used for detection and quantification of aiv-h9n2 (ben shabat et al., 2010) and s1 gene of ibv (callison et al., 2006) . the qrt-pcr reaction volume was 25 μl containing 5 μl of extracted rna, 12.5 μl 2× one-step rt-pcr ready mix, 1.25 μl rt enhancer, 0.25 μl verso enzyme mix, 1 μl of 20 pmol of both forward and reverse primers, 0.25 μl of virus specific probe and 3.75 nuclease free water. the thermal profile included a reverse transcription step at 50°c for 15 min followed by 15 min at 95°c. the pcr cycling was 40 cycles of denaturation at 95°c for 15 s, annealing at 60°c at 60 s, and a final extension at 72°c for 10 min. to determine aiv-h9n2 and ibv viruses shedding titers, a standard curve for each virus was generated using titrated viruses in spf-ece and shedding titers were determined using interpolation (lee and suarez, 2004) . the differences in hi antibody titers and virus shedding titers were estimated using one-way anova with tukey's post-test through graphpad prism version 5.00 for windows (graphpad software, san diego california usa, www.graphpad.com). experimentally infected broilers were observed for clinical signs till 10 dpi. clinical signs varied from mild signs like conjunctivitis, sneezing coughing and head shaking to severe respiratory signs represented in rales and mouth breathing especially with aiv-h9n2 challenged group at 5 dpi, and in mixed aiv-h9n2/ibv vaccine and mixed aiv-h9n2/variant ibv challenged groups at 7 dpi (table 1) . mortality was recorded only in mixed aiv-h9n2/variant ibv challenged group at 9 and 10 dpi (13.3%). gross lesions were variable with slight airsaculitis in all groups. slight mucous was observed in the trachea in aiv-h9n2 and classical ibv single infections, petechial hemorrhage in the trachea of all aiv-h9n2 challenged groups at 2 and 5 dpi. by 7 dpi mucoid plugs at the tracheal bifurcation in single aiv-h9 and mixed aiv-h9n2/variant ibv infected groups were observed ( supplementary fig. 1) . histopathologically, the trachea in aiv-h9n2 single infection showed congestion of blood vessels in the submucosa, deciliation, and mononuclear cell infiltration. variant and classic ibv single infection caused mild degeneration and necrosis of the tracheal mucosal epithelium, mild edema in the submucosa, deciliation and mononuclear cell infiltration. in aiv-h9n2 and ibv mixed infection including the 4/ 91 strain, severe degeneration, and necrosis of the mucosal epithelium, severe congestion of blood vessels with edema in the submucosa, marked deciliation associated with massive lymphocytic cell infiltrations ( supplementary fig. 2) . thymus congestion and petechial hemorrhage were associated with aiv-h9n2 infection as it was only seen in aiv-h9n2 single infection and with variable degrees of severity in mixed aiv-h9n2/ibv infection. thymus atrophy was observed in aiv-h9n2 single infection by 7 dpi (fig. 1) . the negative control, variant and classical ibv challenged groups showed normal thymus histology, while in aiv-h9n2 single infection and aiv-h9n2 with classical, variant ibv or vaccine ibv strain mixed infection the thymus was severely affected and showed congestion and hemorrhage. in some areas, the blood vessels were ruptured leaving blood-filled spaces. later, the thymus appeared slightly or moderately atrophied (fig. 2) . kidney histopathological examination revealed that kidneys were mostly affected in aiv-h9n2 with variant ibv and in aiv-h9n2 with ibv vaccine strain challenged groups with congested blood vessels, mild hemorrhage, massive interstitial infiltration of leucocytes and tubular degenerative changes. interstitial nephritis was frequently seen in some birds. swelling and hypercellularity of the glomeruli were commonly observed (fig. 3) . the shedding titers of aiv-h9n2 virus single infection decreased by 7 dpi from 6.2 to 4.5 log 10 , meanwhile in aiv-h9n2 with ibv mixed infection groups the shedding titer of aiv-h9n2 virus was significantly higher than the aiv-h9n2 single infection group. similarly, the classical ibv shedding titers in the mixed infection group were significantly higher than those of the classical ibv single infection. on the other hand, variant ibv shedding titers did not show significant differences between either single or combined infection (fig. 4) . pre-challenge aiv-h9n2 and ibv hi antibody titers (20 day-old) confirmed the absence of previous exposure before the challenge. at 10 days post challenge, aiv-h9n2 challenged groups (either single or mixed with ibv) showed high hi antibody titers with the most significant increase in aiv-h9n2 plus classical ibv infection. however, no variation between the titers in the single aiv-h9n2 infected group and the titers in mixed aiv-h9n2/variant ibv and aiv-h9n2/vaccine ibv strain (table 2) . non-significant slight increase in ibv post challenge antibody titers against both classical and variant ibv antigens in aiv-h9n2 and ibv combined infections (table). disease outbreaks with variable mortality rates and different clinical manifestations have been increased in egyptian commercial chicken flocks with respiratory affections being the most common complaint (hassan et al., 2016) . respiratory affections represent a great problem to the poultry industry because of their multifactorial nature (bano et al., 2003; roussan et al., 2008) . studies showed that mixed infection, especially with ib and aiv-h9n2 viruses, was the most common condition in the egyptian poultry (hassan et al., 2016) . in this study, clinical manifestations were limited to respiratory signs in all challenged groups under experimental conditions. mild clinical signs were observed in single infection while severe respiratory distresses were common with mixed infection. similar results were previously reported (monne et al., 2013; nili and asasi, 2002) with a comparable mortality rate of 10% in mixed aiv-h9n2 and ibv infection. unlike what is known about low virulence nature of h9n2 subtype, the role of co-infection with other pathogens including ibv, staphylococcus aureus or haemophilus paragallinarum (kishida et al., 2004) , and escherichia coli (bano et al., 2003) in exacerbating h9n2 virus infection in chickens is of major significance. such exacerbation possibly occurs by secretion of trypsin-like proteases by bacterial stimulation of host cells to produce or secrete more protease, or destruction of endogenous cell protease inhibitors (mancini et al., 2005) and suppression of the immune system due to stress (kishida et al., 2004) . petechial hemorrhage at trachea was constantly observed and progressed to congestion and trachitis with the presence of mucoid plug at the tracheal bifurcation in group aiv-h9n2 and in aiv-h9n2 plus ibv vaccine challenge. the mucoid plug was previously reported in aiv-h9n2 single infection (capua and marangon, 2000; hooper and selleck, 1998; nili and asasi, 2002; perk et al., 2006) . this finding further support the hypothesis that ibv, even vaccine strains play a role in increasing severity of h9n2 infection (cook et al., 1976; haghighat-jahromi et al., 2008; nili and asasi, 2002) possibly through impairment of clearance of bacterial pathogens in the respiratory tract of broilers (dwars et al., 2009) . histopathologically, trachea in all mixed infection groups including the aiv-h9n2 with vaccine ibv strain showed severe changes comparable to the milder changes in single classical or variant ibv challenged groups (purcell et al., 1976) . in aiv-h9n2 single infection, congestion is the most predominant lesion with deciliation and leucocytic infiltration, that may explain the formation of mucoid plug by secondary bacterial infections (nili and asasi, 2002) . remarkably, thymus of groups single aiv-h9n2 and aiv-h9n2 plus ibv was greatly affected with severe petechial hemorrhage followed by atrophy as compared to the negative control and single ibv infection groups. in chickens, the presence of aiv-h9n2 infected lymphocytes, during virus dissemination throughout the body via blood or lymph vessels, may explain the thymus pathological lesions (kwon et al., 2008) . however, secondary infection and/or stress due to endogenous glucocorticoid secretion or from the production of specific cytokines cannot be neglected. it was suggested that the immunosuppression predisposing to secondary bacterial infections and subsequent high mortality in field situations in aiv-h9n2 infection could be explained by the atrophy and lymphoid depletion of thymus and probably some other lymphoid organs (hadipour et al., 2011) . k.e. hassan et al. research in veterinary science 115 (2017) 356-362 similar virus tracheal shedding titers were detected in the variant ibv infected birds either single or mixed infections with aiv-h9n2, however, in the current study, the classical ibv shedding significantly increased with aiv-h9n2 mixed infection. elevated virus shedding titers of aiv-h9n2 in presence of ibv infection was previously reported (monne et al., 2013) . similarly, in this study, significant increases of aiv-h9n2 virus shedding with classical ibv infection especially at 2 and 5 dpi and with the variant and vaccine ibv strains co-infections up to 7 dpi were observed. however, the only significant elevation of aiv-h9n2 hi antibody titers was recorded with the classical ibv co-infection at 10 dpi. this may be attributed to the earlier and higher replication of both aiv-h9n2 and classical ibv viruses as indicated by higher viruses shedding titers as early as 2 dpi. these results and previous studies results indicate the possible synergistic mechanism between ibv and aiv-h9n2 possibly by trypsin-like proteases encoded by coronaviruses that enhance the aiv-h9n2 hemagglutinin cleavage (haghighat-jahromi et al., 2008; klenk and garten, 1994; liu et al., 1995; ng and liu, 2000; perk et al., 2004) . the current study results indicate that the high mortalities observed in aiv-h9n2 under field conditions in egypt might be partly attributed to mixed infections with widely spread different ibv strains. further research is needed to elucidate the pathobiological interactions between aiv-h9n2 and ibv viruses in terms of increasing virus replication and pathogenicity. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.rvsc.2017.06.024. the authors would like to thank d. manal a. afifi for providing classical ibv isolate. the authors also thank research and development team at the middle east for veterinary vaccines (me vac) co. for providing purified enzyme treated ibv strains and antisera. this study was supported by the project support and financing unit at beni-suef university, egypt (project title: advanced studies on viral respiratory fig. 4 . the aiv-h9n2 (a) and ibv (b) viruses shedding titers in experimentally infected broilers in both single aiv-h9n2 and aiv-h9n2 with ibv mixed infections. post-challenge log 2 hemagglutination inhibition antibody titers of experimentally infected groups using aiv-h9n2, classical and variant ibv antigens. hi antigen used aiv-h9n2 ag ibv classical ag negative control 0.8 ± 0.8 0.7 ± 0.8 1.1 ± 0.7 variant ibv 0.7 ± 0.9 7.4 ± 0.5 8.4 ± 0.5 classical ibv 0.1 ± 0.4 8.7 ± 0.5 6.6 ± 0.5 aiv-h9n2 4.5 ± 1.6 0.3 ± 0.5 0.6 ± 0.5 aiv-h9n2 + variant ibv 4.6 ± 1.0 7.9 ± 0.4 8.9 ± 0.4 aiv-h9n2 + classical ibv 6.7 ± 0.9 a 8.9 ± 0.4 6.9 ± 0.7 aiv-h9n2 + 4/91 ibv vaccine 4.5 ± 1.4 7.0 ± 0.8 8.7 ± 0.5 a significant difference (p ≤ 0.05). 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viruses during the respiratory disease outbreak in commercial broiler farms of iran coinfection of avian influenza virus (h9n2 subtype) with infectious bronchitis live vaccine prevalence of avian respiratory viruses in broiler flocks in egypt pathology of low and high virulent influenza virus infections risk factors for detection of bronchial casts, most frequently seen in endemic h9n2 avian influenza infection, in poultry flocks in iran evaluation of the hemagglutination-inhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination co-infection of staphyloccocus aureus or haemophilus paragallinarum exacerbates h9n2 influenza a virus infection in chickens host cell proteases controlling virus pathogenicity immune responses and pathogenesis in immunocompromised chickens in response to infection with the h9n2 low pathogenic avian influenza virus application of real-time rt-pcr for the quantitation and competitive replication study of h5 and h7 subtype avian influenza virus identification of a trypsin-like serine proteinase domain encoded by orf 1a of the coronavirus ibv coinfection between influenza virus and flagellated bacteria h9n2 influenza a virus circulates in h5n1 endemically infected poultry population in egypt further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site natural cases and an experimental study of h9n2 avian influenza in commercial broiler chickens of iran avian influenza h9n2 outbreak in iran avian influenza, in manual of diagnostic tests and vaccines for terrestrial animals low pathogenicity avian influenza h9n2 in israel a threat to the poultry industry genetic characterization of the h9n2 influenza viruses circulated in the poultry population in israel the histopathology of infectiousbronchitis in fowls infected with a nephrotropic tstrain of virus molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in jordan natural co-infection caused by avian influenza h9 subtype and infectious bronchitis viruses in broiler chicken farms key: cord-286658-9kco7qad authors: jiang, lei; han, zongxi; chen, yuqiu; zhao, wenjun; sun, junfeng; zhao, yan; liu, shengwang title: characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/ch/ljl/140901-like backbone and an s2 fragment from a 4/91-like virus date: 2018-01-15 journal: virus res doi: 10.1016/j.virusres.2017.11.007 sha: doc_id: 286658 cord_uid: 9kco7qad in this study, we isolated an infectious bronchitis virus, designated i1101/16, from broiler breeders in china. analysis of the s1 gene showed that isolate i1101/16 was genetically close to strain ck/ch/ljl/140901, which belongs to the tw i genotype (also known as lineage gi-7 based on the recent ibv classification), however the s2 gene showed genetic diversity comparing to that of s1 gene. comparison of the genomic sequences showed that the genome of isolate i1101/16 was similar to that of strain ck/ch/ljl/140901 from the 5′ end of the genome to the 5′ end of the s2 gene and from the 5′ end of the 3a gene to the end of the genome, whereas the remaining parts of the genome sequences were more closely related to those of strain 4/91 than those of ck/ch/ljl/140901, thereby suggesting that recombination might have occurred during the origin of the virus. simplot and bootscan analysis of the complete genomic sequence confirmed this hypothesis, where it showed that isolate i1101/16 arose through recombination events between ck/ch/ljl/140901and 4/91-like viruses. isolate i1101/16 and strain ck/ch/ljl/140901 shared identical amino acids in almost all five of their b cell epitopes, but the two viruses had a serotype relatedness value of 65, which is well below 80, i.e., the lower cutoff value for viruses of the same serotype. in addition, pathogenicity tests demonstrated that isolate i1101/16 was more pathogenic to 1-day-old specific-pathogen-free chickens than strain ck/ch/ljl/140901, according to analysis of the clinical signs, whereas strain ck/ch/ljl/140901 exhibited prolonged replication and shedding after challenge compared with isolate i1101/16. this study did not provide evidence that recombination can directly alter the antigenicity, virulence, replication, shedding, and tissue tropism of a virus, but it did show that recombination events are likely to be major determinants of viral evolution. avian infectious bronchitis virus (ibv), the prototype gammacoronavirus species in the family coronaviridae, is a positivesense, single-stranded rna virus with a genome of approximately 27.6 kb (5′-untranslated region (utr)-1a/1ab-s-3a-3b-e-m-5a-5b-n-3′-utr). it causes an acute and highly contagious disease in chickens, which is responsible for high economic losses in the poultry industry. the current control strategies are based mainly on mass vaccination strategies. nevertheless, vaccine-induced immunity generally give poor protection because the current vaccine offers only limited cross protection among strains (cook et al., 2012; de wit et al., 2011) mainly due to the antigenic diversity caused by the variability of the s1 protein. recently, numerous ibv strains have been identified and new genotypes/serotypes have emerged from existing viruses via point mutations, insertions, and deletions in the viral genome, especially in the s1 subunit of the spike protein gene. at least six ibv genotypes together comprise 34 distinct viral lineages and a number of unassigned interlineage recombinants have been recognized worldwide according to a simple and repeatable phylogeny-based classification system that uses the complete nucleotide sequence of the s1 gene and an unambiguous and rationale lineage nomenclature for the assignment of ibvs (valastro et al., 2016; chen et al., 2017; jiang et al., 2017) . there is also considerable variation in the virulence and tropism of ibvs, and in some cases the novel ibv strains emerged from point mutations, insertions, and/or deletions in the s1 gene. another important mechanism that underlies the emergence of novel ibv strains is genomic recombination. in australia, three recently isolated novel subgroup ibv strains were shown to be derived from recombination between subgroup 1 and 2 strains (mardani et al., 2010) . more recently, the complete genome analysis of newly emerged strains found multiple recombination events throughout the genome between wild-type viruses and vaccine strains (quinteros et al., 2016) . in the usa, the emergent virulent strain ark dpi appears to have originated from recombination among four different ibv strains (ammayappan et al., 2008) . in addition, the emergence of new strains in the usa has been reported to have arisen from recombination events between the massachusetts (mass), connecticut (conn), and holte strains as well as field strains (thor et al., 2011) . recently in europe, it has been shown that emergent viruses, such as the xdn-like virus in spain and italy (moreno et al., 2016) , and the γcov/ck/italy/i2022/13 virus in italy (franzo et al., 2015) , may have arisen from recombination events. there have been several episodes of infectious bronchitis (ib) in chinese chicken flocks, and the genotypes/serotypes of ibvs were previously classified based mainly on the nucleotide sequences of genes encoding the s1 subunit of the spike protein (han et al., 2011) , and in some cases based on cross virus-neutralization chen et al., 2017) in china. since 1995, the predominant ibv type in china has been lx4 (also known as qx-like viruses), but molecular studies have shown that new types and variants are emerging continually mo et al., 2013; zhao et al., 2013; liu et al., 2014; zhou et al., 2014; chen et al., 2015; xu et al., 2016; zhang et al., 2015; leghari et al., 2016; chen et al., 2017; zhao et al., 2017; zhou et al., 2017) . it has been suggested that the emergent ibv strains in china may have different origins. it is considered that some of the ibv types circulating in china, such as mass and 793/b, are the most widely distributed types worldwide and they may have infected chinese chickens from an exogenous source, probably due to the use of live vaccines (chen et al., 2015; han et al., 2017) . by contrast, some ibv types, such as lx4 and ck/ch/ldl/97i (q1-like), are believed to have originated in china and spread to other regions of the world (valastro et al., 2016) . the remaining ibv types, such as ck/ch/lsc/99i (liu et al., 2006a,b) , nrtw i , and gi-28 , are considered to be indigenous to china. the origins of most of the ibv types in china are still unknown, although it has been shown that some of these types have arisen from recombination events (chen et al., 2015; xu et al., 2016; chen et al., 2017) . the results of many of these previous studies are based on analyses of the available s1 gene sequences, but it is impossible to fully understand the origins and evolutionary processes related to these emerging viruses by only analyzing this small part of the genome. in this study, we isolated and identified an nrtw i type ibv from an h120 and 4/91 vaccinated chicken flock with respiratory signs of ib. we sequenced the complete genome and compared it with other ibv sequences available in genbank, and we then subjected these sequences to phylogenetic, molecular, and recombination analyses. we also investigated the antigenicity, pathogenicity, replication, and shedding of the nrtw i type ibv in chickens. trachea samples were collected from five suspected ibv-infected broiler breeders and they were submitted to our laboratory in 2016 for routine diagnosis. chickens in this flock had been vaccinated against ibv using the live attenuated h120 vaccine at 7 days of age, and the chickens were then boosted at 25 and 60 days with the live attenuated 4/91 and h120 vaccines, respectively. the birds were also vaccinated with a bivalent, inactivated vaccine, newcastle disease virus la sota/ ibv m41, at 120 days. some of the birds exhibited respiratory signs of ib at 150 days, which was accompanied by decreased egg production and abnormal shell quality in the breeding hens, and the clinical signs disappeared approximately 20 days later. the morbidity was approximately 5% and only a few birds died during this outbreak. gross lesions were mainly associated with tracheitis and proventriculitis, and hepatitis and hydropericardium were observed in some chickens. kidney lesions were not observed in the chickens examined. to isolate the virus, the tracheal samples from the dead chickens were pooled and inoculated into 9-day-old specific pathogen-free (spf) embryonated chicken eggs via the allantoic sac route, as described previously . the virus recovered from the third passage was named γcov/ck/china/i1101/16 (i1101/16) (ducatez and the european unit cost action fa1207, 2016) and it was used in the following experiments. the virus stock was prepared after an additional passage by inoculating the allantoic cavity of 9-day-old embryonated spf chicken eggs. after incubation for 48 h, the allantoic fluid was collected aseptically and clarified by centrifugation at 2500 × g for 20 min, as described previously . fertile white leghorn spf chicken eggs and white leghorn spf chicks were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. the birds were kept in isolation units with negative pressure throughout the experiment, and water and feed were provided ad libitum. the complete genomes of the i1101/16 isolate and the ck/ch/lsc/ 99i strain were sequenced in this study. ibv strain ck/ch/lsc99i was isolated from preventriculus of a layer hen in sichuan province in china in 1999 (liu et al., 2006a,b) . genomic rna was extracted from virusinoculated allantoic fluids using trizol reagent (invitrogen corporation, carlsbad, ca, usa) according to the manufacturer's instructions. first-strand cdna synthesis and subsequent pcr were performed using a primescript™ one-step rt-pcr kit ver. 2 (takara bio inc., shiga, japan) according to the manufacturer's instructions. the complete genomes of the i1101/16 isolate and ck/ch/lsc/99i strain were amplified with primers used for amplifying the complete genomes of other chinese ibv strains . the pcr profiles comprised initial denaturation for 5 min at 94°c, followed by 30 cycles at 94°c for 30 s, 50-60°c (depending on the primer set) for 30 s, and 72°c for 2 min. a 3′/5′ rapid amplification of cdna ends kit (takara bio inc.) was used to determine the 3′ and 5′ ends of the viral genomes according to the manufacturer's instructions . the amplified products were sequenced directly or cloned into the pmd18-t vector (takara bio inc.) according to the manufacturer's instructions. each fragment of the viral genome was sequenced at least three times. the complete genomic sequences of the i1101/16 isolate and the ck/ch/lsc/99i strain were mapped using the genome sequences of the beaudette and ck/ch/ljl/140901 ibv strains, respectively, in order to determine a consensus sequence. 2.4. comparison and analysis of the s1 subunit of the spike and spike protein genes the region coding for the s1 subunit of the spike protein is used for genotyping and classification purposes. comparisons with published sequences were first performed using the nucleotide blast search tool in genbank with the s1 gene nucleotide sequence from the i1101/16 isolate. the s1 nucleotide sequences from 70 ibv reference strains were downloaded from genbank, where most of these ibvs were isolated in china and they represented the previously reported types (valastro et al., 2016; chen et al., 2017) . phylogenetic analyses were conducted based on the s1 gene nucleotide sequences using the neighbor-joining method (jukes-cantor model) with 1000 bootstrap replicates (mega software version 5.0; available at http://www.megasoftware.net/). to analyze the genetic diversity of the i1101/16 isolate, the percentages of similarity were calculated for both the s1 subunit and the entire spike protein at the nucleotide and amino acid levels using the i1101/16 isolate and seven reference strains, which were selected based on the phylogenetic analysis results. the seven reference strains included two vaccine strains, h120 and 4/91, which are used commonly in chicken flocks in china and they represented two genotypes, and five ibv field strains that circulate in chicken flocks in china, where they represented different genotypes (i.e., ck/ch/ljl/140901 [the nrtw i type] , ck/ch/ldl/091022 [the lx4 type] , ck/ch/lgx/111119 [the gi-28 type] , ck/ ch/lsc/99i [the ck/ch/lsc/99i type] (liu et al., 2006a,b) , and ck/ ch/ldl/97i [the ck/ch/ldl/97i type] (liu et al., 2006a) ). in addition, we compared the amino acid sequences of the five conformationdependent neutralizing antigenic sites (epitopes) mapped to the s1 subunit of the spike protein as well as another immunodominant region in the amino-terminal region of s2 lenstra et al., 1989; koch et al., 1990 ) in a pairwise manner in isolate i1101/16 and the seven reference strains. multiple sequence alignment of the complete genome sequences of the i1101/16 isolate and the reference strains was performed with multiple alignment with fast fourier transformation (mafft) v6 (http://mafft.cbrc.jp/alignment/software/). the alignment included seven sequences from the strains mentioned above. to refine the recombination event pattern analysis, similarity plots (simplots) and bootscan analysis were used to evaluate the possible recombinant nature of the isolate using the on-line multiple sequence local alignment (mulan) tool (ovcharenko et al., 2005) with the complete genome sequences of the ck/ch/ljl/140901 and 4/91 strains, where we used a window size of 500 bp and a step size of 20 bp. the analyses were performed using simplot version 3.5.1 (lole et al., 1999) where the complete genome sequence of the ck/ch/lsd/110851 strain (accession number kp118884) was used as the query because it was a representative of 4/91 vaccine sequence and supposed parental recombinant sequence of strain i1101/16. the deduced sequences of the recombination breakpoints and flanking sequences of the i1101/16, ck/ch/ljl/140901, and 4/91 viruses were compared in a pairwise manner to accurately detect the positions of the recombination breakpoints. in addition, we performed pairwise comparisons of the nucleotide sequences of the s2 subunit of the spike gene between i1101/16 isolate, and 4/91 vaccine (genbank accession kf377577) and a pathogenic 4/91 strain (genbank accession jn192154), in order to identify which was the possible parental virus strain of i1101/16 isolate. the ibv i1101/16 isolate and the seven reference ibv strains mentioned above were used in virus-neutralization tests. stocks of these viruses were prepared by passaging them in the allantoic cavity of 9day-old embryonated spf chicken eggs. viral titers were determined by inoculating 10-fold dilutions into groups of five 9-day-old embryonated chicken eggs and the 50% embryo infectious dose (eid 50 ) was calculated using the method of reed and muench (1938) . serotype-specific antisera against all the viruses listed in table 1 were prepared in chickens, and cross virus-neutralization tests were conducted in 9-day-old spf chicken eggs as described previously (jackwood et al., 2007) . the β virus neutralization method with constant virus and diluted serum was employed. titers were calculated using the method of reed and muench (1938) , and relatedness values were calculated using the method of archetti and horsfall (1950) . viruses with an archetti and horsfall relatedness value greater than 50 were considered to be related serotypes. ethical approval for the animal experiments was granted by the ethical and animal welfare committee of the harbin veterinary research institute, china. forty-five 1-day-old spf layer chickens were assigned randomly to three groups each containing 15 birds. the i1101/16 isolate and the ck/ch/ljl/140901 strain were administered by eyedrop inoculation to 1-day-old birds in groups 1 and 2, respectively, at a dosage of 1 × 10 5.5 eid 50 in a 0.1-ml volume, and the remaining groups served as negative controls. five days after challenge all chickens were evaluated blindly for respiratory rales, such as nasal and/or tracheal, by closely listening to each bird. signs were scored as 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). at 5 days post-challenge, five birds from each group were killed and necropsied, and the trachea and kidneys were collected. measurements of infectivity and viral replication levels in the chicks were performed by determining the viral titers in 9-day-old spf embryonated chicken eggs. in addition, the ibv antigen was detected by immunohistochemistry using monoclonal antibody 6d10 . blood samples and nasopharyngeal and cloacal swabs were collected from the birds in each of the groups on days 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 , and 44 post-challenge. the blood samples were used to detect antibodies against ibv with a commercial enzyme-linked immunosorbent assay (idexx corporation, westbrook, me, usa) according to the manufacturer's instructions . the swabs were placed in individual tubes containing sterile phosphate-buffered saline (ph 7.4) with antibiotics and stored at −80°c for virus recovery, as described previously . the chickens were observed for disease symptoms, death, and kidney lesions for 45 days. the s1 gene phylogenetic tree contained seven clusters (fig. 1) , which corresponded to the seven types of ibv, i.e., the gi-28 , lx4 (qx or gi-19) , ck/ch/lsc/99i (gi-22) (liu et al., 2006a,b) , 793/b (gi-13) , ck/ch/ ldl/97i (q1 or gi-16) (liu et al., 2006a) , mass (gi-1), and tw i (gi-7) types . among these, two vaccine strains, h120 and 4/ 91, are used commonly in china and they belong to the mass (gi-1) and 793/b (gi-13) types. the remaining five ibv types are field strains that circulate in chicken flocks in china. the i1101/16 isolate clustered into the tw i group (gi-7), which was genetically close to the ck/ch/ljl/ 140901 strain and slightly distant from other strains in the tw i group. in agreement with the results of the phylogenetic analysis, the i1101/16 isolate shared the highest genetic similarity (95.7% and 95.4% at the nucleotide and amino acid levels, respectively) with the ck/ch/ljl/140901 stain. by contrast, the i1101/16 isolate shared low genetic similarity with the other types of viruses, with nucleotide and amino acid identities ranging among 76.1-80.7% and 76.7-80.6%, respectively. similarly, the i1101/16 isolate also shared the highest similarity with the ck/ch/ljl/140901 strain at both the nucleotide (90.9%) and amino acid (93.5%) levels based on the spike protein gene, whereas it shared no more than 90% similarity with the other strains at either the nucleotide or amino acid levels. interestingly, the genetic similarities between the i1101/16 isolate and the ck/ch/ljl/140901 strain based on the s1 gene were obviously higher than those based on the spike protein gene, which is the opposite result compared with those obtained between the i1101/16 isolate and most of the reference strains (table 1) . pairwise comparisons were performed of the amino acid sequences of b cell epitopes d (residues 24-61), e (residues 132-149), c/a/b (residues 291-398), and f (residues 497-543) in the s1 subunit and the g region (residues 548-574) of the s2 subunit of the spike protein, and the results are summarized in fig. 2 . the i1101/16 isolate shared similar amino acids in all epitopes with the ck/ch/ljl/140901 strain, whereas it exhibited amino acid differences compared with the ck/ch/ lgx/111119, ck/ch/lsc/99i, ck/ch/ldl/97i, conn, 4/91, and h120 strains. the i1101/16 isolate shared similar amino acids with the ck/ ch/ldl/091022 strain in the f and g epitopes, but the amino acid residues differed in the d, e, and c/a/b epitopes of these strains. the genome sequences of the i1101/16 isolate and the ck/ch/lsc/ 99i strain were determined and deposited in genbank under accession numbers ky620116 and ky799582, respectively. the genomes of the viruses comprised 27,674 bp and 27,665 bp, respectively, excluding the poly (a) tail. the complete genome sequences of the i1101/16 isolate and the ck/ch/lsc/99i strain had the typical ibv gene order of 5′-utr-1a-1b-s-3a-3b-e-m-5a-5b-n-3′-utr, and the gene lengths are shown in after comparing the genomic sequence of the i1101/16 isolate with those of the reference strains using the mafft alignment, we found that the genome of the i1101/16 isolate was similar to that of the ck/ ch/ljl/140901 strain from the 5′ end of the genome to the 5′ end of the s2 gene, whereas the sequences differed significantly from that of the ck/ch/ljl/140901 strain from the 5′ end of the s2 gene to the end of the s2 gene. in the i1101/16 isolate, this region was most closely related to the 4/91 strain. interestingly, the sequence of the i1101/16 isolate was more closely related to that of the ck/ch/ljl/140901 strain than that of the 4/91 strain from the 5′ end of the 3a gene to the end of the genome (fig. 3) . simplot and bootscan analyses also confirmed these results (fig. 4) (fig. 5 ). in addition, 12 nucleotides differences were identified in the s2 subunit of the spike protein gene of isolate i1101/16 and the two 4/91 strains; pairwise comparisons demonstrated that isolate i1101/16 shared seven nucleotide identities with the 4/91 vaccine strain and only two with the pathogenic 4/91 strain (table 3) , thereby indicating that this region of the i1101/16 isolate is more similar to that of 4/91 vaccine strain than that of the pathogenic strain. based on the method of archetti and horsfall (1950) , serotype-relatedness values were calculated in cross virus-neutralization studies using embryonating chicken eggs (table 1) , which showed the i1101/ 16 isolate and the ck/ch/ljl/140901 strain were related serotypes with a serotype relatedness value of 65. none of the other ibv strains tested in this study were related serotypically to isolate i1101/16. the pathogenicity of the i1101/16 isolate was tested in 1-day-old spf chickens and compared with that of the ck/ch/ljl/140901 strain, and the results are presented in table 4 . at 5 days post-challenge, clinical signs were observed in 2/10 birds and 1/10 birds inoculated with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. at 11 days post-challenge, 10/10 birds inoculated with the i1101/16 isolate had severe clinical signs including tracheal rales, watery eyes, nasal mucus, and occasional sneezing, whereas 9/10 birds inoculated with the ck/ch/ljl/140901 strain had only mild clinical signs. the mean scores of the clinical signs for the birds challenged with the i1101/16 isolate and the ck/ch/ljl/140901 strain were listed in table 4 . no birds died in any of the groups during the experiments. no clinical signs were observed in the non-challenged birds. at 8 days post-challenge, antibodies against ibv were detectable by an enzyme-linked immunosorbent assay in seven and four birds challenged with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. antibodies were detected in all the challenged birds at 12 days post-challenge. antibodies were not detected in the non-challenged birds. table 1 antigenic relatedness a and s1/s gene similarities between the i1101/16 isolate and seven reference strains. percentage of relatedness/s1 or s similarities archetti and horsfall (1950) with neutralization indices derived from the results of reciprocal virus-neutralization tests performed in 9-day-old embryonated eggs. b the percentages of relatedness for the first 1665 nucleotides starting at the aug translation start codon in the s1 protein genes between the i1101/16 isolate and seven ibv reference strains. c the percentages of relatedness for the amino acid of the s1 subunit in the spike protein between the i1101/16 isolate and seven ibv reference strains. d the percentages of relatedness of the nucleotides encoding the spike protein between the i1101/16 isolated and the seven ibv reference strains. e the percentages of relatedness of amino acids in the spike protein between the i1101/16 isolate and seven ibv reference strains. as illustrated in fig. 6 , replication of the ck/ch/ljl/140901 strain was detected in the trachea and kidneys of chickens, with titers of 3.83-4.83 log 10 eid 50 and 3.5-4 log 10 eid 50 , respectively. by contrast, replication of the i1101/16 isolate was detected in the trachea and kidneys, with titers of 1.7-2.83 log 10 eid 50 and 1.38-1.68 log 10 eid 50 , respectively. in addition, viral antigens were detected by fig. 1 . phylogenetic tree based on the s1 nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of 71 ibv strains, including the i110116 isolate and 70 ibv reference strains representing seven genotypes from china. bars denoting the genotype and lineage designations are shown. the ibv strain and genbank accession number are given for each strain. the scale bar represents the number of nucleotide substitutions per site. the ibv strains used for further comparisons and virus-neutralization testing are highlighted in bold. immunohistochemistry in the trachea and kidneys of the birds challenged with both viruses (fig. 7) . no virus was detected in the trachea and kidneys of birds in the control group. shedding of the challenge viruses in the respiratory and digestive tracts was determined by virus recovery using nasopharyngeal and cloacal swabs from 9-day-old spf chicken eggs. as illustrated in table 4 , the challenge viruses were recovered from the nasopharyngeal swabs of all the birds at 4 days post-challenge, whereas viruses were not detectable in some of the birds at 8 and 12 days post-challenge with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. viral shedding was not detectable in the nasopharyngeal swabs of all the birds at 24 days post-challenge with the ck/ch/ljl/140901 strain. by contrast, viral shedding was still detectable at 28 days post-challenge in the nasopharyngeal swabs from one bird challenged with the i1101/16 isolate. based on the cloacal swabs, viral shedding from some of the birds lasted until 40 and 28 days post-challenge with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. virus was not recovered from the non-challenged control birds. ib is among the most important diseases in both vaccinated and non-vaccinated chickens in china, and it has been monitored continuously in chicken flocks since it was first identified. an outbreak possibly caused by a respiratory pathogen occurred at the end of 2016 in a broiler breeder flock in neimenggu province in china, and only a very small number of diseased chickens died during the outbreak. however, the egg production decreased and the egg quality was adversely affected. the main lesions were tracheitis and proventriculitis at the early stage of the outbreak, and severe bacterial infections were observed at the late stage, but no obvious kidney lesions were observed in the dead chickens throughout the outbreak. an ibv strain designated as i1101/16 was isolated from the tracheas of the dead chickens. both the ib live vaccines h120 and 4/91 as well as an inactivated vaccine were used to vaccinate the chicken flock before the occurrence of the ib outbreak, so we initially sequenced the s1 gene to identify, differentiate, and genotype the ibv isolate. based on the s1 gene analysis, the i1101/16 isolate clustered most closely with reference strains of the ntw i type , and it has been diverging as an independent genetic clade with the ck/ch/ljl/140901 strain (fig. 1) . our analysis showed that the i1101/16 isolate and the ck/ch/ljl/ 140901 strain were more closely related serologically compared with other ibv serotypes (table 1) , but the serotype relatedness value was only 65 between these strains, which is greater than 50% but well below 80%, i.e., the lower cutoff value for the same serotype and the fig. 2 . multiple alignment of the deduced amino acid sequences of the five conformation-dependent neutralizing antigenic sites (epitopes) mapped to the s1 subunit of the spike protein and another immunodominant region in the amino-terminal region of the s2 protein using the i1101/16 isolate and the seven reference strains. the sequences of the i1101/16 isolate are listed; only the amino acids that differed from those of the i1101/16 isolate are depicted and the amino acids that were same as those of the i1101/16 isolate are represented by dots. deleted amino acid residues are represented by dashes. 3 . alignment of the complete genome sequences of the i1101/16 isolate and the seven reference strains performed using mafft. the genome sequence of the ibv i1101/16 isolate was set as the reference sequence. the reference sequence was represented by black and gray gaps in the reference sequence indicated the nucleotide insertion occurred in the genomes sequence of other viruses. the nucleotide sequences of other viruses which disagreement to the reference sequence at indicated positions were represented by black, while the nucleotide sequences of other viruses which agreement to the reference sequence at indicated positions were represented by gray. the genbank accession numbers for these genome sequences are the same as those in fig. 1. maximum value defining serotype relatedness, respectively (wadey and faragher, 1981) . the low antigenic relatedness between the i1101/16 isolate and the ck/ch/ljl/140901 strain was not due to differences in their b cell epitopes because the sequences of the b cell antigenic epitopes located in the spike protein were nearly identical in both viruses, although it has been reported that only a few amino acid changes in the s1 subunit of the spike protein can result in serologically distinct viruses (cavanagh et al., 1992a,b) . hence, we focused on the entire spike gene of the two viruses, especially the s2 subunit of the spike gene. interestingly, there was greater genetic diversity in the spike gene (90.9%) between the i1101/16 isolate and the ck/ch/ljl/140901 strain compared with that of s1 gene (95.7%), thereby indicating the greater divergence of the s2 region, which led us to conclude that possible recombination events may have occurred in the genome of the i1101/16 isolate. next, recombination events were analyzed using the complete genomic sequences of these strains. according to mafft, only the s2 gene of the i1101/16 isolate was highly similar to that of the 4/91 strain, whereas the remainder of the genome was highly similar to that of the ck/ch/lhlj/140901 strain. this suggests that for the emergent i1101/16 isolate, the donor of the s2 gene was probably a 4/91-like virus, whereas the remainder of the genome came from a ck/ch/lhlj/ 140901-like virus. the first prerequisite for recombination is considered to be co-infection. ibv strain ck/ch/lhlj/14090 is one of the deduced parental viruses of the i1101/16 isolate and it has an nrtw serotype, where it originated from recombination events between tw i-like and lx4-like viruses in china around 2009 . in the commercial poultry industry in china, ibv is controlled by the use of live attenuated and inactivated vaccines. vaccine 4/91 is another deduced parental virus of the i1101/16 isolate and it has been used widely in china for a long time. previously, we found that vaccination with the 4/91 strain cannot provide complete protection against nrtw viruses , but vaccination with the 4/91 strain allows prolonged replication and shedding of the vaccine virus . hence, it is reasonable to speculate that vaccination with the 4/91 vaccine produces an environment where co-infections between field and vaccine strains can occur, which may enhance the likelihood of recombination. high frequencies of recombination between vaccines (such as the 4/91 strain) and field strains have been reported frequently in china and other parts of the world (cavanagh et al., 1992a,b; jackwood et al., 2012; liu et al., 2013; han et al., 2017; jiang et al., 2017) . it has been suggested that recombination may play an important role in viral evolution. the exchange of part of a region of the genome allows viruses to rapidly explore areas of sequence space, potentially leading to the emergence of variants with different features in terms of their virulence, cross-protection, and cell and host tropisms (simon-loriere and holmes, 2011) . it is considered that the emergence of the australian n1/88 strain was driven by antigenic differences between the virus and other circulating ibvs, but also by the enhanced capacity of the recombinant virus to replicate in chickens (quinteros et al., 2016) . we found that the recombinant i1101/16 isolate was serologically related to but not the same as strain ck/ch/lhlj/140901. we cannot conclude that this antigenic change was due to recombination because the b cell antigenic epitopes located in the spike protein (including that located in the s2 subunit of the spike gene) were nearly identical in the two viruses. in addition, it is very interesting to note that the/i1101/16 isolate exhibited decreased replication levels in both the tracheal and kidney tissues (two target tissues for most ibvs) compared with one of its parental viruses (the ck/ch/lhlj/140901 strain, which does not cause severe clinical disease in spf chickens), but it exhibited prolonged replication and shedding post-challenge in a table 3 pairwise comparisons of the nucleotide sequences of the s2 subunit of the spike genes between the 4/91 vaccine strain, i1101/16 isolate, and pathogenic 4/91 strain a . a nucleotides shared between the i1101/16 isolate and 4/91 vaccine strain are shown in gray. b nucleotide positions corresponding to those in the spike gene in the 4/91 vaccine strain. the genbank accession number of the pathogenic 4/91 strain is jn192154. the genbank accession numbers of the other viruses are the same as those shown in fig. 1 . similar manner to one of its parental viruses, the 4/91 vaccine . meanwhile, the high viral titer but low clinical score of chickens infected with ck/ch/ljl/140901 was also observed in this study although the exact mechanism was unknown and needed to be further investigated. it is believed that the coronavirus s1 domain is involved with host cell receptor binding and that the conserved s2 domain mediates fusion between the virus and cellular membranes (bosch et al., 2003) . the s2 4 8 12 16 20 24 28 4 8 12 16 20 24 28 32 36 40 44 i1101/16 10/10 3 10/10 b 9/10 7/10 6/10 2/10 1/10 1/10 7/10 8/10 10/10 7/10 7/10 5/10 6/10 6/10 6/10 2/10 0/10 ck/ch/ljl/ 140901 9/10 0.9 10/10 10/10 6/10 2/10 2/10 0/10 -c 10/10 10/10 10/10 10/10 6/10 6/10 6/10 0/10 ---negative 0/10 0 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 a two procedures were used for virus recovery after challenge. first, lesions were observed in individual tissue samples (trachea or kidneys) from inoculated embryos. second, rt-pcr was conducted using the oligonucleotide primers n(+) and n(−) on rna recovered from the allantoic fluid in the same eggs. the results obtained using the two procedures were identical. b number of chickens yielding virus/number of chickens tested. c not done. fig. 6 . replication of the i1101/16 isolate and ck/ch/ljl/140901 strain in the trachea and kidneys of chickens. one-day-old spf layer chickens were inoculated with a dosage of 1 × 10 5.5 eid 50 of the i1101/16 isolate or the ck/ch/ljl/140901 strain in a 0.1-ml volume, and the trachea and kidneys from each bird were collected at 5 days post-challenge for virus titration in eggs. each bar represents the virus titer from an individual bird. membrane fusion unit of the ectodomain contains two heptad repeat regions, which interact to form the coiled-coil structure of the stalk (de groot et al., 1987; wickramasinghe et al., 2011) , and a putative fusion peptide. after endocytosis, conformational changes in the s protein are triggered by exposure to acidic ph in the endosomes (chu et al., 2006) , thereby resulting in fusion of the viral envelope with the cellular membrane. the s2 domain is not involved mainly with binding to the host cell receptor, but the interplay between s1 and s2 might synergistically determine the avidity and specificity of viral attachment (de hann et al., 2006; promkuntod et al., 2013) . it is unknown whether the changes in the spike protein caused by the recombination event altered the antigenic and pathogenic features of the i1106/16 isolate, or whether it is more likely that these changes as well as other changes in polymerase peptides are jointly rather than individually responsible for the differences in the features of the two ibv strains. however, it has been reported that recombination is likely to have a high fitness cost because of the destruction of optimized intra-and inter-protein interactions (simon-loriere and holmes, 2011), where the fitness of recombinant ibv strains must be superior to that of the parental strains if they are to emerge and spread in the environment. thus, the fitness of recombinant ibvs requires further investigation. this study does not provide evidence that recombination can directly alter the antigenicity, virulence, replication, shedding, and tissue tropism of a virus, but because the backbone of the investigated virus differs substantially, we showed that gene(s) exchange between ibvs is likely to be the major determinant of viral evolution. our previous results liu et al., 2014; chen et al., 2015) and the results obtained in this study provide evidence that recombination events are occurring frequently in the field in china. high chicken densities in poultry farms and the co-circulation of multiple ibv strains in a given flock as well as the use of different live vaccine strains may explain the high frequency of recombination. researchers should be aware of the effect of virus recombination, especially recombination events between field and vaccine strains, which may cause changes in viral features. in addition, the currently applied vaccination strategy for protection against the ntw i ibv type should be improved and implemented properly. complete genomic sequence analysis of infectious bronchitis virus ark dpi strain and its evolution by recombination persistent antigenic variation of influenza a viruses after incomplete neutralization in ovo with heterologous immune serum the coronavirus spike protein is a class i virus 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and evolution of lx4 genotype infectious bronchitis coronavirus in china complete genome sequence of a novel infectious bronchitis virus strain circulating in china with a distinct s gene identification of a novel recombinant virulent avian infectious bronchitis virus this work was supported by grants from the china agriculture research systerm (no. cars-40-k18) and national "twelfth five-year" plan for science and technology support (2015bad12b03). the authors declare that they have no competing interests. key: cord-291510-jh2fdks4 authors: jiang, yi; cheng, xu; zhao, xiumei; yu, yan; gao, mingyan; zhou, sheng title: recombinant infectious bronchitis coronavirus h120 with the spike protein s1 gene of the nephropathogenic ibyz strain remains attenuated but induces protective immunity date: 2020-02-11 journal: vaccine doi: 10.1016/j.vaccine.2020.01.001 sha: doc_id: 291510 cord_uid: jh2fdks4 infectious bronchitis (ib) is a highly infectious viral disease responsible for major economic losses in the poultry industry. a reverse genetic vaccine is a safe, rapid, and effective method of achieving ib prevention and control. in this study, we constructed the recombinant strain, rh120-s1/yz, using a reverse genetic system, based on the backbone of the h120 vaccine strain, with the s1 gene replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/2011, isolated in china. the results of dwarf chicken embryos, growth kinetics, and viral titration in the embryos demonstrated that the biological characteristics of the recombinant virus remained unchanged. like the rh120-infected group and in contrast to the ribyz-infected group, no mortality, clinical signs, or lesions were observed in the lungs or kidneys of young chickens inoculated with rh120-s1/yz. the viral loads in various tissues, cloacal, and oral swabs was lower in most types of samples, indicating that the rh120-s1/yz strain was highly safe in chicks. compared to rh120 vaccination group, when the efficacy of this strain was evaluated against the qx-like ibv strain, better protection, with 100% survival rate and no disease symptom or gross lesion was observed in the chickens vaccinated with rh120-s1/yz. increased levels of ibv-specific antibodies were detected in the serum of the rh120-s1/yz-vaccinated animals 14 days post-vaccination. collectively, our results suggest that the recombinant strain, rh120-s1/yz, may represent a promising vaccine candidate against qx-like ibvs. infectious bronchitis (ib) was first described as a respiratory disease affecting chicks in the us in 1931 as an acute and highly contagious viral disease, and continues to cause major economic loss within the poultry industry worldwide [1] [2] [3] . as a result of the damage to the respiratory system, the clinical signs of this disease include depression, coughing, head-shaking, as well as nasal and ocular discharge [1] . since the virus causing ib replicates in many non-respiratory epithelial surfaces (e.g., kidney and gonads), nephritis, reduced egg production, and egg quality is also observed [2, 3] . the causative agent of ib is infectious bronchitis virus (ibv), a non-segmented, positive-sense, single-stranded rna virus, which belongs to the genus gammacoronavirus, family coronaviridae, in the order nidovirales [4] . the full length rna genome of ibv is 27.6 kb in length and encodes non-structural proteins, four accessory proteins, and four structural proteins (spike [s], matrix [m], nucleocapsid [n] , and envelope [e] ). the s protein is cleaved into s1 and s2 as two subunits at the s1/s2 cleavage site. the s1 protein contains the receptor-binding domain and mediates viral attachment to host cells, whereas s2 is responsible for membrane fusion [5, 6] . errors generated during the genomic replication of ibv and the selective pressure following the use of live attenuated vaccines or multiple infections with different ibv serotypes has led to the emergence of numerous ibv variants [7, 8] . in particular, small change as little as 5% in the amino acid composition of s1 gene may lead to alteration in cross protection among closely related serotypes [9] . in china, ibv was first observed during the early 1980s, and outbreaks have since been frequently reported [7] . the qx-like genotype is thought to have originated during the mid-1990s and continues to be the predominant strain, whereas the ldt3, 4/91, and taiwan subtypes have also recently been frequently isolated in china [10] [11] [12] [13] . severe nephritis, false layer syndrome, and high mortality are observed in chickens infected with this viral genotype, which represents a major problem in the poultry industry, particularly in china. using a live attenuated or inactivated vaccine is typically considered to be the most cost-effective means of controlling viral infections [14] . unfortunately, inactivated ib vaccines are not effective if used alone, which could induce little or no protection against egg loss [15, 16] , as well as impaired ciliary activity in the trachea [17] ; thus, birds must continue to be given one or a series of vaccinations with live attenuated ib vaccines to provide broad heterologous protection [1] . commercial attenuated live vaccines used against ibv in china include the h120, ldt3, and 4/91 strains [7] ; however, phylogenetic analysis indicates that the qx-like genotype is genetically distant from the strains described above, which may explain the poor cross-protectivity against infection in chickens immunized with these classical vaccines [18, 19] . recently, there has been increasing research on live attenuated vaccines against the qx-like ibv strains. different strains of qxlike ibvs were serially passaged in embryonated eggs or primary chicken kidney cells (ck) to obtain vaccines that are less virulent and exhibit high immunogenicity against the qx-like epidemic ibv strains [20] [21] [22] [23] [24] . however, in addition to causing tissue damage or secondary bacterial infections in young vaccinated chicks [25, 26] , live attenuated vaccines of rna viruses may not be genetically stable or they may be associated with the tendency to revert back to a virulent form [9, 14] . moreover, the potential for recombination between vaccine and virulent strains can lead to the creation of new virulent virus [27] . furthermore, it takes a substantial amount of time to prepare a extensively passaged attenuated vaccine strain, which may not be able to effectively solved the loss problem caused by the rapid variation exhibited by prevalent strains on the flocks. in considering the limitations of the live attenuated and inactivated ibv vaccines, reverse genetic ibv vaccines have been developed in recent years as they display increased safety and efficacy [28] [29] [30] . as reverse genetic technology is primarily based on the h120 and beaudette strains [31] [32] [33] , both of which are passagegenerated attenuated strains, the mechanism of virus virulence attenuation remains unclear, thereby posing difficulties in the development of attenuated vaccines using this technology. in addition to the function of host invasion and increasing genetic diversity, the ibv s1 subunit can induce virus neutralizing and cross-reactive antibodies [34] . therefore, some researchers have created an attenuated ibv vaccine by replacing the s1 or s genes from other virulent strains using reverse genetic technology to generate protection against strains of the corresponding serotype [5, 28, 30] . in the present study, we constructed a recombinant strain, using the reverse genetic system. rh120-s1/yz is based on the backbone of the vaccine strain h120, and the s1 gene was replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/2011, which was isolated in china. our results demonstrate that this recombinant strain is safe and provides effective protection in young chickens against qx-like ibv challenge. the h120 vaccine strain is currently a widely used vaccine strain, created from 120 serial passages of the h strain isolated in 1956. the ck/ch/ibyz/2011 strain (referred to as ibyz, genbank kf663561.1) used in this study is classified as a qx-like ibv isolated from a flock presenting with ib symptoms by our research group in 2011 in jiangsu province, china. both of these strains were recovered from a full-length clone using reverse genetics, as previously described [35, 36] . fertilized specific pathogen free (spf) chicken embryos were obtained from beijing merial vital laboratory animal technology co., ltd. (beijing, china) and hatched in our lab (lab of chicken infectious disease protection and control, poultry institute, chinese academic of agriculture sciences, yangzhou, china). for hatching, one-day-old spf chickens from the spf chicken embryos as described previously were incubated at 37°c in a relative humidity of 55-65%. all chickens were maintained in isolators under negative pressure, and food and water were provided ad libitum. the ribv rh120-s1/yz strain used in this study is described in the schematic illustration presented in fig. 1 . the genome rna was synthesized in vitro by t7 rna polymerase and transfected into bhk-21 cells, as previously described [35, 36] . the cell supernatants were harvested 48 h following transfection and propagated in 10-day-old spf chicken embryos. the allantoic fluid was harvested for rt-pcr, whole genome sequencing, and was then stored at à80°c until further use. to determine the recombinants' pathogenicity in chicken embryos, 0.2 ml of rh120, ribyz, or rh120-s1/yz virus (diluted to 1:100 in normal saline) was inoculated into the allantoic cavities of five 10-old-day embryonated spf chicken eggs, respectively. chick embryo lesions were examined for lesions at 144 h postinoculation at 37°c. to examine the viral growth ability in chicken embryos, a realtime reverse transcription quantitative polymerase chain reaction (rt-qpcr) method was established. according to the sequences of ibv from genbank, primers were designed based on conservative area in the 5 0 -utr. the upstream primer was 5 0 -ccgttgctt gggctacctagt-3 0 , and the downstream primer was 5 0 -cgcctac cgctagatgaacc-3 0 . the amplification product was cloned to pmd18-t vector (takara) as a positive plasmid and its concentration was measured. a gradient dilution of 5 â 10 2 -5 â 10 8 copies/ll of the plasmid was used as template for quantitation test. by plotting the cycle threshold (ct) values against the copies of the plasmid, the standard curve was generated. 18 10-day-old embryonated spf chicken eggs (six eggs/group) were inoculated with rh120, ribyz, and rh120-s1/yz at a dose of 10 7 viral rna copies/100 ll, and used for growth curve experiments. the allantoic fluids were collected separately by syringe from the six inoculated embryonated eggs of each group at 12, 18, 24, 36, 48, 72 , 96 h per inoculation. rna was extracted from the allantoic fluids using trizol reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions. cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral copies were measured by absolute quantitative method of realtime pcr, which was performed using sybr ò premix ex taq tm ii (takara, otsu, shiga, japan) on an applied biosystems 7500 fast real-time pcr system. the standard curve was plotted against the log of the template copy number. all of the assays were run in triplicate and the copy number of each virus was calculated according to the standard curve. serial 10-fold dilutions from 10 5 copies to 10 9 copies per 0.1 ml of virus of the recombinant strain, rh120-s1/yz, and its parental strains (rh120 and ribyz) were inoculated into the allantoic cavity of 10-day-old embryonated spf chicken eggs. for each dilution, 0.1 ml of the virus solution was injected into each egg and 10 eggs were used for each dilution. pbs was used as a negative control. after a 24 h incubation, the dead embryos were considered nonspecific deaths and discarded. the embryos were examined for the presence of specific lesions caused by the virus after a 144-h incubation. dead and live embryos that displayed ibv infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples. the 50% embryo infectious dose (eid 50 ) of these ribvs was calculated using the reed-muench method [37] . the chickens were separately housed in isolators under consistent conditions, and food and water was provided ad libitum. a total of 120 one-day-old spf chickens were randomly divided into four groups (n = 30 per group). the chickens in the experimental groups were intranasally inoculated with 200 ll allantoic fluid per chick containing 10 5 eid 50 of the rh120, ribyz, and rh120-s1/ yz strains. the control group (n = 30) was inoculated with 200 ll sterile pbs via the same route. the morbidity and mortality was schematic diagram for the construction of the chimeric s gene and production of a full-length cdna of rh120-s1/yz. (a) replacement of the h120 s1 fragment by the corresponding sequence of ibyz for construction of pyzs1h120s2. the plasmids pibyzs and ph120s contained the s gene of ibyz strain and h120 strain, respectively, were constructed during the establishment of reverse genetic system. primers ps1f and ps1r were used to amplify the s1 fragment of ibyz and vector fragment, while primers ps2f and ps2r were used to amplify the s2 fragment of h120 strain. by overlapping pcr, the pyzs1h120s2 was constructed which contained a chimeric s gene. (b) strategy for the construction of full-length cdna clones of rh120-s1/yz. ten cdna fragments covering the entire genome of h120 strain was amplified by rt-pcr. unique bsa i sites were inserted at the junctions between each clone, a unique t7 start site was inserted at the 5 0 end of clone tm1, and a 28-nucleotide t tail was inserted at the 3 0 end of clone tm10. the s fragment in the pmdtm8 plasmid of h120 strain was replaced by chimeric s gene. by using appropriate ligation strategy, the genomic cdna of rh120-s1/yz was assembled by in vitro ligation using appropriate restriction sites as indicated. followed-up for 14 days. all experimental groups were monitored daily for clinical signs related to ib infection for 14 days postinfection (dpi), including coughing, sneezing, and tracheal rales. dead chickens were examined for gross tracheal, lung, and kidney lesions. a total of 100 1-day-old spf chickens were assigned to four groups (n = 25 per group). the birds were inoculated with 200 ll allantoic fluid containing 10 5 eid 50 of the rh120, ribyz, or rh120-s1/yz strains, respectively via eye drop and the intranasal route and 200 ll pbs per chick was administered to the control group (n = 25) via the same route. the tracheas, lungs, kidneys, and bursa from the 5 inoculated birds per group were harvested at 1, 3, 5, and 7 dpi, weighed, and collected into 1 ml pbs per sample and frozen at à80°c. after grinding the samples, the viral rna was extracted using trizol, and the cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral rna copies from each of the different samples were detected by real-time pcr as described above. all assays were run in triplicate and the copy number for each virus was calculated according to the standard curve. after inoculation with 10 5 eid 50 dose of the rh120, ribyz, and rh120-s1/yz strains, three infected chickens per group were randomly selected to be euthanized by bleeding, and the tissue pathology was examined in the different groups at 3, 5, 7, 10 dpi. the tracheas, lungs, kidneys, and bursas of three dead or other randomly-selected chickens from the experimental and control groups (inoculated with pbs) were collected at 5 or 7 dpi and further processed for histopathology. the samples were fixed in formalin, embedded in paraffin, cut into 5 lm sections, and stained with hematoxylin and eosin. the slides were examined under light microscopy for the presence of lesions. to determine the level of viral shedding of the different recombinants, 10 birds per group were inoculated with 200 ll allantoic fluid containing 10 5 eid 50 of the rh120, ribyz, or rh120-s1/yz strains, respectively. 200 ll pbs per chick was administered to the control group via eye drop and the intranasal route. . oral and cloacal swabs were collected from each bird into 1 ml pbs at 7, 14, 18, 22, 26, and 30 dpi and stored at à80°c. after freezing and thawing three times and centrifuging at 8,000 â g for 5 min at 4°c, relative amount of virus present in 200 ll of supernatants of each sample was quantified by rna extraction, reverse transcription, and real-time pcr as described previously. 2.8. efficacy of rh120-s1/yz vaccination 2.8.1. viral challenge in total, 30 one-day-old spf chickens were divided into three groups of 10 chickens. the three groups were intranasally vaccinated with the rh120 and rh120-s1/yz strains at a dose of 10 4 eid 50 /200 ll, and the control group received pbs. two weeks post-vaccination, the chickens in each group were challenged with the qx-like strain, ribyz, at 10 6 eid 50 /200 ll via eye drop and the intranasal route. any clinical signs, as well as the percentage of morbidity and mortality were recorded for 14 days. the dead chickens were also examined for gross tracheal, lung, and kidney lesions. 45 1-day-old spf chickens were divided into three groups (n = 15 per group). after vaccinating with the rh120 and rh120-s1/yz strains at a dose of 10 4 eid 50 /200 ll, the sera of 10 birds from each group were randomly collected and antibodies were tested using an elisa created in our lab at 7, 14, 21, and 28 days post-vaccination (dpv). elisa plates were coated with antigen of culture supernatants of inactivated ibv rmj , a vero cells adaption strain domesticated from its parental strain of ibyz in our lab. individual chicken sera collected from different groups diluted to 1:200 were loaded and the plates were incubated at 37°c for 60 min. ibv-specific igg was detected with anti-chicken igy (igg) (whole molecule)-peroxidase antibodies produced in rabbits (sigma-aldrich, germany). after incubated with 3,3 0 ,5,5 0 -tetrame thylbenzidine(tmb) substrate for 15 min and terminated with 2 mol/l h 2 so 4 solution, the absorbance at 490 nm was measured using a model 680 microplate reader (bio-rad, usa). graphpad prism 7 software (graphpad software inc., la jolla, ca, usa) was used for the statistical analyses. for the antibody test, growth kinetics, tissue tropism, and viral shedding test, the collected data were analyzed using a two-way anova to determine whether there was a significant difference between the different groups. the significance was considered as follows: significant at p 0.05 (*); highly significant at p 0.01 ( ** ); and extremely significant at p 0.001 ( *** ). characterization of the rh120-s1/yz strain after being inoculated with the recombinant strains, typical ib lesions in the chick embryos from each group were observed, including amniotic membrane thickening, dwarfing, stunted growth, curling, or death of the embryo (fig. 2a) . the eid 50 of the virus was calculated according to the reed-muench method. the titers of the rh120, ribyz, and rh120-s1/yz strains were determined to be 10 7.3 eid 50 /ml, 10 6.8 eid 50 /ml, and 10 8.4 eid 50 /ml, respectively (fig. 2b) . the growth kinetics of the ribvs were assessed by inoculation with 10 7 copies of virus per egg in 6 eggs from each group. the relative viral load was determined at 12, 18, 24, 36, 48, 72, and 96 h post-inoculation by quantitative real-time rt-pcr. the growth curves of all three strains reached a relatively stable plateau at 36-48 h after infection; the level of viral rna for the rh120-s1/ yz strain was significantly higher than that of the ribyz strain for the majority of the time points (fig. 2c ). the earliest death was observed at 3 dpi in the qx-like strain ribyz group, which virus was highly pathogenic to one-day-old spf chickens; the final mortality of this group was 63.3% (fig. 3a) . the morbidity of the infected chickens in the ribyz group reached higher than 80%, and the diseased chickens exhibited respiratory symptoms (e.g., sneezing, coughing, as well as tracheal and bronchiolar rales), and the severe cases presented with additional signs of listlessness, huddling, and ruffled feathers. at necropsy, lesions were detected both in the respiratory and urinary system, including mucus, hyperemia, and hemorrhage in the trachea, as well as swelling and urate deposition in the kidney. edema and congestion were observed in the lungs in 20% of cases (fig. 3b) . however, following inoculation with the rh120 or rh120-s1/yz strains, the morbidity rates of the chickens was less than 20%, with moderate respiratory signs of coughing in some of the chickens. all of the infected chickens presented no visible lesions in the kidney and the survival rate was 100% through 14 dpi in both the rh120 and rh120-s1/yz groups ( fig. 3a and b) . no obvious clinical signs, gross lesions, or death attributable to ibv was observed in the control group. for the chickens inoculated with the qx-like strain ribyz, lesions in the trachea at 5 dpi were characterized as cilia necrosis and loss, epithelial cell congestion, hemorrhage, and lymphoid infiltration (fig. 4c ). in the bronchial and capillary lumen of ribyz-infected lungs, congestion, hemorrhage, as well as erythrocyte and inflammatory cell infiltration was observed in some severe cases from 7 to 10 dpi (fig. 4g) . moreover, erythrocyte and lymphocyte infiltration, as well as tubular dilation was observed in the kidney of ribyz-inoculated chickens (fig. 4k) . similar to the rh120 group, chickens inoculated with rh120-s1/yz presented with mild pathology in the trachea at the early infection time points of 3, 5 dpi. lesions in the trachea consisted of the loss of epithelial cells and cilia, decreased number of secretory cells, and thickening of the mucosa (fig. 4b and d) . in contrast, there were no lesions observed in the lungs and kidneys of rh120 and rh120-s1/yz-infected chickens during the infection period (fig. f , h, j, and l). no histopathological evidence of damage was detected in the bursa of all the groups and no ibv-associated lesions were found in the tissue samples of the control chickens (fig. 4a , e, i, m-p). the time-dependent levels of viral replication in the different tissues determined by real-time rt-pcr presented in fig. 5 are consistent with the tissue histopathology results. compared to rh120 and rh120-s1/yz groups, one day after inoculation with the qxlike strain, ribyz, the viral load in all organs began to rise rapidly, except at 5 dpi in the trachea. the viral load in tracheal samples in the rh120-s1/yz group increased and peaked at 5 dpi. the level of viral rna was significantly lower than the rh120 group both at 1 dpi and 7 dpi (fig. 5a) . the viral load in lung samples from all infection groups increased starting at 3 dpi, and the viral rna in the lungs of the rh120-s1/yz group was significantly lower compared with both the rh120 and ribyz groups (fig. 5b) . the viral load in the kidney of all ibv-inoculated groups was detected at 1 dpi. in contrast to the rh120 and rh120-s1/yz groups, the viral growth in kidney of ribyz-infected chickens exhibited an extremely significant increase at 3 dpi and peaked at 5 dpi. the viral 50 of the recombinant ibvs, rh120, ribyz, and rh120-s1/yz in spf chicken embryonated eggs. (c) the multicycle growth kinetics of the recombinant ibvs, rh120, ribyz, and rh120-s1/yz in spf chicken embryonated eggs. all data are presented as the mean ± standard deviation (sd). some of the error bars are too small to be seen. the qrt-pcr was carried out with three replicates. markers of statistical difference were acquired by comparing between rh120 group and rh120-s1/yz group. *** indicates an extremely significant difference at p 0.001; ** indicates a highly significant difference at 0.001 < p 0.01; *indicates a significant difference at 0.01 < p 0.05. rna copies of the rh120-s1/yz group peaked at 1 dpi, after which they gradually decreased (fig. 5c) . no viral load was detected in the control samples at any of the time points. in all ibv infection groups, the viral load in both the oral and cloacal swabs generally showed a gradual decline from 7 dpi to 26 dpi. compared with the rh120 group, the reduction in viral load between 14 or 18 dpi was significantly greater for chickens infected with rh120-s1/yz than for chickens infected with rh120, and the viral rna of the qx-like strain ribyz was lower than that of the rh120 group at 7, 14, and 18 dpi but with no statistically significance (fig. 6a) . in contrast, the viral load in the cloacal swab of the ribyz group was significantly higher than that of the rh120 and rh120-s1/yz groups at 7 dpi. compared with other groups, the viral rna in the cloacal swab of the rh120-s1/ yz group was significantly lower at 7, 14, and 22 dpi (fig. 6b) . no virus was detected in the control group at any time point. the rh120-s1/yz group exhibited effective protection against challenge with the qx-like strain ribyz. the mortality rate in the rh120-s1/yz group was 0%, and no clinical signs or lesions were observed during the ribyz challenge period (fig. 7a) . in contrast, chickens vaccinated with the rh120 strain began to exhibit clinical signs (e.g., coughing and nasal discharge) at 3 days post-challenge (dpc), and some chickens showed severe signs of listlessness, hudfig. 3 . pathogenicity of the ribv rh120, ribyz, and rh120-s1/yz strains. (a) percent survival of spf chickens infected at 1-day-of age during the 14-day observation period. no mortality was observed in rh120, rh120-s1/yz, or the control groups. (b) gross lesions on the lungs and kidneys of the infected chickens at 5 or 7 dpi. fig. 4 . histopathological changes in the trachea, lungs, kidneys, and bursa of spf chickens infected with the recombinant rh120, ribyz, and rh120-s1/yz strains at 1-day-ofage. black arrows in the trachea indicate cilia and necrosis loss. black arrows in the lungs indicate congestion and hemorrhage. trachea samples were collected at 3, 5 dpi (a-d); lung and bursa samples were collected at 7, 10 dpi (e-h; m-p); and kidney samples were collected at 5, 7 dpi (i-l). black arrows in kidneys indicate tubular dilation, congestion, and inflammation. scale bar = 100 lm or 200 lm. fig. 5 . viral load in the different organs at 1, 3, 5, and 7 dpi of spf chickens infected with the recombinant strains rh120, ribyz, and rh120-s1/yz at 1-day-of age. the bars indicate means ± standard deviations. *** indicates an extremely significant difference at p 0.001; ** indicates a highly significant difference at 0.001 < p 0.01; *indicates a significant difference at 0.01 < p 0.05. dling, and ruffled feathers, with the mortality rate reaching 50% during the observation period. at necropsy, the dead animals displayed typical kidney lesions characterized by swelling and urate deposition in the tubules and ureters. in contrast to chickens vaccinated with rh120-s1/yz, the chickens in the unvaccinated control group exhibited clinical signs at 3 dpc, and a 60% mortality rate; the gross lesions of the dead birds in this group were similar to that of the rh120 group (fig. 7b ). antibodies against the ibvs were measured using an elisa (fig. 7c) . none of the birds in the rh120 or rh120-s1/yz groups showed a positive antibody response at 7 dpv, whereas 100% of the birds had an antibody response detectable at 14 dpv, with the antibody level gradually increasing during the observation period. however, there was no significant difference in the level of serum antibodies between rh120-vaccinated chickens and rh120-s1/yz-vaccinated chickens. no positive serum antibody response was detected in the non-vaccinated controls. vaccines, particularly live-attenuated vaccines, remain the most effective means of protection against ibv challenge (e.g., the mass serotype strains h120, h52, ma5, and w93, which are still widely used in the poultry industry) [38] [39] [40] . however, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the ib endemic in china cannot be controlled [22] . genetic mutations and immune pressure during the replication process of the single stranded rna virus often results in the emergence of ibv variants [41, 42] . to date, at least six genotypes of ibv strains have been identified in china, of which the fig. 6 . viral loads at 7, 14, 18, 22, and 26 dpi in the oral and cloacal swabs of chickens infected with the recombinant rh120, ribyz, and rh120-s1/yz strains. all data are presented as the mean ± standard error of the mean (sem); *** indicates an extremely significant difference at p 0.001; ** indicates a highly significant difference at 0.001 < p 0.01; *indicates significant difference at 0.01 < p 0.05. fig. 7 . efficacy of the rh120 and rh120-s1/yz vaccines. (a) percent survival of the vaccinated chickens challenged with the qx-like ibv ribyz strain during the 14-day observation period. no mortality was observed in the chickens vaccinated with the rh120-s1/yz strain. (b) gross lesions observed on the kidneys of the vaccinated chickens challenged with the ribyz strain at 5 or 7 dpc. (c) the mean antibody od value at 7, 14, 21, and 28 dpv. all data are presented as the mean ± standard error of the mean (sem). qx-type (first isolated in 1999) remains the most prevalent genotype [10, 42, 43] . the poor relationship between the qx-type strains, which are abundant and display variable virulence in various parts of china, and the mass-type vaccine strains could explain the failure of the mass-type vaccination programs to control ibv in these flocks [19, 39] . consequently, the development of novel vaccines against circulating ibv strains in china using local ibv strains is required [20, 43, 44] . optimal vaccination against circulating ibv strains in china requires the development of attenuated vaccines designed from local strains [20] [21] [22] 45] . however, due to the particularity of rna virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [27, 46, 47] , which represents a substantial hidden concern to the poultry industry. to reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., beaudette strains carrying the s1 gene of the h120 vaccine strain, virulent m41 strain, or qx-like strain) [30, 48] . however, the titer of the beaudette strains carrying the s1 gene of the vaccine h120 strain only reaches 10 6.13 ± 0.23 eid 50 [49] , which is lower than that of the h120 backbone strain. therefore, we aimed to develop a recombinant rh120-s1/yz strain based on the h120 vaccine strain that carries the s1 gene of the ck/ch/ibyz/2011 strain (a chinese qx-like nephoropathogenic strain) using reverse genetic technology [35, 36] . in the present study, we isolated a nephoropathogenic strain (ibyz) in 2011. this strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of ibv emerged in jiangsu province. chickens infected with the ibyz strain showed severe kidney damage and a high mortality rate (fig. 3b) . the complete sequence has been deposited in the genbank database under the accession number of kf663561. the s1 gene sequencing data indicated that the ibyz strain belonged to the qx-like genotype (fig. 8) , with a nucleotide homology with the attenuated vaccine h120 strain of only 77.4%, and amino acid homology of 77.1%. moreover, the s1/s2 cleavage site of ibyz is hrrrr/s, which differed from rrfrr/s of the h120 vaccine strain. after wholegenome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed ribyz. in addition, another strain used in this study is the live attenuated ibv vaccine, h120, and the molecular clone strain, rh120, which was constructed using the same method. the spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [6, 50] . however, replacing the s gene of beaudette strain by the virulent strains does not make it virulent [51, 52] . the s protein can be cleaved into s1 and s2 subunits at the s1/s2 cleavage site [53] , the s1 subunit is responsible for receptor binding to the host cells [54] . whether the s1 gene has an effect on the virulence of the virus is unknown. in this study, the recombinant strain, rh120-s1/yz, expressed the s1 gene of the qx-like strain, which retains the s1/s2 cleavage site of h120, maintained the ability to replicate and exhibited pathogenicity in embryos. however, providing ibyz s1 sequences to h120 did not increase its virulence to that of ribyz. ibvs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [2, 6] . the qx-like ribyz strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. in addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points postinfection. the ability of ibv to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of ibv to host cells is dependent on sialic acid on the cell surface being recognized by the s1 subunit [6, 54, 55] . however, the binding of virus to the host cell is the first step in determining tropism and s2 is responsible for membrane fusion [56] . thus, to further explore the different tissue tropism between rh120-s1/yz and ribyz, we examined the viral loads and histopathology in the tissues of the rh120-s1/yz-infected group. it was found that rh120-s1/yz remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the ribyz strain. this finding suggests that both the s2 and s1 genes might play an important role in viral tissue tropism as also suggested by others [57] . another possibility is that the lack of viral replication and damage in kidney following rh120-s1/ yz is due to lower replication ability of backbone except s2 gene from its parental rh120 strain. the viral load of the respiratorytype strain, rh120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of ribyz, which indicates that the ability of the virus to attached to trachea epithelial cells of the rh120 strain was stronger than that of the ribyz strain. however, during the later time points post-infection, the level of the rh120 strain was significantly lower than ribyz, which suggests that the virulence of ibv depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6, 29, [58] [59] [60] . the reduction of the viral load at 5 dpi in the group infected with the ribyz strain may be associated with the necrosis and shedding of the trachea epithelium. moreover, the ability to replication in the different organs of chickens infected with rh120-s1/yz as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the ribyz strain, which demonstrated that the s1 subunit was not the only determinant of viral tropism that was consistent with results by others [57, 61, 62] , and the significant difference in viral load was related to differences in the viral backbone. however, when comparing the rh120-s1/yz and rh120 strains which carry the same backbone, it was found that the viral rna of rh120-s1/yz in the lungs and kidneys was significantly lower than that of rh120, which may due to the replacement of the ibyz s1 gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the s protein of coronaviruses. although cryo-em structure of ibv spike protein has been determined, the relation between structure and function of different ibv s1 genes is unknown. [63] . in addition to the growth in epithelial tissues observed during the early infection period, ibv can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to ibv control [1, 6] . in this study, we detected viral rna in the oral and cloacal swabs isolated from infected chickens at 7, 14, 18, 22, and 26 dpi by real-time pcr. compared with rh120 and ribyz, the viral load in both the oral and cloacal swabs of the rh120-s1/yz group at different time points after infection was significantly decreased. however, while it has been reported that the persistent virus will be re-excreted at the point of lay [1] , the persistence of rh120-s1/yz strain in the egg-laying chickens needs to be further verified. in a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rh120-s1/yz can be considered a safe attenuated vaccine candidate for young chickens. in addition to its role in tissue tropism, the s1 glycoprotein also has an important function in inducing a neutralizing antibody response [64, 65] , and small differences in the s1 contribute to poor cross protectionby neutralization test [64, 66] . to evaluate the protective efficacy of this candidate vaccine, we selected the qx-like virulent ribyz strain to infect chickens at 14 dpv that were vaccinated with either the rh120-s1/yz strain or rh120 strain. we found that little protection against ribyz virulent strain challenge in the rh120 vaccinated group was induced, with a 50% survival rate and severe clinical symptoms associated with ib. this finding could be explained by differences in the antigenicity between rh120 and ribyz. no death, clinical signs, or lesions were observed in the rh120-s1/yz-vaccinated group, indicating that this strain could provide effective protection against ribyz challenge in young chickens. since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [67, 68] , the level of specific igg antibodies remains an important standard for evaluating the immune response to an ibv vaccine. in the present study, the humoral antibody level of chickens vaccinated with the rh120-s1/yz strain was observed to gradually increase compared to the non-vaccinated group. however, although the rh120 strain was able to induce similar levels of humoral antibodies, it provided poor protection against ribyz, suggesting that protection against ibv infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [69] [70] [71] [72] . thus, the detection of mucosal and cellular immunity in rh120-s1/yz-vaccinated animals requires further exploration. nevertheless, some studies have shown that a peptide located near the amino terminal end of s2 can be recognized by neutralizing monoclonal antibodies [73] , and the s2 domain also plays an important role in inducing protective immunity [28, 61, 74] . because the s2 domain plays an important role in cell tropism [57] , we also considered that expressing the s1 subunit would provide greater safety than the entire s gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required. in conclusion, we constructed the recombinant strain, rh120-s1/yz, which was based on the backbone of the h120 vaccine strain, and replaced the s1 gene with that of the qx-like nephropathogenic strain, ck/ch/ibyz/2011, isolated from china using the reverse genetic system. the safety results showed that this recombinant strain was not lethal to one-day-old chicks and it had not gained the ability to tissue invasion or replication in the kidneys and lungs of infected animals. moreover, the viral load of the chickens was significantly decreased compared with the rh120 and ribyz strains. furthermore, rh120-s1/yz was found to provide effective protection against challenge with the qx-like nephropathogenic strain in young chickens. the level of antibody production in the serum of the vaccinated chickens detected by elisa continued to increase after 7 dpi. thus, rh120-s1/yz may be considered a potential live vaccine candidate for protection against chinese qx-like nephropathogenic ibv infection. all of the animals were cared for in accordance with the animal ethics guidelines and approved protocols, and the experimental protocols were performed with the approval of the animal welfare conceptualization, methodology, software, investigation, validation, visualization, resources, writing -original draft, writing -review & editing, visualization, funding acquisition. xu cheng: investigation, data curation. xiumei zhao: visualization, investigation. yan yu: investigation, software. mingyan gao: investigation. sheng zhou: conceptualization, validation, resources, writing -original draft the 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identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus we would like to thank all members of the poultry institute, chinese academy of agricultural sciences (yangzhou, china) for their contribution to this study. yi jiang designed the study and conducted all the experiments, analysis and interpretation of the data, and wrote the manuscript. sheng zhou helped perform the recombinant ibv construction. xu cheng, xiumei zhao, yan yu, and mingyan gao participated in the whole animal experiment in different groups. xu cheng conducted the experiments involving viral shedding. all authors read and approved the final manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.01.001. key: cord-318400-l9kwxsq7 authors: chhabra, rajesh; kuchipudi, suresh v; chantrey, julian; ganapathy, kannan title: pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses date: 2016-01-15 journal: virology doi: 10.1016/j.virol.2015.11.011 sha: doc_id: 318400 cord_uid: l9kwxsq7 to establish a characteristic host response to predict the pathogenicity and tissue tropism of infectious bronchitis viruses (ibv), we investigated innate immune responses (iir) and apoptosis in chicken embryo kidney cells (cekc) and tracheal organ cultures (toc) infected with three ibv strains. results showed nephropathogenic ibv strains 885 and qx induced greater apoptosis in cekc than m41, which induced greater apoptosis in tocs compared to 885 and qx. elevated iir is associated with tissue tropism of different ibv strains. compared to m41, 885 and qx caused greater induction of toll like receptor 3 (tlr3), melanoma differentiation associated protein 5 (mda5) and interferon beta (ifn-β) in cekc. in contrast, m41 infection caused greater expression of these genes than 885 or qx in tocs. in summary, greater levels of apoptosis and elevated levels of tlr3, mda5 and ifn-β expression are associated with increased pathogenicity of ibv strains in renal and tracheal tissues. infectious bronchitis (ib) is an acute and highly contagious disease caused by a gamma coronavirus that affects chickens of all ages and is characterized by lesions in respiratory and urogenital organs (cavanagh, 2007; dolz et al., 2006) . avian infectious bronchitis virus (ibv) continues to cause serious economic losses to global chicken production. along with highly pathogenic avian influenza (hpai) and velogenic newcastle disease (nd), ib is the most economically important viral respiratory disease affecting poultry industry worldwide (cook et al., 2012) . vaccination has been considered as the most reliable approach for ibv control (meeusen et al., 2007) however current vaccines have proved to be inadequate due to constant emergence of new variant viruses (de wit, 2000; de wit et al., 2011) . concurrent circulation of both classic and variant ibvs has been identified in most parts of the world, raising major challenges to the current ibv prevention and control strategies. current ibv isolates present high antigenic diversity (hofstad, 1975) , and emergent strains that differ in antigenic properties, tissue tropism and pathogenicity are continuously being reported across the world (gelb et al., 1991; jackwood, 2012; shaw et al., 1996; zanella et al., 2000) . while all ibv strains appear to initially infect chickens via the respiratory tract, vireamia enables spread to secondary sites for further replication and persistence. an example is ibv strain m41, which replicates primarily in the respiratory tract and subsequently spreads and replicates in a range of other tissues. in contrast, strains of ibv such as strain qx and is/885/00 are primarily nephropathogenic (benyeda et al., 2009; meir et al., 2004) . is/885/00, referred to as a nephropathogenic infectious bronchitis virus (nibv), was first isolated from a severe outbreak of renal disease in several broiler farms in israel (meir et al., 2004) and has been detected in many other middle east countries (abdel-moneim et al., 2012; awad et al., 2014; mahmood et al., 2011) . ibv qx was first isolated in china from chickens with proventriculitis (yudong et al., 1998) but was later found to cause renal, (ganapathy et al., 2012; liu and kong, 2004; terregino et al., 2008; worthington et al., 2008) , respiratory and reproductive lesions (ducatez et al., 2009; terregino et al., 2008) in chicken flocks in europe, asia, africa and middle east (beato et al., 2005; domanska-blicharz et al., 2006; gough et al., 2008; jackwood, 2012) . differences in tissue tropism and thus differences in the pathogenicity of ibv strains have been hypothesized to be associated with differences in the binding properties of their spike proteins (casais et al., 2003; wickramasinghe et al., 2011) . while the ability to bind to susceptible host cells is the first step in the viral life cycle, host innate immune responses could also be a major contributing factor to the pathological outcome of ibv infection. variant ibvs continually emerge and the host determinants of ibv pathogenicity are not yet fully understood. early cellular and innate immune responses of virus infected cells in vitro could act as useful indicators for predicting the pathological outcome of viral infection in vivo. for example, the three different genotypes of newcastle disease viruses (ndv) produce distinct host response patterns in chicken spleenocytes, which is useful to differentiate the ndv genotypes (hu et al., 2012) . in order to establish the characteristic host response to predict the tissue tropism and pathogenicity of ibvs, we investigated apoptosis and innate immune responses in chicken embryo kidney (cek) cells and tracheal organ cultures (tocs) following infection with is/885/00-like, qx-like and m41 ibv strains. cek cells infected with ibv strains 885, qx or m41 were subjected to immuno-cytochemical staining of viral nucleoprotein (np) at 6 h post-infection (hpi). the dose (moi 1.0) used resulted in a similar level of infection in cek cells across all the three virus strains (fig. 1 ). cek cells and tocs were infected with ibv strains 885, qx, m41 or mock infected, cell metabolic activity and percentage of apoptotic cells were evaluated at 24 and 48 hpi by mtt assay and annexin v binding assay, respectively. a significant (po0.05) reduction in cell metabolic activity was found in ibv infected cek cells, compared with mock infected cells, both at 24 and 48 hpi ( fig. 2a) . a significantly (po0.05) greater level of apoptosis was also found in ibv infected cek cells at 24 and 48 h after virus infection (fig. 2b) . total apoptotic cells in ibv or mock infected tocs were evaluated by tunel assay (fig. 3) . it was found that ibv infection resulted in significant increase in total apoptotic cells in tocs when compared with mock infected controls at 24 and 48 hpi (fig. 3c) . notably, infection of cek cells with ibv strains 885 and qx resulted in significantly (po0.05) greater level of cell death, as shown by reduced metabolic activity ( fig. 2a ) and increased apoptosis (fig. 2b ) when compared to m41 infected cells both at 24 and 48 hpi. in contrast, significantly higher levels of apoptosis were observed in m41 infected tocs compared with those infected with 885 or qx (fig. 2c) . infection with ibv strains 885 or qx resulted in significantly (po0.05) lower infectious virus production from cek cells at 24 and 48 hpi when compared with m41 infected cells (fig. 2d ). in sharp contrast, significantly (po0.05) lower infectious virus production was observed from m41 infected tocs compared with 885 or qx infected tocs (fig. 2e ). cek cells were infected with ibv strains 885, qx, m41 or mock infected at a moi of 1.0, and expression of lgp2, mda, tlr1, tlr2, tlr3 and tlr7 were analysed at 9 and 24 hpi. it was observed that expression of lgp2 (fig. 4a ) and mda5 (fig. 4b ) was significantly upregulated at 9 hpi but down-regulated at 24 hpi in ibv infected cek cells compared with mock infected cells (po0.05). however, at 9 hpi, infection with ibv strains 885 and qx resulted in significantly (po0.05) greater mda5 expression than m41 in cek cells (fig. 4b ). there was no significant change in expression level of tlr1 (fig. 4c ). tlr2 (fig. 4d ) was observed at 9 hpi (po0.05), but the expression of these genes was significantly (po0.05) down-regulated at 24 hpi in ibv infected cek cells, compared to mock infected cells ( fig. 4c and d) . a significant (po0.05) up-regulation of tlr3 expression was detected at 9 hpi but compared with mock infected cells, was either unchanged or significantly (po0.05) down-regulated at 24 hpi in ibv infected cek cells (fig. 4e) . interestingly, ibv strains 885 and qx infection resulted in significantly greater tlr3 expression than m41 infection in cek cells at 9 hpi (fig. 4e ). in summary, infection of cek cells with ibv strains 885 and qx, compared with m41, resulted in significantly greater tlr3 and mda5 expression in the early stage of infection (9 hpi). notably, infection of cek cells with ibvs 885 and qx resulted in significantly (p o0.05) greater ifn-β expression than m41 at 9 hpi ( fig. 5b ). however, ifn-β expression was significantly (p o0.05) down-regulated at 24 hpi in all ibv infected cek cells compared with mock infected cells (fig. 5b ). the significant (p o0.05) up-regulation of il-6 ( fig. 5c ) expression was observed at 9 hpi in 885 and qx infected cek cells, but was not significantly (p o0.05) affected in m41 infected cells. there was no significant (p o0.05) change at 9 hpi but a significant (p o0.05) down-regulation at 24 hpi in the levels of ifn-α ( we investigated apoptosis and innate immune responses in cek cells and tocs following infection with ibv strains 885, qx and m41. infection of cek cells with ibv strains 885 and qx that predominantly cause renal lesions in chickens resulted in significantly greater levels of apoptosis when compared to the m41 strain that causes mainly respiratory lesions. similarly, infection of tocs with m41 resulted in greater apoptosis than with the other two nephropathogenic strains. a variety of viruses have been shown to induce apoptosis in infected host cells (clarke and tyler, 2009; shen and shenk, 1995; teodoro and branton, 1997) . ibvs are known to induce apoptosis in infected cells and b-cell lymphoma 2 (bcl-2) family proteins modulate ibv-induced apoptosis (li et al., 2007; liu et al., 2001; zhong et al., 2012a zhong et al., , 2012b . the modulation of bcl-2 family proteins during ibv infection has also been postulated to be under the regulation of signalling pathways such as endoplasmic reticulum (er) stress and mitogen-activated protein kinase/extracellular signal-regulated kinase (mapk/erk) pathways (fung and liu, 2014; zhong et al., 2012a) . furthermore, ibv infection results in a growth-inhibitory effect by inducing cell cycle arrest at s and g 2 /m phases (li et al., 2007) , both of which results in apoptosis. our findings suggest a strong association between apoptosis in cek cells or tocs to the ability of ibv strains to cause renal or respiratory lesions respectively in chickens. nephropathogenic ibv strains 885 and qx resulted in significantly greater up-regulation of innate immune sensing genes namely tlr3 and mda5 along with greater ifn-β mrna levels in cek cells at 9 h of infection when compared to m41 infection. in contrast, these genes were significantly up-regulated in m41 infected tocs than those infected with 885 or qx at 9 hpi. concurrent with our findings, a previous study observed a significant increase of tlr3 mrna expression in both the tracheas and lungs of ibv infected chickens, when compared to uninfected controls (kameka et al., 2014) . we did not identify any particular change in regulation pattern of other innate immune genes, namely tlr1 and tlr2. tlr3 and tlr7 are well known for recognition of rna virus encoded pathogen associated molecular patterns (pamps) (akira, 2001) . tlr7 was not significantly affected by ibv infection in cek cells. the end products of the tlr3 signalling pathway are the production of anti-viral type i interferon (ifn)-α and -β, whereas tlr7 activation results in the production of pro-inflammatory cytokines (guillot et al., 2005) . concurrent with tlr3 activation, we found a significant up-regulation of ifnβ, but not ifnα mrna expression, at 9 h after ibv infection in cek cells. likewise, the lack of tlr7 induction correlated with no change in the expression of pro-inflammatory cytokines, namely il-1β, il-8 and nlrp3 in ibv infected cek cells. in contrast to our findings, a recent study found that ifn-β transcription was not found in cek cells until 12 h after infection with ibv m41 at moi of 0.1 (kint et al., 2015) . this difference may have been due to variation in the moi used and origin of cells. notably, expressions of all the innate immune genes tested were down-regulated at 24 h after ibv infection in cek cells. this could be a consequence of host shut-off induced by ibv virus replication. corona viruses (covs) are known to down-regulate host gene expression and increased host mrna degradation in order to suppress host innate immune responses (kamitani et al., 2006; tohya et al., 2009) . corona virus nucelocapsid (n) protein has been shown to interfere with the 2 0 ,5 0 -oligoadenylate synthetase/rnasel (2 0 -5 0 oas) activation, which is responsible for type i ifn induction and can also inhibit the production of various pro-inflammatory cytokines and chemokines via global translational shutdown (ye et al., 2007; zhong et al., 2012b) . however, mda5 and ifn-β expression was also up-regulated in tocs at 24 h after ibv infection suggesting a sustained immune response in tocs compared with cek cells. we found a similar virus replication pattern in ceks and tocs with a comparable virus output. higher levels of apoptosis and ifn-β expression is also associated with significantly lower infectious virus particle production from both cek cells and tocs. this could be due to increased antiviral state induced by ifn-β and/or increased apoptosis. apoptosis of cultured cells have previously been shown to be associated with reduced production of infectious influenza virus in duck cells (kuchipudi et al., 2012a) . several in vitro and ex vivo studies have suggested a key role of tlr3 in viral detection (alexopoulou et al., 2001; bowie and haga, 2005; finberg and kurt-jones, 2004; tabeta et al., 2004) . however, recent evidence suggests tlr3 contributes to pathogenic effects of viral infections. tlr3 mediates west nile virus entry into the brain, causing lethal encephalitis (wang et al., 2004 ) and contributes to a detrimental inflammatory response to influenza virus infection in mice, resulting in acute pneumonia (le goffic et al., 2006) . a recent study has shown that mda5, but not tlr3, is involved in the sensing of ibv (kint et al., 2015) . however, higher mrna levels of mda5 and tlr3 correlated with the in vivo pathogenicity of the ibv strains used in this study, such that the nephropathogenic and tracheotropic ibvs induced much higher levels in cek cells and tocs, respectively. it is possible that mda5 activation is a protective antiviral host response against the virus whereas tlr3 contributes to some detrimental effects from viral infection. the precise role of mda5 and tlr3 in host defence against ibv infection and/or disease pathogenesis is not yet fully understood and hence, warrants further in-depth studies. we found that lgp2 expression was significantly increased at 9 h but down-regulated at 24 h after ibv infection in cek cells. lgp2 functions as a negative regulator by interfering with the recognition of viral rna by rig-i and mda5 (yoneyama et al., 2005) . mda5 is ifn-inducible (kang et al., 2004) , so once an ifn response is triggered, this innate antiviral loop initiates autoamplification of mda5 until the natural inhibitor lgp2 is induced (yoneyama et al., 2005) . however, we found that the expression of lgp2, mda5 and ifn-β mrna levels were down-regulated at 24 h after ibv infection, possibly due to the virus induced host gene shutoff. in agreement with our findings, a recent study also reported a down-regulation of tlr3, il-1β and ifn-γ expression in ibv infected chicken tracheas at 12 hpi (kameka et al., 2014) . in this study, we infected cells at moi of 1.0; however, infection at a lower moi could be helpful to profile the effect of lgp2 on mda5 and ifn-β regulation in ibv infected cells. there have been conflicting reports on the difference in virulence among ibv strains using this in vitro system. cook et al. (1976) did not find marked differences when compared three strains of ibv on the basis of their effect on tracheal cilia. raj and jones (1996) also reported little difference among several ibv strains using measurement of ciliary activity as a criterion for damage to the tracheal epithelium. however, abd el rahman et al. (2009) has revealed that the strains differed in their efficiency to infect the tracheal epithelium. infection by beaudette and qx resulted in a larger number of infected cells compared to itlay-02 and 4/91 strains, when tracheal organ cultures (tocs) were infected with the same amount of virus (abd el rahman et al., 2009) . in summary, our study found that greater levels of apoptosis and elevated expression of tlr3, mda5 and ifn-β is associated with increased pathogenicity of ibvs in kidney (cek cells) and respiratory (tocs) tissues. the findings of this study using cek cells and tocs raise a strong possibility that host innate immune response could aid generation of a predictive prognosis for the tissue tropism of novel ibv strains. however, further studies are needed to confirm the association between pathogenicity and host response in vivo and to establish the mechanisms underlying such responses. relative mrna expression was determined by real-time pcr normalised to 18s rrna. graph values are the mean of three biological replicates with error bars as standard error, and are expressed as fold change relative to the mocked-infected group. different letters indicate that the differences between groups at that time point are significant (po 0.05). monolayers of primary chicken kidney (cek) cells were prepared from kidneys of specific-pathogen-free (spf) chicken embryos after 18 day incubation (lohmann, germany). kidneys removed aseptically were collected in minimum essential medium (mem), supplemented with 0.5% fetal bovine serum (sigma) and 1% antibiotics (penicillin and streptomycin). the kidneys were minced into small pieces using sterile scissors, followed by trypsinization (0.25% trypsin) for 30 min at 37°c and filtered through a 100 mm mesh to remove tissue debris. after centrifugation at 1500 g for 10 min, cell pellet was re-suspended in the mem medium and seeded at 1 â 10 5 cells/cm 2 in cell culture plates. we used a traditional ibv strain m41, and two variant ibv strains namely is/885/00-like (885) (awad et al., 2013) and qx-like strain kg3p (ganapathy et al., 2012) were used. is/885/00 causes mortality, poor weight gain and severe renal damage, while qx causes renal and reproductive problems. allantoic fluid containing these ibv strains were propagated in tocs prepared from 19 to 20-day old spf chicken embryos (cook et al., 1976) . for the cek cell infection study, all ibv strains were titrated on cek cells using an immuno-cytochemical focus assay described previously (kuchipudi et al., 2012a) . in brief, serial 2-fold dilutions of a known volume of toc supernatants were used to infect cek cells in 96-well culture plates (cellstar, grenier bio-one, uk). cells were washed after a 2 h incubation with virus, followed by a further 4 h incubation, and then fixed in 1:1 acetone:methanol for 10 min. cells were subjected to viral nucleoprotein detection by anti-ibv nucleoprotein monoclonal antibody (prionics, uk) followed by visualization with envisionþsystem-hrp (dab; dako, ely, uk). cells expressing viral nucleoprotein (np) were counted, and the mean number of positive cells in five fields was used to calculate focus-forming units of virus per microlitre of inoculum. for toc infection study viruses were titrated in chicken embryo tocs and expressed in median ciliostatic doses (cd 50 )/ml (cook et al., 1976) . in brief, for each dilution of virus, three toc rings with 100% of the cilia beating were used. tocs were inoculated with viruses (0.1 ml) in serial dilutions before being incubated at 37.0°c in a rotating incubator (lab thermal equipment, greenfield, nr oldham) at 8 rpm. the tocs were observed for ciliary activity post-infection, a reduction in at least 5% mean activity compared with mock infected were considered as positive for ibv infection (cook et al., 1976) and further confirmed by rt-pcr followed by sequencing . cek cells were seeded on cell culture plates (cellstar, grenier bio-one, uk) at a density of 1 â 10 5 cells/cm 2 . after incubation at 37°c for 48 h, 80-90% confluent monolayers were washed three times with phosphate-buffered saline (pbs), and then either infected with different ibv strains at multiplicity of infection (moi) 1.0 or mock infected with cell culture medium. triplicate wells were used for each virus or mock infection for each time point. cells were analyzed at 24 and 48 h post-infection (hpi) to determine the effect of virus infection on cell metabolic activity and induction of apoptosis. cell culture supernatant was titrated for infectious virus in toc expressed as cd 50 /ml (cook et al., 1976) . at 9 and 24 hpi cell pellets were collected in rlt buffer (qiagen, uk) for total rna extraction and cell metabolic activity was analysed at 24 and 48 hpi using celltiter 96, a nonradioactive cell proliferation assay (promega, madison, wi, usa), according to the manufacturer's instructions. tocs were infected 5 days after preparation to allow the early inflammatory responses of the tissue to subside (reemers et al., 2009) . tocs were infected with ibv strains using a dose of 4 log 10 cd 50 per individual ring, or mock-infected with toc medium without virus. three rings were used for each virus or mock infection for each time point. the infection dose of 4 log 10 cd 50 was chosen based on a preliminary study wherein this dose caused 100% cilliostasis in toc by day 3 after infection. samples of toc supernatant or complete rings were collected at 9, 24 and 48 hpi. toc rings were analysed for apoptotic cells using in situ terminal deoxynucleotidyl transferase dutp nick end labelling (tunel) assay, and supernatants were titrated for quantification of infectious virus at 24 and 48 hpi. total rna extracted from toc rings collected at 9 and 24 hpi were used for the quantification of host gene expression analysis. at 24 and 48 h after virus or mock infection, cek cells in 24well tissue culture plates were analysed to quantify the percent of apoptotic cells. briefly, cells were washed in pbs and removed from the tissue culture plate by trypsinization, followed by inactivation with growth medium containing serum. the cells were then washed, resuspended in binding buffer (clontec, ca), and subsequently analysed by staining with annexin v-fitc and pi according to the manufacturer's instructions (apoalert s , annexin v-fitc apoptosis kit, clontech, ca) using flow cytometer (bd accuri c6, san jose, ca). fluorescence was quantified using cflow software (bd accuri). the single-dye positive controls were prepared by staining representative cells with fitc and pi dye individually and used for estimation of proper colour compensation. unstained cell samples were used as a negative control to adjust the threshold. apoptotic cells were detected and quantified by the tunel assay using the peroxidase (pod) in situ cell death detection kit (roche diagnostics, indianapolis, in, usa) according to the manufacturer's instructions. a total of five sections were analyzed for each time point. briefly, cells were fixed in paraformaldehyde and permeabilized with 0.1% triton x-100 at room temperature. after equilibration, specimens were overlaid with 50 μl tunel reaction mixture or 50 μl label solution of the no-enzyme control. after initial incubation for 1 h at 37°c in a dark humidified atmosphere, reactions were then developed using peroxidase substrate kit dab (vector laboratories, burlingame, usa). sections were counterstained with haematoxylin (merck, germany) and mounted with dpx (dibutyl phthalate, polystyrene granules and xylene) aqueous mounting medium (vwr international, leuvan). for each sample, the total number of positive cells/400 â microscopic field was calculated. supernatants from virus infected cek cells and tocs were titrated for infectious virus and expressed as cd 50 /ml (cook et al., 1976) . in brief, tocs were inoculated with 0.1 ml of supernatant in serial dilutions, and three rings with 100% of the cilia beating were used for each dilution and incubated at 37.0°c in a rotating incubator (lab thermal equipment, greenfield, nr oldham) at lowest rotation speed. the tocs were observed for ciliary activity; those exhibiting less than 5% mean activity were recorded as ibv positive. total rna from cell pellets in rlt buffer (qiagen, uk) and toc ring was extracted using rneasy plus mini-qiashredder kit (qiagen, uk) following the manufacturer's instructions. rna concentration was quantified by nanodrop s nd-1000 (nanodrop, wilmington, de/ nanodrop nd-1000; peqlab). the cdna was generated from 1 mg of rna using the superscript iii first-strand cdna synthesis system (life technologies) with random primers as per the manufacturer's recommendations. the generated cdna samples further diluted to 1:50 in nuclease-free water were used in quantitative reverse transcription pcr (qrt-pcr) analysis. qrt-pcr was performed on the lightcycler s 480 (roche, uk), using lightcycler 480 sybr green i master mix (roche, uk). primers were designed with primer express version 2.0 (applied biosystems), based on previously reported sequences (table 1) . all primers were provided by eurofins genomics (edersberg, germany). the cycling conditions were 10 min at 95°c, 45 cycles of 10 s at 95°c, 10 s at 60°c annealing temperature and 10 s at 72°c, finally followed by a melting curve analysis to ensure the specificity of the sybr green pcr. qrt-pcr data was normalised using a relative standard curve method to 18s ribosomal rna (18srrna) expression (kuchipudi et al., 2012b) and the data were presented as fold difference in gene expression of virus versus mock infected samples. the data were analysed using one-way anova, followed by the post-hoc lsd multiple comparison test using graphpad prism version 6 software. differences between groups at that time point were considered significant at p o0.05. table 1 primers used in the study. primer sequences: sense (s) and anti-sense (as) 18s rrna (18s ribosomal rna ) (kuchipudi et al., 2012b) (s) tgtgccgctagaggtgaaatt (as) tggcaaatgctttcgcttt lgp2 (laboratory of genetics and physiology 2) g.k., s.v.k, and r.c conceived and designed the study. g.k supervised r.c who carried out all the experiments. s.v.k and r.c analyzed the data and wrote the manuscript with editorial contributions from all other authors. comparative analysis of the sialic acid binding activity and the tropism for the respiratory epithelium of four different strains of avian infectious bronchitis virus emergence of a novel genotype of avian infectious bronchitis virus in egypt toll-like receptors and innate immunity recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 detection of variant infectious bronchitis viruses in broiler flocks in libya immunopathogenesis of infectious bronchitis virus related to is/1494/06 and is/885 in specific pathogen-free chicks evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in 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recent progress in studies of arterivirus-and coronavirus-host interactions rajesh chhabra is a commonwealth scholar, funded by the uk government (incs-2012-180). key: cord-329245-6tj2k1yn authors: corse, emily; machamer, carolyn e title: the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction date: 2003-07-20 journal: virology doi: 10.1016/s0042-6822(03)00175-2 sha: doc_id: 329245 cord_uid: 6tj2k1yn abstract virus-like particle (vlp) formation by the coronavirus e and m proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. we studied interactions between the infectious bronchitis virus (ibv) e and m proteins using in vivo crosslinking and vlp assembly assays. we show that ibv e and m can be crosslinked to each other in ibv-infected and transfected cells, indicating that they interact. the cytoplasmic tails of both proteins are important for this interaction. we also examined the ability of the mutant and chimeric e and m proteins to form vlps. ibv m proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support vlp formation, regardless of their ability to be crosslinked to ibv e. interactions between the e and m proteins and the membrane bilayer are likely to play an important role in vlp formation and virus budding. enveloped viruses acquire their lipid membranes from those of the host cell in a process known as budding, in which the virus envelope proteins accumulate at the appropriate membrane and cooperate with the nucleocapsid and other viral components to induce membrane curvature and pinching (garoff et al., 1998) . most well-studied enveloped viruses, such as influenza and retroviruses, bud from the plasma membrane of cells, which results in the release of virus particles from the host cell as soon as the pinching process is complete. however, the envelopes of other viruses are derived by budding into the lumen of intracellular secretory compartments, such as the endoplasmic reticulum (er) and golgi apparatus (griffiths and rottier, 1992) . intracellular budding is a seemingly more complex way of obtaining a lipid envelope, since virus particles are contained within intracellular membrane-bound compartments as a result of budding. the newly budded viruses presumably exploit the cellular secretory pathway to exit the cell for a new round of infection. the reasons that some enveloped viruses have evolved to bud inside the cell are not known. several clinically important viruses bud into intracellular membranes. viruses of the family flaviviridae, which include hepatitis c virus and kunjin virus, derive their lipid envelopes by budding into the lumen of the er (dubois-dalcq et al., 1984; mackenzie and westaway, 2001) . as another example, the hemorrhagic fever-causing hantaviruses, members of the family bunyaviridae, bud into the membranes of the golgi complex (hung et al., 1985; pettersson, 1991) . the advantage provided by intracellular budding remains an interesting and important question in the biology of these and other viruses that obtain their envelopes from intracellular membranes. coronaviruses are positive-stranded rna viruses that acquire their membrane envelope by budding into the lumen of a pre-golgi compartment or cis-golgi network (cgn) (klumperman et al., 1994) . accumulating evidence indicates that the e and m proteins are instrumental in this process. rottier and colleagues made the initial observation that mouse hepatitis virus (mhv) e and m proteins are sufficient to produce membrane-bound particles very similar to virions in size and shape (vennema et al., 1996) , and this result has been confirmed with the e and m proteins of several other coronaviruses (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000) . these observations suggest that coronavirus budding is directed by envelope proteins alone, which is an unusual type of enveloped virus assembly (garoff et al., 1998) . the coronavirus e protein appears to play a critical role in the budding step, since mutations introduced into the cytoplasmic tail of mhv e by targeted rna recombination result in elongated virions (fischer et al., 1998) . indeed, it has been shown that both the mhv e and the infectious bronchitis virus (ibv) e proteins are sufficient for formation of the virus-like particles (vlps) described above (corse and machamer, 2000; maeda et al., 1999) , although the efficiency probably varies with cell type and protein expression system. we are interested in understanding the mechanisms by which the avian coronavirus ibv selects a bud site and assembles in cgn membranes. we have previously shown that the ibv m and e proteins independently localize to the golgi region of transfected cells (corse and machamer, 2000; machamer and rose, 1987) and characterized the golgi targeting signals in the two proteins that direct this localization (corse and machamer, 2002; machamer et al., 1993) . in the study described here, we examined interactions between the ibv e and m proteins in transfected ost7-1 cells using the chemical crosslinker dithiobis[succinimidyl propionate] (dsp). using mutant e and m proteins that are correctly targeted to the golgi complex, we found that the cytoplasmic tails of both proteins are required for their interaction. we found that ibv e and m proteins can be efficiently assembled into vlps in ost7-1 cells, and we investigated which domains of ibv e and m are required for this process. interestingly, the interaction of m and e, as measured by crosslinking, was not sufficient for vlp formation. it is likely that interactions of e and m with each other as well as the membrane bilayer are important for vlp formation and virus budding. although the ibv e protein is distributed throughout the golgi stack when expressed alone machamer, 2000, 2001) , the ibv e and m proteins exactly colocalize in ibv-infected vero cells examined by indirect immunofluorescence at early times postinfection (fig. 1a) . we wanted to investigate the possibility that this precise colocalization was indicative of a physical interaction between the two proteins. since attempts to coimmunoprecipitate e and m proteins from ibv-infected vero cells in a variety of detergent conditions were unsuccessful, we employed the cellpermeable chemical crosslinker dsp, which is a thiol-cleavable molecule that contains two amine-reactive groups separated by a 12 å spacer arm. ibv-infected vero cells were radiolabeled at 45 h postinfection and treated with dsp prior to lysis and immunoprecipitation with anti-e or anti-m antibodies. when we analyzed the immunoprecipitates by sds-page, we found that m was present in the anti-e immunoprecipitations and vice versa (fig. 1b, lanes 4 and 8) . this association was dependent on the presence of dsp since it was not seen in samples that were mock-treated (lanes 2 and 6). immunoprecipitation with irrelevant antibodies yielded no visible bands (not shown). in the absence of ␤-mercaptoethanol, which cleaves the disulfide bond of dsp, the unseparated crosslinked e and m proteins were present as high molecular weight aggregates and migrated at a high position on the gel (not shown). the crosslinking and coimmunoprecipitation was not quantitative, since only a fraction of the total e protein (as measured by the amount of e in the anti-e immunoprecipitation) was found in the anti-m immunoprecipitation. this could be due to the inherent inefficiency of chemical crosslinking. alternatively, this result could reflect the small e:m ratio in virions (liu fig. 1 . ibv e and m colocalize and interact in ibv-infected vero cells. (a) ibv-infected vero cells were fixed for immunofluorescence at 6 h postinfection and double labeled with rat anti-e antibody (a) and rabbit anti-m antibody (b). secondary antibodies were fluorescein-conjugated goat antirat igg and texas red conjugated donkey anti-rabbit igg. panel c is a differential interference contrast (dic) image of the labeled cells. bar, 15 m. (b) ibv-infected vero cells were labeled with [ 35 s]methionine-cysteine at 45 h postinfection, treated with dsp as indicated, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-15% page, in the presence or absence of ␤-mercaptoethanol (␤me) as indicated, and fluorography. ibv e is coprecipitated with ibv m after crosslinking and vice versa. these data are representative of at least three independent experiments. and inglis, 1991), which is presumably determined by the stoichiometry of envelope protein interactions. we conclude that the ibv e and m proteins interact in infected cells. ibv-infected cells contain other viral proteins which might be required for interaction of e and m. to determine if the ibv e and m proteins could interact directly, we coexpressed them in ost7-1 cells. we used indirect immunofluorescence to verify coexpression and correct intracellular localization of the two proteins (fig. 3a , panels a-c). as expected, the ibv e and m proteins colocalized in a compact perinuclear region characteristic of the golgi complex in this cell type, as seen previously in bhk-21 cells (corse and machamer, 2000) . when we treated ost7-1 cells coexpressing ibv e and m with dsp and analyzed anti-e and anti-m immunoprecipitates as described above, we found that m protein was present in the anti-e immunoprecipitate (fig. 2, lane 4) and that e protein was present in the anti-m immunoprecipitate (fig. 2, lane 8) , suggesting that the two proteins directly interact in transfected cells. detection of the interaction required crosslinking (compare lanes 3 and 7 to lanes 4 and 8) and antisera specific for e or m. even though less e was expressed in transfected cells (fig. 2 ) compared to ibv-infected cells (fig. 1b) , the ratio of e:m crosslinks (as seen in the relative amounts of e and m in the anti-m and anti-e immunoprecipitations, respectively) was similar, suggesting that there is a precise stoichiometry of interaction. to investigate which domains of the e and m proteins interact, we made use of a panel of mutant e and m proteins that were generated to study the golgi targeting signals present in the two proteins (corse and machamer, 2002; machamer and rose, 1987) . although many mutant e and m proteins have been generated, in this study, we used only those that are properly targeted to the golgi region. since these mutant proteins had previously been localized in bhk-21 or cos-7 cells, we first confirmed that they were properly targeted in ost-7 cells. cells coexpressing wildtype m protein and e mutants were analyzed by indirect immunofluorescence microscopy (fig. 3a) . eg3 (panels d-f) contains the transmembrane domain from the plasma membrane protein vesicular stomatitis virus (vsv) g; cte (panels g-i) is an n-terminal truncation of ibv e consisting only of its cytoplasmic tail, and get (panels j-l) is a chimeric protein containing the lumenal and transmembrane domains of vsv g and the cytoplasmic tail of ibv e. in all cases, the e mutant proteins were correctly localized to the golgi region of ost7-1 cells (including the partial transmembrane replacement mutants eg1 and eg2, not shown), consistent with our previous studies in bhk-21 cells (corse and machamer, 2002) . fig. 3b shows cells that are coexpressing wild-type e protein and the m protein deletion mutants mct⌬1 (panels a-c), which is missing cytoplasmic tail amino acid residues 103-203, mct⌬2 (panels d-f), which is missing cytoplasmic tail residues 119 -203, mct⌬3 (panels g-i), which is missing cytoplasmic tail residues 103-118, or m⌬m2,3 (panels j-l), which is missing the second and third transmembrane domains (amino acid residues 43-101). the m mutants, especially mct⌬3 (fig. 3b , panel h) and m⌬m2,3 (fig. 3b , panel k), were incompletely localized to the golgi region, since some er staining was observed. however, they all reached the golgi to some extent, as indicated by the juxtanuclear spot. we asked whether the e transmembrane replacement mutants eg1, eg2, and eg3 (in which the first third, the first two-thirds, or the entire e transmembrane domain, respectively, were replaced with the corresponding regions of the vsv g transmembrane domain) (corse and machamer, 2002) could be crosslinked to wild-type m protein. the appropriate proteins were coexpressed and immunoprecipitated with anti-e or anti-m antibodies after treating the cells with dsp as described above (fig. 4a ). all three e transmembrane replacement mutants were crosslinked to m protein, suggesting that the e transmembrane domain is not required for e and m interaction. however, there was less m in the anti-e immunoprecipitation of eg3 (fig. 4a , lane 4), suggesting that the interaction of m with this mutant could be decreased. perhaps the e and m transmembrane domains interact, or the conformation of the cytoplasmic fig. 2 . ibv e and m interact in transfected ost7-1 cells. ost7-1 cells expressing e and m proteins alone or together were labeled with [ 35 s]methionine-cysteine, treated with dsp as indicated, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. all immunoprecipitates in this and the following figures were deglycosylated by treating with n-glycanase to collapse the m protein to a single band. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. these data are representative of at least three independent experiments. tail of e is altered when the complete transmembrane domain is replaced (see below). to evaluate the role of the e cytoplasmic tail in interaction with m, we tested whether the chimeric protein get (which consists of the cytoplasmic tail of ibv e and the transmembrane and lumenal domains of vsv g; see eg3 and wild-type m protein (d-f), cte and wild-type m protein (g-i), or get and wild-type m protein (j-l) were fixed for immunofluorescence and double labeled with rat anti-e antibody (a, d, g, and j) and rabbit anti-m antibody (b, e, h, and k). secondary antibodies were fluorescein-conjugated goat anti-rat igg and texas red conjugated donkey anti-rabbit igg. the third image in each row (c, f, i, and l) is a dic image of the labeled cells. in the diagrams of the e mutant and chimeric proteins, ibv e sequence is shown in black and vsv g sequence is shown in gray. bar, 10 m. (b) ost7-1 cells expressing wild-type e protein and mct⌬1 (a-c), mct⌬2 (d-f), mct⌬3 (g-i), or m⌬m2,3 (j-l) were fixed for immunofluorescence and double labeled with rat anti-e antibody (a, d, g, and j) and rabbit anti-m antibody (b, e, h, and k). secondary antibodies were fluorescein-conjugated goat anti-rat igg and texas red conjugated donkey anti-rabbit igg. the third image in each row (c, f, i, and l) is a dic image of the labeled cells. in the diagrams of the deletion mutant m proteins, the thin lines indicate deleted sequence. bar, 10 m. ost7-1 cells expressing wild-type m protein and wild-type e protein or the e transmembrane replacement mutant proteins eg1, eg2, or eg3 were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. (b) ost7-1 cells coexpressing the get chimera and wild-type m protein were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-15% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. (c) ost7-1 expressing cte protein alone or with m protein were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. these data are representative of at least three independent experiments. the e cytoplasmic tail and thus the get chimera. the reciprocal immunoprecipitation with anti-m antibody contained get protein (lane 3). we conclude that the cytoplasmic tail of ibv e protein is sufficient for interaction with m protein. indeed, the n-terminal truncation mutant cte, which consists only of the cytoplasmic tail of ibv e (corse and machamer, 2002) , was coprecipitated with m after crosslinking (fig. 4c, lane 2) . we previously found that partial or complete removal of a 50 amino acid region within the 80 amino acid e cytoplasmic tail resulted in transport of the get chimera to the cell surface (corse and machamer, 2002) . therefore, we did not attempt to find a specific region within the ibv e cytoplasmic tail that interacts with ibv m, because colocalization of the ibv e and m proteins is likely to be required for their interaction. to determine which regions of the ibv m protein were involved in interaction with ibv e, we tested how well the m deletion mutants mct⌬1, mct⌬2, mct⌬3, and m⌬m2,3 could be crosslinked to wild-type e protein compared to wild-type m protein (fig. 5) . m⌬m2,3 is missing the second and third transmembrane domains of ibv m; since it was crosslinked to e protein as well as wild-type m (lanes 5 and 10), we conclude that these membrane-spanning regions are not involved in interactions with e. a large portion of the m cytoplasmic tail is deleted in mct⌬1 (see fig. 3b ), and this protein was not crosslinked efficiently to e, compared to wild-type m protein (fig. 5, lanes 2 and 7) , although a small amount of e protein is visible in the mct⌬1 immunoprecipitation (fig. 5, lane 7) . this result indicates that the m cytoplasmic tail is involved in interaction with e. the portion of the m cytoplasmic tail removed by the mct⌬1 deletion is split into parts by the mct⌬2 and mct⌬3 deletions. note that the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wild-type m and the mct⌬3 and m⌬m2,3 mutants (compare lanes 7 and 8 with lane 6) because they have fewer methionines and cysteines due to their deletions, which could explain why a reciprocal amount of mct⌬1 is not seen in the anti-e immunoprecipitation (fig. 5, lane 2) . mct⌬3, which is missing amino acids 103-118 of the m cytoplasmic tail, was crosslinked to e (fig. 5, lanes 4 and 9) , while mct⌬2, which is missing amino acids 119 -203 of the m cytoplasmic tail (see fig. 2b ), was not (fig. 5, lanes 3 and 8) . this indicates that the amino acids removed by the mct⌬2 deletion (119 -203) are required for crosslinking of the ibv m protein to the ibv e protein. the e and m proteins of several coronaviruses are released from cotransfected cells in membrane-bound particles that are morphologically similar to virions (vlps), suggesting that interactions between these proteins are an integral part of coronavirus assembly (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000; vennema et al., 1996) . we initially attempted to study vlps formed in bhk cells from ibv e and m proteins expressed by coinfection with recombinant vaccinia viruses encoding each protein. however, we found that vlp formation was extremely inefficient under these conditions and estimated that only 0.01% of the cellular e and m proteins were released into the supernatant as vlps (corse and machamer, 2000) . since we were interested in using vlp formation to delineate portions of the ibv e and m proteins that are important in virus assembly, we employed the expression system used by rottier and colleagues in their initial description of mhv vlps (vennema et al., 1996) . these authors infected ost7-1 cells (elroy-stein and moss, 1990) , which are osteosarcoma cells that stably express phage t7 rna polymerase, with a vaccinia virus encoding t7 polymerase (vtf7-3) (fuerst et al., 1986) to provide higher levels of t7 rna polymerase. they then transfected the cells with plasmids encoding mhv e and m behind the t7 promoter. when we expressed the ibv e and m proteins this way, we found that vlps were formed efficiently (fig. 6, lanes 3 and 14) . the supernatant side of the gel (lanes 12-22) shown in this figure was exposed 20 times longer than the cell side to allow the visualization of e and e transmembrane mutant proteins (lanes 1-11). we have consistently observed at least 10%, and frequently more, of the cellular m protein being released into the supernatant during the 3 h chase (data not shown). this level of vlp release was dependent on e protein, since less than 1% of the cellular m protein was found in the supernatant in the absence of e. e protein was not visualized in the supernatants when expressed alone (fig. 6, lane 12) . we believe that a small amount of e is released into the supernatant when it is expressed alone, and that it is simply not visualized unless a large amount of supernatant is loaded on the gel, as in our previous experiments (corse and machamer, 2000) . our results differ from the data of makino and colleagues, who observed a substantial amount of mhv e being released into the supernatant when expressed by itself (maeda et al., 1999) ; this discrepancy could be explained by the different expression system used to produce the e protein in their study. we favor the hypothesis that vlp formation involves a lattice of m protein within the membrane with curvature induced by small amounts of the e protein (see discussion). this would account for the small amount of ibv e protein relative to ibv m protein that is found in vlps. we found that the partial or complete replacement of the ibv e transmembrane domain (mutants eg1, eg2, and eg3) had no effect on e's ability to induce vlps as measured by the amount of ibv m protein released into the supernatant (fig. 6 , compare the m protein bands in lanes 14, 16, 18, and 20). we did notice that more eg2 and eg3 proteins were incorporated into vlps compared to wildtype e and eg1 proteins (fig. 6 , compare the e protein bands in lanes 18 and 20 with those in lanes 14 and 16). it is possible that conformational changes induced by replacement of the transmembrane domain facilitates increased incorporation of these mutants into vlps. the eg2 and eg3 mutants did not induce vlps more efficiently than wild-type e, because an increased amount of m protein was not released into the supernatant. we examined the ability of the e cytoplasmic tail to induce vlps by using the chimeric protein get. when expressed by itself, we observed that a negligible amount of this protein was released into the supernatant (fig. 6 , lane 21; recall that the supernatant side of the gel was exposed 20 times longer than the cell side) and that expression of get with ibv m did not increase the amount of m that was released when m was expressed alone (fig. 6, lanes 22 and 13) . we conclude that the get chimera does not induce vlps. since both the eg3 and the get chimeras contain the transmembrane domain of vsv g and the cytoplasmic tail of ibv e, it seems likely that the vsv g lumenal domain is incompatible with vlp formation, either because it is too large or because it cannot be incorporated into the m protein lattice for some other reason (see discussion). thus, it is difficult to predict if the e cytoplasmic tail alone is capable of inducing vlps. we also did not observe vlp formation with the ibv e n-terminal truncation mutant cte, which consists only of the e cytoplasmic tail (data not shown). however, this result is inconclusive since the cte protein is less stable than wild-type e protein and thus accumulates to significantly lower levels (corse and machamer, 2002) . we evaluated the incorporation of the ibv m deletion mutants mct⌬1, mct⌬2, mct⌬3, and m⌬m2,3 into vlps. as shown in fig. 7 , none of the deletion mutants were able to support vlp formation at a detectable level, regardless of whether they could be crosslinked to ibv e. the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wildtype m and the mct⌬3 and m⌬m2,3 mutants (compare lanes 5,6,7, and 8 with lanes 2 and 3) because they have fewer methionines and cysteines due to their deletions. however, we would still expect to see a detectable signal in the supernatants after a long exposure if these mutants supported vlp formation at a level comparable to wild-type m. since we have never seen such a signal, we conclude that these mutants do not support vlp formation. these results indicate that both the cytoplasmic tail and the transmembrane regions of ibv m are involved in vlp formation, which is consistent with the results of rottier and colleagues, who found that mutations in all domains of the mhv m protein abrogated vlp formation (de haan et al., 1998) . these results suggest that a direct interaction between ibv e and m proteins is not sufficient for vlp formation and that this process may depend on how the two proteins interact with the membrane bilayer, both separately and together. we have demonstrated an interaction between the ibv e and m proteins using both in vivo chemical crosslinking and vlp formation as assays. using ibv e and m protein mutants that were generated in the course of golgi targeting studies, we examined the domains in the ibv e and m proteins that are involved in their interaction. we found that the cytoplasmic tails of both proteins mediate their interaction, as measured by crosslinking. interestingly, however, we observed that reciprocal crosslinking of ibv e and m protein mutants did not correlate with vlp formation, suggesting that interaction between ibv e and m is not sufficient for vlp formation. here we compare our results to those reported by other groups and discuss the implications of our findings for coronavirus assembly. lim and liu previously used a coimmunoprecipitation assay to detect ibv e and m protein interactions (lim and liu, 2001) . they also concluded that the cytoplasmic tail of ibv e is important for interaction with ibv m, since dele-tion of a 20 amino acid region within the e cytoplasmic tail (amino acids 37-57) prevented coimmunoprecipitation of the e and m proteins and vlp formation. as mentioned above, we did not attempt to find a region within the e tail that mediates its interaction with ibv m, because we previously found that partial or complete replacement of a 50 amino acid region in the ibv e tail results in transport of a reporter chimera from the golgi complex to the cell surface, which indicates that the cytoplasmic tail of e contains golgi targeting information (corse and machamer, 2002) . our subcellular localization results differ from those of lim and liu, who reported that ibv e is localized to the endoplasmic reticulum of transfected cells (lim and liu, 2001) , possibly because they examined cells overexpressing epitope-tagged ibv e protein. the m protein is by far the most abundant envelope protein of coronaviruses and has been estimated to account for ϳ40% of the mass of ibv and mhv particles (stern et al., 1982; sturman et al., 1980) . thus the physical nature of the coronavirus envelope must be largely determined by the characteristics of the m protein. interestingly, however, the m protein cannot drive coronavirus budding by itself, but displays a strict requirement for a small amount of e protein for vlp formation (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000; vennema et al., 1996) . we have observed this low e protein:m protein ratio in ibv vlps (fig. 6, lane 14) ; measurements of the amount of e and m proteins in ibv virions also reveal a relatively low amount of e protein (liu and inglis, 1991) . we favor the hypothesis that vlp formation involves a specific membrane curvature that is induced by the proper ratio of m and e proteins. the mechanism by which a small amount of e protein interacts with a large amount of m protein to induce invagination and pinching of the membrane to form vlps fig. 7 . cytoplasmic and transmembrane region deletion mutants of ibv m do not support vlp formation. ost7-1 cells expressing the indicated proteins were labeled with with [ 35 s]methionine-cysteine for 1 h and chased for 3 h, and the supernatants and cells were harvested and immunoprecipitated with anti-e and anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page and fluorography. the supernatant samples (lanes 12-22) were exposed to film 13 times longer than the cell samples (lanes 1-11) . the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wild-type m and the mct⌬3 and m⌬m2,3 mutants because they have fewer methionines and cysteines due to their deletions. these data are representative of at least three independent experiments. remains unclear, but its elucidation is likely to be essential for a complete understanding of coronavirus assembly. several studies have addressed the question of how the coronavirus e and m proteins interact in vlp formation. rottier and colleagues (de haan et al., 1998) studied the primary sequence requirements for incorporation of the mhv m protein into vlps. they found that all regions of mhv m seem to be important for vlp formation, since mutations in the short lumenal domain, the transmembrane region, and the cytoplasmic tail all impaired vlp formation to some extent. these results are consistent with our observation that deletions in both the cytoplasmic and the transmembrane domains of ibv m prevent vlp formation. interestingly, the extreme c-terminus of the cytoplasmic tail mhv m was found to be especially important, since deletion of only the c-terminal threonine residue prevented vlp formation (de haan et al., 1998) . we were unable to examine the role of the extreme c-terminus of the ibv m protein in vlp formation because our antibody to ibv m recognizes this sequence. laude and colleagues (baudoux et al., 1998) showed that coexpression of the e and m proteins of transmissible gastroenteritis virus (tgev) and of bovine coronavirus (bcv) resulted in vlp production. in this study vlps were also produced with a bcv-tgev chimeric m protein. interestingly, the chimeric m protein was able to form vlps with tgev e, bcv e, and bcv-tgev chimeric e proteins. since tgev and bcv are in different coronavirus groups based on serological and genomic sequence relationships, this suggests that vlp formation does not depend on interactions between e and m proteins that are based on strict sequence requirements. well-conserved three-dimensional characteristics of the e and m proteins may be instrumental in vlp formation. our results suggest that vlp formation involves more than an interaction between e and m proteins, since m protein mutants that could be crosslinked to e protein (mct⌬3 and m⌬m2,3; see fig. 5 ) are not incorporated into virus-like particles. it is possible that vlp formation requires interactions between m proteins, as proposed by rottier and colleagues . these workers demonstrated homotypic interactions among mhv m protein molecules by coimmunoprecipitation and incorporation of vlp assembly-incompetent mhv m mutants into vlps via their interaction with assembly-competent mhv m proteins. these authors hypothesize that the vlp (and virion) envelope mainly consists of an m protein lattice, with a few e proteins interspersed within this lattice. although we have not examined whether homotypic interactions between ibv m proteins exist, we think it possible that such interactions may be important in ibv vlp assembly. we did observe a small amount of sds-resistant ibv m protein dimers in our anti-m immunoprecipitations (indicated by asterisks in figs. 2, 4 , 5, and 6), although we have no direct evidence that these dimers are physiologically relevant. we also showed that the get chimera could be crosslinked to ibv m protein (fig. 4b ), but did not induce vlp formation (fig. 6 ). since the get chimera contains the same transmembrane domain and cytoplasmic tail as the transmembrane replacement mutant eg3, which was able to induce vlp formation as well as wild-type e protein (fig. 6) , a likely explanation is that the vsv g ectodomain prevents induction of vlps. one possibility is that this large ectodomain does not fit well into the lattice of m protein in the membrane. a similar observation was reported by rottier and colleagues , who showed that foreign proteins such as wild-type vsv g protein, equine arteritis virus m protein, and cd8 were effectively excluded from mhv vlps. likewise, it is possible that the m deletion mutants we tested in the vlp assay are not in the right conformation to fit into such a lattice of m protein, and thus, do not support vlp formation. as mentioned above, we have previously shown that the cytoplasmic tail of the ibv e protein is sufficient to localize it to the golgi complex and to mediate its redistribution with the golgi scaffold proteins gm130 and p115 during brefeldin-a treatment (corse and machamer, 2002) . since these and other golgi scaffold proteins are known to be part of a complex which is thought to mediate vesicle tethering during intra-golgi transport (barr et al., 1998; seemann et al., 2000a,b) , this led us to hypothesize that the ibv e protein may be targeted to the golgi apparatus via a direct interaction with one or more golgi scaffold proteins. furthermore, interaction of ibv e with the golgi scaffold could result in modulation of vesicular traffic, which might be advantageous for collecting coronavirus envelope proteins in a specific golgi compartment for assembly (corse and machamer, 2002) . since we have shown here that the cytoplasmic tail of ibv e is also important for interactions with ibv m, this raises the question of whether the e cytoplasmic tail can interact with m and the golgi scaffold simultaneously. if not, then it is possible that two functional pools of ibv e protein exist in an infected cell. one pool could interact with ibv m and the membrane at the assembly site to induce virus budding, while the other could associate with the golgi scaffold to influence vesicular traffic through the golgi complex. the latter interaction might aid in collecting viral membrane proteins for assembly at golgi membranes. since the small amount of ibv e protein found in virions (liu and inglis, 1991) and vlps (shown here) suggests that not much e protein is required to induce virus budding, maybe a second function of e protein, such as modulation of golgi transport, accounts for the apparent excess of ibv e protein in infected cells (corse and machamer, 2000) . ost7-1 cells (elroy-stein and moss, 1990) and vero cells were maintained in dulbecco's modified eagle's medium (dmem) containing 10% fetal calf serum (fcs) and antibiotics. the adaptation of ibv (beaudette strain) to vero cells has been described (machamer and rose, 1987) . the recombinant vaccinia viruses encoding phage t7 polymerase (vtf7-3), ibv m (vvibvm), and wild-type and mutant versions of ibv e (vvibve, vveg3, vvcte) have been described machamer, 2000, 2002; fuerst et al., 1986; machamer and rose, 1987) . growth and titering of recombinant vaccinia viruses were done as described (weisz and machamer, 1994) . the pbs/ibv e, pbs/eg1, pbs/eg2, pbs/eg3, pbs/ get, par/m and par/m⌬m2,3 plasmids have been described machamer, 2000, 2002; machamer and rose, 1987) . ibv m was subcloned into the bamhi site of pt7/t3-18 (brl) to create pt7/m. the m cytoplasmic tail deletion mutant plasmids par/mct⌬1, par/mct⌬2, and par/mct⌬3 were constructed by oligonucleotide-directed mutagenesis as described (machamer and rose, 1987) . mct⌬1 is missing amino acids 103-203, mct⌬2 is missing amino acids 119 -203, and mct⌬3 is missing amino acids 103-118. all three of these cytoplasmic tail deletion mutants contain the ibv m c-terminal 22 amino acids that are recognized by the polyclonal anti-m antibody (see fig. 3b ). par/e and par/cte were made by pcr addition of bamhi sites at both ends of the e and cte inserts. pbs/ibv e and pbs/cte (corse and machamer, 2002) , respectively, were used as templates for pcr. the bamhi-digested pcr products were cloned into the bamhi site of par2529, and subclones were screened for correct orientation by indirect immunofluorescence. the pt7/mc⌬1, pt7/mct⌬2, and pt7/mct⌬3 plasmids used in vlp experiments were made by pcr addition of ecori and xbai restriction sites, with the corresponding ar plasmids as templates. the t7/ m⌬m2,3 plasmid used in vlp experiments was made by subcloning the par/m⌬m2,3 bamhi fragment into the the bamhi site of pt7/t3-18. the polyclonal anti-m antibody and its affinity purification for use in immunofluorescence has been previously described (machamer and rose, 1987) . the rat and rabbit polyclonal antibodies to the c-terminal 14 amino acids of ibv e have been reported (corse and machamer, 2000) . the rabbit anti-vsv polyclonal antibody used to immunoprecipitate radiolabeled g and get proteins has been described (weisz et al., 1993) . texas red conjugated donkey anti-rabbit immunoglobulin g (igg) and fluorescein-conjugated goat anti-rat igg were from jackson immunoresearch laboratories, inc. (west grove, pa). vero cells were plated in 35-mm dishes 1 day before being infected with ibv (passage 12), as previously described (machamer and rose, 1987) . the cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (pbs) for 20 min at room temperature, permeabilized with 0.5% triton x-100, and stained as previously described (swift and machamer, 1991) with rat anti-e antibody and rabbit anti-m antibody. ost7-1 cells were plated on cover slips in 35-mm dishes 2 days before infection with vtf7-3, vvibve, vveg3, or vvcte at a multiplicity of infection of 20. adsorption was for 1 h at 37°c, and cells infected with vtf7-3 were transfected with 5 g of each expression plasmid using 20 l of lipofectin (invitrogen, carlsbad, ca) as directed by the manufacturer. at 3.5 to 4 h postinfection the cells were fixed and stained as described above. ost7-1 cells were plated in 35-mm dishes 2 days before being infected with vtf7-3 and transfected with the appropriate constructs as described above. in samples transfected with only a plasmid encoding ibv e or ibv m alone, an equal amount of empty vector plasmid was added so that an equal amount of plasmid dna was added to all samples. at 4 h postinfection, the cells were labeled with 100 ci of 35 s-promix (amersham pharmacia biotech, piscataway, nj) in 0.5 ml methionine-and cysteine-free medium for 1 to 1.5 h at 37°c. vero cells were plated in a 10-cm dish 1 day before being infected with ibv and labeled at 45 h postinfection for 1 h at 37°c with 500 ci of 35 s-promix. after being rinsed twice in room temperature pbs, cells were scraped into pbs, transferred to microfuge tubes, and the thiol-cleavable crosslinker dsp (pierce, rockford, il) was added to 1 mm after being prepared just before use as a 20 mm stock solution in dmso. for mock dsp treatments an equivalent amount of dmso was added. the cells were incubated with dsp for 10 min at room temperature, and the dsp was quenched by adding glycine to 40 mm. the cells were lysed in detergent solution [62.5 mm edta, 50 mm tris (ph 8), 0.4% deoxycholate, 1% np-40] containing protease inhibitors, and the postnuclear supernatants were immunoprecipitated with the appropriate antibodies in the presence of 0.2% sds as described (machamer and rose, 1987) . treatment of immunoprecipitates with n-glycanase was as previously described (machamer et al., 1990) . the samples were subjected to sds-page in the presence of ␤mercaptoethanol (except for the indicated samples in fig. 1b ) and the labeled proteins were visualized by fluorography. appropriate constructs were expressed in ost7-1 cells by infection with vtf7-3 and transfection as described above. at 3 h postinfection the cells were shifted from 37 to 32°c for 1 h and then radiolabeled at 32°c with 35 s-promix for 1 to 1.5 h as described above. after labeling the cells were chased in 1 ml of growth medium for 3 h at 32°c. the medium was microcentrifuged at 14,000 rpm for 20 min at 4°c to remove cellular debris, and the supernatants were immunoprecipitated with rat anti-e and rabbit anti-m antibodies in the presence of 1% triton x-100 and 0.2% sds. the cells were harvested by rinsing in pbs and lysing in detergent solution. the postnuclear supernatants were immunoprecipitated with anti-e and anti-m antibodies in the presence of 0.2% sds. the immunoprecipitates were treated with n-glycanase as described (machamer et al., 1990) . mapping the interaction between grasp65 and gm130, components of a protein complex involved in the stacking of golgi cisternae coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles infectious bronchitis virus envelope protein targeting: implications for virus assembly the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting coronavirus particle assembly: primary structure requirements of the membrane protein assembly of the coronavirus envelope: homotypic interactions between the m proteins assembly of enveloped rna viruses cytoplasmic expression system based on constitutive synthesis of bacteriophage t7 rna polymerase in mammalian cells analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase virus maturation by budding. microbiol assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein cell biology of viruses that assemble along the biosynthetic pathway morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome (hfrs). i. some peculiar aspects of the morphogenesis of various strains of hfrs virus coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins association of the infectious bronchitis virus 3c protein with the virion envelope retention of a cis golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain the e1 glycoprotein of an avian coronavirus is targeted to the cis golgi complex a specific transmembrane domain of a coronavirus e1 glycoprotein is required for its retention in the golgi region assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively release of coronavirus e protein in membrane vesicles from virus-infected cells and e protein-expressing cells protein localization and virus assembly at intracellular membranes matrix proteins can generate the higher order architecture of the golgi apparatus the role of the tethering proteins p115 and gm130 in transport through the golgi apparatus in vivo structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid a golgi retention signal in a membrane-spanning domain of coronavirus e1 protein nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes use of recombinant vaccinia virus vectors for cell biology oligomerization of a membrane protein correlates with its retention in the golgi complex this work was supported by national institutes of health grants gm42522 and gm64647. we thank the members of the machamer lab for critical reading of the manuscript. key: cord-329429-ur8g68vp authors: miłek, justyna; blicharz-domańska, katarzyna title: coronaviruses in avian species – review with focus on epidemiology and diagnosis in wild birds date: 2018-12-10 journal: j vet res doi: 10.2478/jvetres-2018-0035 sha: doc_id: 329429 cord_uid: ur8g68vp coronaviruses (covs) are a large group of enveloped viruses with a single-strand rna genome, which continuously circulate in mammals and birds and pose a threat to livestock, companion animals, and humans. covs harboured by avian species are classified to the genera gammaand deltacoronaviruses. within the gamma-covs the main representative is avian coronavirus, a taxonomic name which includes the highly contagious infectious bronchitis viruses (ibvs) in chickens and similar viruses infecting other domestic birds such as turkeys, guinea fowls, or quails. additionally, ibvs have been detected in healthy wild birds, demonstrating that they may act as the vector between domestic and free-living birds. moreover, covs other than ibvs, are identified in wild birds, which suggests that wild birds play a key role in the epidemiology of other gammacovs and deltacovs. development of molecular techniques has significantly improved knowledge of the prevalence of covs in avian species. the methods adopted in monitoring studies of covs in different avian species are mainly based on detection of conservative regions within the viral replicase, nucleocapsid genes, and 3’utr or 5’utr. the purpose of this review is to summarise recent discoveries in the areas of epidemiology and diagnosis of covs in avian species and to understand the role of wild birds in the virus distribution. among the most abundant viruses infecting a wide variety of animals, including birds and humans are representatives of the large coronaviridae family. their virions contain the largest single-stranded positive sense rna (ssrna) genome, the feature which distinguishes them from other known viral rna genomes (19) . similarly to other rna viruses, coronaviruses (covs) are characterised by high genetic diversity driven by mutation and recombination, which can lead to the emergence of new viruses. such new pathogens can have new features which even enable them to switch to new hosts (49) . these newly created viruses can acquire zoonotic potential, as witnessed by the severe acute respiratory syndrome (sars), the epidemic from southern china in 2003 caused by sars-covs. this disease, termed "atypical pneumonia", was diagnosed in humans in 29 countries and had a nearly 10% mortality rate. in 2012, there emerged a subsequent disease caused by a novel coronavirus, the so-called middle east respiratory syndrome (mers) with even higher mortality rates. both sars-and mers-covs crossed the species barrier from bats to humans through civet cats and camels as intermediate organisms (17) . wild bird species serve as a natural reservoir of many emerging zoonotic pathogens and thus have a significant impact on public health. they are also the source of pathogens dangerous to domestic animals, and such infections could have socio-economic consequences. this is the reason why over the years wild birds have been under epidemiological surveillance. among the viruses transmitted by wild birds, the most well-known are influenza a viruses (9) . wild birds are also implicated in the spread of the west nile virus, borrelia burgdorferi, and other bacterial infections such as salmonella or campylobacter, and with them also resistance genes to antibiotics (35) . studies from the last 10 years have also shown the presence of covs in wild birds (22, 24, 25, 53) . among factors which make birds an excellent reservoir of various pathogens and also a bioreactor contributing to their variability, there are the high biodiversity of bird species, their ecological traits such gathering/grouping during feeding and roosting, but most importantly their capability to fly long distances (8) . in this paper, we will focus more on different aspects related to covs identified in wild birds. however, acknowledging the gaps in understanding of the biology of these viruses, we will also refer to the most attention-worthy representatives of avian coronavirus species, i.e. infectious bronchitis viruses (ibv). taxonomic classification. covs belong to the family coronaviridae, subfamily coronavirinae, and order nidovirales. initially, classification of members of this subfamily was based on their serological relationships as opposed to the new taxonomic revision based on a threshold level of sequence identity of a few replicase regions (the pp1ab polyprotein and the orf1ab gene). according to these criteria, coronavirinae are divided into four genera: alpha-, beta-, gamma-, and deltacoronavirus, replacing the traditional division into antigenic groups 1, 2, and 3 (4) . the final list of species proposed by the international committee on taxonomy of viruses (ictv) including the genus identified in birds, is presented in table 1 . generally, alpha-and betacovs infect humans and domestic animals, while gamma-and deltacovs are largely associated with avian hosts although they were also detected in marine mammal species as well as in some asian carnivores (32, 52) . the main representative of the gammacoronavirus genus is avian coronavirus. this taxonomic name includes ibv which causes a highly contagious disease of chickens, and genetically similar viruses isolated from other domestic galliformes: turkey coronavirus (tcov), responsible for turkey enteritis, and the more recently detected guinea fowl coronavirus (gfcov), the aetiological factor of fulminating disease in this species (2, 6, 27) . analogous viruses were also detected in pheasants, peafowl, and quails, but also in nongalliformes, namely columbiformes, pelecaniformes, ciconiiformes, psittaciformes, and anseriformes (12, 13, 22, 24, 30) . there are plenty of ibv variants with divergent molecular and biological properties. due to the lack of a clear method for ibv classification, new rules based on the spike gene fragment (s1) sequence were recently proposed. it distinguished and named 32 lineages, categorised into six genotypes (gi to gvi) (42) . the difference between other members of the avian coronavirus family, namely tcov and gfcov, is also in the s gene structure. interestingly, all other regions of the genome were found to be similar to ibv, suggesting that they come from the same ancestor (2, 27) . in 2008, surprising information about a new gammacov species from the white beluga whale appeared, which challenged the prevailing opinion of the specificity of gammacovs only to birds (32) . further studies identified another coronavirus in ducks in 2015 whose genome fulfilled the official ictv criteria required to distinguish a new species in the gammacoronavirus genus. ictv approval is still awaited for this new species designation (54) . coronaviruses identified in 2009 in birds of the passeriformes order, namely munia, bulbul, and thrush, appeared to be similar to each, but distinct from known coronaviruses, and they formed a unique cluster in the phylogenetic tree which was the basis for generation of a novel genus: deltacoronavirus (53). interestingly, deltacovs were also identified in pigs in hong kong and the united states (31) . additionally, these viruses clustered with previously unclassified coronaviruses detected in the asian carnivores, the asian leopard cat, and ferret badger (14) . currently, the deltacoronavirus genus comprises eight species, including seven avian and one swine coronaviruses. it is suspected that covs appeared over 300 million years ago, corresponding in time to the coevolution and codivergence of bat and avian species (43) . their subsequent diversification was a product of differences in alimentation, reproduction, and roosting ecology. genomic organisation of avian coronaviruses. the coronavirinae subfamily is characterised by an exceptionally large rna genome (19) . the genome size among viruses, included under the taxonomic species name avian coronavirus, is about 27,500 nucleotides (nt), the smallest being the 27,231 nt of 3575/98 ibv strain (genbank no kx266757), and the largest the 27,718 nt of ck/ch/lgd/120724 (kc119407). duck cov (genbank no. km454473), a candidate for a separate species within the gammacoronavirus genus, has a similar size of 27,754 nt. the complete genomic sequences of 18 deltacov strains, identified in wild birds available in genbank, are about 1,000 nucleotides shorter, their sizes ranging from 26,041 nt in white-eye cov hku (nc016991) to 26,689 nt in magpie-robin cov hku18 (nc016993) strains. the genomes of all coronaviruses have similar structures and organisation, but also display unique groups or even strain-specific genomic structures, including accessory genes. generally, the genome is contained between the 5'capped end and poly(a) tail at the 3'-end, comprising short untranslated regions (utrs). about two-thirds of the genome is occupied by two overlapping large open reading frames (orfs), encoding replicase (rna-dependent rna polymerase (rdrp)) polyproteins 1a and 1 ab. these two large polyproteins are formed by a ribosomal frameshift mechanism and subsequently cleaved by viral proteases into the nonstructural proteins (nsp) (19) . the number of nsps of viral replicase in the most complex of the family of positive-strand rna viruses and in alphaand betacovs is 16, whereas gamma-and deltacovs have 15 nsps because they lack nsp1. part of these replicase domains, the seven most conserved, which are nsp3, nsp5, and nsp12-16, serve as species demarcation among coronaviruses (4) . the other third of the genome includes four structural protein genes organised in the following, canonical way: s, e, m, and n (19) . among these four genes, there are a set of lowmolecular accessory proteins the presence of which could be strain-dependent, and these are 3a, 3b, 4b, 4c, 5a, 5b, and 6b, this being the most recently identified in some ibv and tcov strains and only orf downstream of n protein, -6b (2, 20) . it is believed that accessory proteins are not essential for virus replication, but could play a role in virus virulence as 3b protein does. in addition to these four canonical structural proteins s, e, m, and n, deltacovs seem to have a smaller number of nonstructural accessory proteins. they contain nonstructural protein ns6 and a number of proteins located downstream of the n protein, designated ns7a, 7b, and sometimes also 7c and 7d (53) . the functions of nonstructural proteins of gamma-and deltacovs are largely unknown. viral proteins. the name "coronavirus" aptly represents their appearance of solar corona in negatively stained electron micrographs, which displays characteristic club-shaped spike (s) proteins on the envelope of the virion. in addition to protruding s proteins, the envelope is formed by the most abundant membrane (m) proteins and non-glycosylated envelope proteins (e) present in lower quantities. such an envelope surrounds the viral genomic rna which forms the complex with a few copies of the nucleocapsid (n) protein (7). information about the role of virus proteins in the course of infection comes from studies on ibv and tcov. the crucial stage in the virus life cycle relies on interaction of a viral attachment protein with a particular host cell receptor and then release of the genome inside the cell through the fusion with this cell membrane. the key player in both stages is the s protein, which is therefore recognised as a determinant of tissue and cell tropism and pathogenesis. this protein consists of two subunits: the n-terminal s1 subunit which forms a globular head structure and the c-terminal s2 subunit which is a transmembrane stalk. the s1 subunit is responsible for recognising and binding the receptor cell to the host, and the s2 domain specialises in the fusion process (44) . coronaviruses identify their various specific receptors and co-receptors, which may be proteins and sugars. the ibv has a primary affinity for the respiratory systems of chickens, but its variants could have tropism also to other organs such as kidneys, oviduct, testes, bursa of fabricius, caecal tonsils, or the alimentary system (7). the main attachment factor for respiratory ibv is α2,3-linked sialic acid glycan, widely distributed on host tissue, and this explains why such strains can also have affinity to other organs. additionally, the diversity of the s1 domain sequence as high as 20%-30% among different ibv variants could further contribute to the binding capacity of these viruses. it is suspected that nephropathogenic ibv strains could use a different or additional receptor for tissue binding. studies on enteric coronaviruses (tcov and gfcov) also revealed their different receptor specificity, as binding of their s protein to the host glycan receptor is independent of the presence of sialic acid residues and recognises poly-lacnacon complextype n-glycans. the difference in receptor binding observed between respiratory ibvs and enteric gammacovs could be concluded from the difference between their s1 coding regions which is as high as 64% (45) . the function of the s protein in avian deltacov seems to be analogous to ibv or tcov, but nothing is known about their receptor specificity. diagnosis. information about the range of avian species infected with covs and the prevalence of these infections has been accumulated from the increasing number of studies based on the use of virus-specific molecular tests. however, such detection methods depend on the specificity of the primers used. the method which was applied for the first time in a systemic monitoring of coronaviruses in wild birds amplified 251 bp of the replicase gene of all coronaviruses known at that time (37) . this allowed the identification of novel coronaviruses infecting graylag geese, feral pigeons, and mallards (24) . next, the methods targeting the conserved regions of ibv viruses, i.e. 3'utr, 5'utr, or even s1 gene fragments (1, 3, 5) were used for monitoring purposes. additionally, in a molecular survey of avcov in ducks the viral nucleocapsid (n) gene was amplified in a reverse-transcription polymerase chain reaction (rt-pcr) (10) . the viruses identified in such way were designated ibv-like. the summary of monitoring studies of coronaviruses in wild birds is given in table 2 . however, this approach changed after a few findings; firstly, it turned out that other gammacovs such as tcov or gfcov have genomes closely related to ibv, and the only diverged gene between them is the s gene; secondly, recombination events were discerned to be a quite common phenomenon in these groups of viruses; and the most important discovery was that of deltacov and determination of the criteria which enable discrimination between gamma-and deltacov (2, 23, 27) . more recently, primers specific to the appropriate conserved site of the replicase gene have commonly been applied (11, 33, 51) . all these primers are within a region of about 840 nt occupied by the nsp12 of orf1ab which is instrumental in coronavirus species differentiation and contains different numbers of degenerative nucleotides to detect the broader spectrum of coronavirus strains. the position of the used primers, aimed at the replicase gene, is shown in table 3 . epidemiology. it is well known that ibv is ubiquitous in most parts of the world in regions with intensive poultry production, where it causes huge economic losses. ibv strains are responsible for diseases of the respiratory, urogenital, and digestive tracts of domestic fowl (gallus gallus). however, there are many reports of ibv presence in other bird species, which indicate that the virus can cross the species barrier. recently, ibvs homologous to vaccine h120 and field ibv strains were found in healthy domestic teal (anas) and peafowl (pavo cristatus), respectively. this indicates that the virus can replicate in these bird species without clinical signs (30) . moreover, a virus similar to field ibv inoculated into specific-pathogen-free chickens caused nephritis and high mortality, in contrast to h120-like ibv which revealed itself to be apathogenic. these findings indicate the possible role of these birds as asymptomatic carriers of ibv and the possibility of virus transmission to susceptible chicken populations. similar conclusions were drawn from chinese and brazilian studies which showed the presence of mass-like ibv strains in wild peafowl and pigeons (16, 38) . the detection of virus closely related to the h120 ibv vaccine strain in faeces of free living ducks and whooper swans also suggested cross-species infection from a poultry population to synanthropic birds (22) . the presence of gammacov was also found in wild birds sampled in poland. ibv-like strains were identified in anseriformes, charadriiformes, and galliformes, and the detected gene fragments were highly similar to the most frequently detected lineages of ibv in this geographical region, i.e. mass, 793/b and qx (13) . similarly, ibv strains were identified in studied samples from wild birds of the corvidae, ardeidae and anatidae families in egypt, and some of them had s1 gene fragments highly homologous to the ma5 vaccine strain. such findings suggest the possibility for vaccine strains to spillover to wildlife which may serve as the asymptomatic host, enabling these strains to undergo some genetic changes. such modified virus could then spillover in the reverse direction back to a poultry population, and the possibility of its higher pathogenicity could not be ruled out (36) . recently, interesting results for the presence of both gamma-and deltacoronaviruses in quails were reported (39) (40) (41) . the identified gammacovs revealed a similarity to ibv strains unique to south america (40) . furthermore, in some cases, both genera of the viruses were identified simultaneously (41) . the summary of monitoring studies on the occurrence of coronaviruses in wild birds is presented in table 2 . the rate of positives of gamma-and deltacovs differs greatly from 0.3% to 50%, depending on temporal/seasonal and spatial features and depending on applied detection methods. however, even using the same method, the rate of positives varies from 0.95% to 15%. among factors influencing the prevalence of cov in wild birds, there could also be the age of the birds sampled, bird order/species, and their behaviour (migratory versus resident, water birds or land-dwelling birds). very interesting is the identification of betacovs in south american wild birds, these being viruses which were previously detected only in mammals (15) . it seems, however, that these studies may suffer from methodological deficiencies and require a thorough re-analysis. there is much evidence that cov could be transmitted from poultry to wild birds and from wild birds to poultry. in this way, the virus could spread over long distances. wild birds are suspected of spreading different ibv variants into new geographical regions such as variant qx (gi-19 lineage) from china to europe or var2 (gi-23 lineage) from the middle east to poland (18, 29) . if the hosted wild bird acquires infections of various coronaviruses, it could be an excellent environment for recombination events, which may lead to the emergence of a new disease dangerous to humans as evidenced by sars-or mers-covs. that is the reason why wild birds have to be continuously studied for the presence of various coronaviruses. the authors declare that there is no conflict of interests regarding the publication of this article. financial disclosure statement: this publication was possible due to the polish ministry of science and higher education (grant no. know2015/cb/pro1/33 within the scientific consortium "healthy animal -safe food"). animal rights statement: none required. phylogenetic analysis of partial s1 and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity 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betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus genomic analysis and surveillance of the coronavirus dominant in ducks in china key: cord-307304-irji8owi authors: britton, paul; evans, sharon; dove, brian; davies, marc; casais, rosa; cavanagh, dave title: generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection date: 2004-11-05 journal: j virol methods doi: 10.1016/j.jviromet.2004.09.017 sha: doc_id: 307304 cord_uid: irji8owi a reverse genetics system for the avian coronavirus infectious bronchitis virus (ibv) has been described in which a full-length cdna, corresponding to the ibv (beaudette-ck) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cdnas [casais, r., thiel, v., siddell, s.g., cavanagh, d., britton, p., 2001. reverse genetics system for the avian coronavirus infectious bronchitis virus. j. virol. 75, 12359–12369]. the method has subsequently been used to generate a recombinant ibv expressing a chimaeric s gene [casais, r., dove, b., cavanagh, d., britton, p., 2003. recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. j. virol. 77, 9084–9089]. use of vaccinia virus as a vector for the full-length cdna of the ibv genome has the advantage that modifications can be made to the ibv cdna using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. we describe the use of homologous recombination as a method for modifying the beaudette full-length cdna, within the vaccinia virus genome, without the requirement for in vitro assembly of the ibv cdna. to demonstrate the feasibility of the method we exchanged the ectodomain of the beaudette spike gene for the corresponding region from ibv m41 and generated two recombinant infectious bronchitis viruses (ribvs) expressing the chimaeric s protein, validating the method as an alternative way for generating ribvs. avian infectious bronchitis virus (ibv), a group three member of the genus coronavirus (order nidovirales, family coronaviridae), is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct (cavanagh, 2001; cavanagh and naqi, 2003; cook et al., 2001) . genetically very similar coronaviruses cause disease in turkeys and pheasants (cavanagh et al., , 2002 . coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, 5 -capped and 3 -polyadenylated, single-stranded, positive-sense rna genome of 28-32 kb (de vries et al., 1997; lai and cavanagh, 1997; siddell, 1995) . all coronavirus envelopes contain at least three membrane proteins, the spike glycoprotein (s), a small membrane protein (e) and an integral membrane protein (m). in addition, the coronavirus virion also contains a nucleocapsid protein (n) that interacts with the grna. molecular analysis of the structure and the role of individual genes in pathogenesis of rna viruses has been advanced by the availability of full-length cdnas, for the generation of infectious rna transcripts that can replicate and result in infectious viruses from permissive cell lines (boyer and haenni, 1994) . such reverse genetics systems have resulted in the recovery of a number of infectious positive-stranded rna viruses including, picornaviruses, caliciviruses, alphaviruses, flaviviruses, arteriviruses and closterovirus, whose rna genomes range in size from 7 to 20 kb in length (agapov davis et al., 1989; racaniello and baltimore, 1981; rice et al., 1987 rice et al., , 1989 satyanarayana et al., 2001; sosnovtsev and green, 1995; sumiyoshi et al., 1992; van dinten et al., 1997) . recently full-length cdnas capable of producing infectious rna transcripts for several coronaviruses, viruses with the largest rna genomes, transmissible gastroenteritis virus (tgev; (almazán et al., 2000; yount et al., 2000) ), human coronavirus 229e (hcov; ), ibv (casais et al., 2001) , murine hepatitis virus (mhv; (yount et al., 2002) ), and severe acute respiratory syndrome coronavirus (sars-cov; yount et al., 2003) have been produced. the assembly of full-length cdnas corresponding to the coronavirus genomes was hampered due to some cdnas, derived from regions of the replicase gene, being unstable in bacteria. however, despite this, three methods have been described for the assembly of full-length cdnas of the genomic rna of coronaviruses. the first method involved the assembly of a tgev full-length cdna in a bacterial artificial chromosome (bac), immediately downstream of a viral rna polymerase ii promoter. transfection of the bac dna into susceptible cells resulted in synthesis of infectious rna and the recovery of infectious virus (almazán et al., 2000) . the second system involved the in vitro assembly of full-length cdnas using a series of contiguous cdnas with engineered specific restriction sites. bacteriophage t7-rna polymerase-derived rna transcripts of the cdnas were used to generate infectious virus (yount et al., 2000 (yount et al., , 2002 (yount et al., , 2003 . the third strategy involved the in vitro assembly of contiguous cdnas followed by direct cloning into the genome of vaccinia virus. infectious rna transcripts were synthesised in vitro from the vaccinia virus dna, using bacteriophage t7 rna polymerase, and transfected into cells resulting in the recovery of infectious virus . in an alternative strategy infectious ibv was recovered following transfection of restricted vaccinia virus dna, containing the ibv full-length cdna, into cells infected with recombinant fowlpox virus expressing t7 rna polymerase (casais et al., 2001) . the vaccinia virus system has the advantage that the coronavirus full-length cdna is present in a non-bacterial system and offers the opportunity of modifying the coronavirus cdna by homologous recombination. falkner and moss (1990) devised a general method for modifying the vaccinia virus genome, involving insertions, deletions and specific mutations, termed transient dominant selection (tds). the method relies on a two-step procedure. in the first step, a complete plasmid sequence is introduced into the vaccinia virus genome as a result of a single cross-over event involving homologous recombination between vaccinia virus sequences in the plasmid dna and virus genome. the plasmid sequence contains the sequence being modified and a dominant selectable marker gene, escherichia coli guanine xanthine phosphoribosyltransferase (ecogpt) (mulligan and berg, 1981) , under the control of the vaccinia virus p 7.5k promoter. recombinant viruses expressing the gpt gene are selected for resistance against mycophenolic acid (mpa) in the presence of xanthine and hypoxanthine. in the second step, the mpa-resistant viruses are grown in the absence of selection, resulting in loss of the ecogpt gene due to a single homologous recombination event between duplicated sequences, present as a result of the integration of the plasmid dna. the second step results in one of two recombination events; return to the original input virus sequence, without modification to the virus or introduction of the modified sequences into the vaccinia virus genome. both events should occur with equivalent efficiencies if the homology regions are of equal length. we have utilised the in vitro ligation and direct cloning into the vaccinia virus genome method of generating ribvs for the recovery of a ribv expressing a chimaeric s gene (casais et al., 2003) . the ribv expressed an s gene in which the ectodomain of the protein was derived from a different strain of ibv. having demonstrated that it was possible to produce a ribv expressing a modified s gene we decided to investigate an alternative, quicker and simpler, procedure for modifying the full-length ibv cdna within the vaccinia virus genome for the production of ribvs containing the chimaeric s gene. in this paper, we describe the generation of two homologous ribvs, ribv-m41s-a and ribv-m41s-b, in which the ectodomain sequence of the beaudette s gene in vaccinia virus vnoti-ibv fl (casais et al., 2001) was exchanged with the corresponding region from the m41 strain of ibv using the tds method. our results demonstrated that ribvs can be generated following direct modification of the full-length ibv cdna by homologous recombination within the vaccinia virus genome. the growth of ibv in either 11-day-old embryonated domestic fowl eggs or chick kidney (ck) cells was as described previously (pénzes et al., 1994 stirrups et al., 2000) . the ibv isolates used were: (1) beaudette-ck (beau-ck; (cavanagh et al., 1986) ), a virus adapted for growth in ck cells that can grow on, but has not been adapted for growth on, vero cells an african green monkey cell line; (2) beau-r, a recombinant ibv (ribv) produced from an infectious rna transcribed from a full-length cdna of beau-ck (casais et al., 2001) ; (3) m41-ck, a virus adapted from m41 (darbyshire et al., 1979) for growth on ck cells but not vero cells; and (4) beaur-m41(s), a ribv expressing a m41-ck/beau-ck chimaeric s gene, produced following in vitro assembly of a full-length ibv cdna in vaccinia virus, that grows on ck but not vero cells (casais et al., 2003) . beaudette and m41 belong to the same, massachusetts, serotype. vaccinia viruses were titrated on monkey kidney fibroblast cells (cv-1) grown in dulbecco's-modified eagle medium (d-mem) supplemented with 0.37% (w/v) sodium bicarbonate, l-glutamine, 10% foetal calf serum and an-tibiotics. baby hamster kidney cells (bhk-21) cells were grown in glasgow medium supplemented with 0.37% (w/v) sodium bicarbonate, tryptose phosphate broth, l-glutamine, 10% foetal calf serum and antibiotics and used for the propagation of vaccinia viruses for isolation of virus dna. recombinant fowlpox virus rfpv-t7 (fpeflt7pol; , expressing the bacteriophage t7 rna polymerase under the direction of the vaccinia virus p 7.5 early/late promoter, was grown in chick embryo fibroblast (cef) cells (casais et al., 2001) . recombinant dna techniques used standard procedures (ausubel et al., 1987; sambrook et al., 1989) or were used according to the manufacturers' instructions. all ibv-related nucleotide and amino acid residue numbers refer to the positions in the ibv beau-r genome (casais et al., 2001) , accession no. aj311317. the complete beau-ck s gene, within a 5.6 kb stui and bamhi fragment, was removed from pfrag-3 (casais et al., 2001) and inserted into hincii and bamhi digested pgpt-neb193 (a gift from dr. m. skinner iah) (boulanger et al., 1998) , resulting in pgpt-ibv-stui-bamhi ( fig. 1) . plasmid pgptneb193 contains the e. coli guanine xanthine phosphoribosyltransferase (ecogpt) gene under the control of the vaccinia virus p 7.5 early/late promoter and a separate multiple cloning site. most (94%) of the beau-ck s gene was removed from pgpt-ibv-stui-bamhi by digestion with paci and styi followed by end repair of the dna and ligation, resulting in pgpt-ibv-s (fig. 1) . the m41-ck/beau-ck chimaeric s gene, within a 4.5 kb paci and bamhi fragment, was removed from pfrag-3-m41s (casais et al., 2003) and used to replace the beau-ck s gene sequence in pgpt-ibv-stui-bamhi, resulting in pgpt-m41s (fig. 2) . recombinant vaccinia viruses were generated as a result of transient dominant selection (tds; (falkner and moss, 1990) ) using the ecogpt gene as the transient selectable marker. cv-1 cells were infected with vaccinia virus vnoti-ibv fl , containing the full-length ibv cdna derived from beau-ck, at a multiplicity of infection (moi) equivalent to 0.2 plaque-forming units (pfu) per cell, and subsequently transfected with pgpt-ibv-s in the presence of lipofectin (invitrogen). recombinant viruses expressing gpt, from the ecogpt gene, were selected by three rounds of plaque purification in cv-1 cells in the presence of selection medium, cv-1 growth medium containing 25 g/ml mpa, 250 g/ml xanthine and 15 g/ml hypoxanthine (fig. 3) . several mpa-resistant vaccinia viruses were subsequently plaque purified (x3) in the absence of mpa resulting in the spontaneous loss of the ecogpt gene (fig. 3) . several recombinant vaccinia viruses that had a gpt-negative phenotype were analysed by pcr and found not only to lack the ecogpt gene sequence but also, as expected, to have the beau-ck s gene sequence deleted from the ibv cdna. one such recombinant vaccinia virus, vnoti-ibv-s fl , was used in subsequent experiments. the tds method was used to insert the m41-ck/beau-r chimaeric s gene into the ibv cdna within vnoti-ibv-s fl using pgpt-m41s and involving ecogpt selection in the presence of mpa followed by loss of the ecogpt gene in the absence of mpa (fig. 4) . recombinant vaccinia viruses that had a gpt-negative phenotype were analysed by pcr and found to lack the ecogpt gene sequence. further analysis identified a recombinant vaccinia virus, vnoti-tdsr-m41s fl , that contained the m41-ck/beaudette-ck chimaeric s gene sequence within the full-length ibv cdna. vaccinia virus vnoti-tdsr-m41s fl was grown in 13 t150 flasks of bhk-21 cells until a cpe was evident. the infected cells were harvested, resuspended in 10 mm tris-hcl ph 9.0, 1 mm edta lysed by three cycles of freeze thawing, sonicated and the cell nuclei pelleted by centrifugation at 750 × g for 5 min at 4 • c. virus particles were partially purified by centrifugation, through a cushion of 30% sucrose in 10 mm tris-hcl, ph 9.0, at 14,000 rpm for 1 h at 4 • c, using a sw28c rotor in a sorvall otd65b ultracentrifuge and resuspended in 10 mm tris-hcl, ph 9.0, 1 mm edta. the semi-purified virus was incubated in 100 mm tris-hcl ph 7.5, 5 mm edta, 0.2% sds, 200 mm nacl containing 0.1 mg/ml proteinase k, incubated at 50 • c for 2 h, extracted with phenol/chloroform and the dna ethanol precipitated. the vaccinia virus dna was digested with asci (1 u/g of dna) and analysed by pulse field gel electrophoresis using 1% agarose gels (casais et al., 2001) . ck cells, grown to 50% confluence (approximately 2 × 10 6 cells) in 60 mm diameter plates, were infected with rfpv-t7 at an moi of 10. after 45-60 min, the cells were transfected with 10 g of ascirestricted vnoti-tdsr-m41s fl dna and 5 g of pci-nuc (hiscox et al., 2001) using 30 g of lipofectin (invitrogen) (casais et al., 2001 (casais et al., , 2003 . the transfected cells (p 0 cells) were incubated at 37 • c for 16 h after which the transfection medium was replaced with fresh maintenance medium (pénzes et al., 1994) . at 2.5 days post transfection, the culture medium, potentially containing ribv (v 1 ), was removed, centrifuged for 3 min at 2500 rpm, filtered through a 0.22 m filter, to remove any rfpv-t7 , and used for serial passage on ck cells. two independently rescued the recombination vector is based on a gpt expressing plasmid, pgptneb193, in which a segment of the ibv genome, corresponding to the ibv s gene and flanking sequences of approximately equal length, was introduced. the majority of the s gene was then removed, leaving only the transmembrane and c-terminal domains within the recombination vector, pgpt-ibv-s. the flanking regions were included to allow homologous recombination events to occur between the ibv sequences in pgpt-ibv-s and the corresponding sequences in the ibv full-length cdna in the vaccinia virus (vnoti-ibv fl ) genome. the flanking regions were of approximate equal length to allow unbiased homologous recombination events. the various ibv gene sequences and relevant restriction sites are indicated; s represents the spike glycoprotein gene, 3a, 3b, 3c (e) represent the ibv gene 3 products; m represents the integral membrane protein; 5a, 5b represent the ibv gene 5 products; n represents the nucleocapsid protein; 3 utr represents the ibv 3 untranslated region; h␦r represents the hepatitis delta ribozyme sequence; t7 term represents the t7 termination sequence; ecogpt represents the e. coli gpt gene and vv p 7.5 represents the vaccinia virus early/late p 7.5 promoter. both types of recombination events have an equal chance of occurring. the various ibv genes are as described in the legend to fig. 1 rep represents the end of the ibv replicase gene, s indicates that the signal sequence and ectodomain are deleted with the transmembrane and c-terminal domain sequences remaining in the partial s gene sequence in the ibv cdna; 3 and 5 represent the ibv gene 3 and 5 sequences, respectively. ribvs, ribv-m41s-a and ribv-m41s-b, were obtained and characterised. oligonucleotides gpt-forw (5 -atgagcgaaaaat-acatcgtc-3 ) and gpt-rev1 (5 -ttagcgaccgga-gattgg-3 ) were used to determine the presence and absence of the ecogpt gene sequence in the intermediary vaccinia virus recombinants and in the vaccinia virus recombinants, potentially containing the modified ibv sequences; a 459 bp product was indicative of the ecogpt gene. oligonucleotides bg-44 (5 -20941 gctggtggacctata-act 20958 -3 ) and bg-134 (5 -1417 agcaattgaaactg-aaagtg 21398 -3 ) were used to analyse vnoti-ibv-s fl and vnoti-tdsr-m41s fl dna to determine the absence of the beau-r or presence of the m41-ck s gene sequences, respectively; a 476 bp product was indicative of the m41-ck s gene sequence. sequence analysis was used to confirm that the pcr products were derived from the m41-ck s gene sequence. total cellular rna was extracted from ck cells infected with the ribvs, ribv-m41s-a and ribv-m41s-b, by the rneasy method (qiagen) and analysed by rt-pcrs, using ready-to-go tm rt-pcr beads (amersham pharmacia biotech) and a variety of oligonucleotide pairs. the rt-pcr products were sequenced to confirm the sequence of the s gene present in the two ribvs. confluent monolayers of ck and vero cells in 60 mm dishes were infected with 7.5 × 10 4 pfu of m41-ck, beaur-m41(s), ribv-m41s-a and ribv-m41s-b or 1 × 10 4 pfu of beau-r. a lower multiplicity of infection for beau-r was used to further enhance the difference of growth on vero cells of ibvs expressing either a beaudette-derived or m41derived s protein ectodomain. following adsorption, for 1 h at 37 • c, the cells were washed three times with phosphatebuffered saline (pbs) to remove residual virus and incubated at 37 • c in 5 ml of ck media. samples of media were, at selected times over a 48 h period, analysed in triplicate for progeny virus by plaque assay in ck cells. sequence analysis of plasmid dna, pcr products from the ibv cdna sequences within vnoti-ibv-s fl and vnoti-tdsr-m41s fl and from rt-pcr products generated from rna isolated from ribv-m41s-a and ribv-m41s-b infected ck cells, was done using an abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems) or ceq tm dtcs quick start kit (beckman coulter). oligonucleotide primers used for the sequencing reactions were derived from the beau-ck sequence (boursnell et al., 1987) . sequences were determined on an applied biosystems 377 dna sequencer or a ceq tm 8000 capillary sequencer. pregap4 and gp4 of the staden sequence software programs (bonfield et al., 1995) were used for sequence entry, assembly and editing. we have developed a reverse genetics system for the avian coronavirus ibv in which the full-length ibv cdna was assembled by in vitro ligation followed by insertion into the vaccinia virus genome (casais et al., 2001) . we decided to investigate an alternative procedure, utilising homologous recombination by tds (falkner and moss, 1990) , for modifying the full-length ibv cdna within the vaccinia virus genome to circumvent the necessity of assembling the ibv cdna in vitro and subsequent insertion into the vaccinia virus genome. to test the tds-based method we proposed to replace the ectodomain of the beau-ck s gene, within the full-length ibv cdna in vnoti-ibv fl (casais et al., 2001) , with the ectodomain of the m41-ck s gene. however, to replace the 3.4 kb beau-ck s sequence with the corresponding m41-ck s sequence an intermediary recombinant vaccinia virus, vnoti-ibv-s fl , containing the beau-ck cdna with the s gene deleted had to be produced. this was to avoid re-combination events occurring within the s gene sequence if attempts were made to directly exchange the two sequences. plasmid pgpt-ibv-s ( fig. 1) with 94% of the beau-ck s gene sequence deleted and beaudette-derived sequences, 1188 bp proximal and 1155 bp distal to the deleted region of the s gene sequence, for homologous recombination with the ibv cdna in vnoti-ibv fl , was used for the tds process for generation of vnoti-ibv-s fl (fig. 3) . pcr and sequence analysis of the ibv cdna within vnoti-ibv-s fl confirmed that the beau-ck s gene had been deleted (data not shown). the m41-ck/beau-ck chimaeric s gene sequence for insertion into the ibv cdna within vnoti-ibv-s fl was identical to the sequence we had previously used for the generation of ribv beaur-m41(s) (casais et al., 2003) . the chimaeric s gene consisted of the signal sequence, ectodomain and transmembrane regions derived from ibv m41-ck and the cytoplasmic tail domain from beau-ck. the transmembrane domains are identical between the two sequences but the cytoplasmic tail domain of the m41-ck s protein is truncated by nine amino acids when compared to the beau-ck s protein. we had previously indicated that we retained the beau-ck cytoplasmic tail domain because this region of the s protein has been demonstrated to interact with other virus proteins (godeke et al., 2000) . the chimaeric s gene sequence, from pfrag-3-m41s (casais et al., 2003) , was used to replace the corresponding beau-ck s gene sequence in pgpt-ibv-stui-bamhi, resulting in pgpt-m41s (fig. 2) . plasmid pgpt-m41s, contained the chimaeric s gene with beaudette-derived sequences, 1188 bp proximal and 1155 bp distal to the chimaeric s sequence, for homologous recombination with the beau-ck-derived ibv cdna in vnoti-ibv-s fl , to insert the chimaeric s gene sequence into vnoti-ibv-s fl (fig. 4) . a recombinant vaccinia virus, vnoti-tdsr-m41s fl , was isolated and pcr and sequence analysis confirmed that the ibv cdna within the vaccinia virus genome contained the chimaeric s gene sequence. two ribvs, ribv-m41s-a and ribv-m41s-b, were independently recovered from ck cells, previously infected with rfpv/t7, to provide t7 rna polymerase, and co-transfected with asci-restricted vnoti-tdsr-m41s fl dna and pci-nuc (casais et al., 2001 (casais et al., , 2003 . the vnoti-tdsr-m41s fl dna was prepared from semi-purified vaccinia virus and pci-nuc, a plasmid expressing the ibv n protein under control of both the t7 and cmv promoters, was required for the successful recovery of ribv (casais et al., 2001) . the transfected ck cells (p 0 ) were incubated for 2.5 days post transfection, until they showed a cytopathic effect (cpe), the medium was filtered to remove any rfpv/t7 and any potential ibv passaged on fresh ck cells (p 1 ). the two independently rescued ribvs, isolated from the p 1 cells, were partially analysed and found to contain the chimaeric s gene and subsequently virus rna derived from p 3 ck cells, was sequenced to confirm the presence of the chimaeric s gene. the ibv strains, beau-ck and m41-ck, have different cell tropisms, both viruses replicate to similar titres in ck cells but only beau-ck produces infectious virus on vero cells (casais et al., 2003) . previous results using beaur-m41(s), containing the chimaeric s gene following assembly of the full-length ibv cdna in the vaccinia virus genome, showed that the ribv had a phenotype like that of m41-ck rather than beau-ck for growth on vero cells (casais et al., 2003) . therefore, we analysed the growth characteristics of ribv-m41s-a and ribv-m41s-b, containing the chimaeric s gene generated as a result of the tds method, on ck and vero cells for comparison with beaur-m41(s). beau-r, m41-ck, beaur-m41(s), ribv-m41s-a and ribv-m41s-b were used to infect ck and vero cells and the titre of progeny virus determined over a 48 h period. all five viruses displayed similar growth profiles on ck cells; progeny viruses were detectable 8 h post infection with peak titres between 5 × 10 7 and 8 × 10 8 pfu/ml 30 h post infection (fig. 5a ). all the viruses caused observable cpe within 24 h on the ck cells. analysis of the growth profiles of the five viruses on vero cells (fig. 5b) showed that only beau-r replicated, with a maximum titre of 10 5 pfu/ml by 48 h post infection. in contrast, both ribv-m41s-a and ribv-m41s-b together with m41-ck and beaur-m41(s) showed significantly lower titres on vero cells in comparison to beau-r (fig. 5b) . overall, our results showed that ribv-m41s-a and ribv-m41s-b had similar phenotypes to those observed for beaur-m41(s), indicating that all three ribvs, expressing the chimaeric s glycoprotein, were similar irrespective of the method used to modify the ibv cdna used to generate the ribvs. the results presented in this paper demonstrate the feasibility of producing ribvs from ibv cdnas within the vaccinia virus dna that had been modified by homologous recombination. our results affirmed that the tds method is a suitable method for modifying the ibv full-length cdna within the vaccinia virus genome, resulting in isogenic variants that differ only in the introduced sequences. this method for generating ribvs will be useful for generating isogenic ribvs for determining the effect of the modifications on gene functions. we also demonstrated that an intermediary form of the ibv cdna in the vaccinia virus, containing a deleted region of the ibv genome, can be used for swapping large regions of the ibv genome avoiding the generation of chimaeric sequences. noncytopathic sindbis virus rna vectors for heterologous gene expression engineering the largest 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arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys infectious bronchitis protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures in vitro synthesis of infectious venezuelan equine encephalitis virus rna from a cdna clone: analysis of a viable deletion mutant the genome organisation of the nidovirales: similarities and differences between arteri-, toro-and coronaviruses utilizing fowlpox virus recombinants to generate defective rnas of the coronavirus infectious bronchitis virus transient dominant selection of recombinant vaccinia viruses assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus the molecular biology of coronaviruses selection for animal cells that express the e. coli gene coding for xanthine-guanine phosphoribosyl transferase characterization of a replicating and packaged defective rna of avian coronavirus infectious bronchitis virus replication and packaging of coronavirus infectious bronchitis virus defective rnas lacking a long open reading frame cloned poliovirus complementary dna is infectious in mammalian cells transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation production of infectious rna transcripts from sindbis virus cdna clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants molecular cloning: a laboratory manual, second ed. cold spring harbor laboratory amplification of citrus tristeza virus from a cdna clone and infection of citrus trees the coronaviridae rna transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require vpg for infectivity leader switching occurs during the rescue of defective rnas by heterologous strains of the coronavirus infectious bronchitis virus infectious japanese encephalitis virus rna can be synthesized from in vitro-ligated cdna templates an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 this work was supported by the department of environment, food and rural affairs (defra) project code od0712, european communities specific rtd program quality of life and management of living resources qlk2-ct-1999-00002 and the biotechnology and biological sciences research council (bbsrc). key: cord-321545-3gzj09mr authors: pohuang, tawatchai; chansiripornchai, niwat; tawatsin, achara; sasipreeyajan, jiroj title: detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand date: 2009-09-07 journal: j vet sci doi: 10.4142/jvs.2009.10.3.219 sha: doc_id: 321545 cord_uid: 3gzj09mr thirteen field isolates of infectious bronchitis virus (ibv) were isolated from broiler flocks in thailand between january and june 2008. the 878-bp of the s1 gene covering a hypervariable region was amplified and sequenced. phylogenetic analysis based on that region revealed that these viruses were separated into two groups (i and ii). ibv isolates in group i were not related to other ibv strains published in the genbank database. group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with ibvs published in the genbank database. this group likely represents the strains indigenous to thailand. the isolates in group ii showed a close relationship with chinese ibvs. they had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with chinese ibvs (strain a2, sh and qxibv). this finding indicated that the recent thai ibvs evolved separately and at least two groups of viruses are circulating in thailand. infectious bronchitis (ib), caused by the ib virus (ibv), is a highly contagious disease and results in a significant economic loss to the commercial chicken industry. the disease frequently causes respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. in layer fowls, respiratory distress and a decrease in egg production have been reported [10] . in addition, some strains have been associated with kidney lesions [18, 19, 22] . ibv, the causative agent of ib, belongs to the genus coronavirus in the family coronaviridae [6] . it is an enveloped virus and has a positive-sense, single-stranded, rna genome, approximately 27 kb in length. the virion has three major structural proteins namely the nucleocapsid (n) protein, the membrane (m) protein, and the spike (s) glycoprotein. the s glycoprotein is post-translationally cleaved into the s1 and s2 subunits [6] . the s1 subunit, located on the outside of the virion, is responsible for the fusion between the virus envelope and the host cell membrane. moreover, it is responsible for neutralizing serotype-specific antibodies in chickens [4] . the s1 subunit demonstrates more sequence variability than s2 [14] . neutralizing and serotype-specific epitopes are associated with the defined hypervariable region (hvr) in the s1 subunit; therefore, the molecular characterization of ibv is based on analysis of the s1 gene [12] . during 1953 during -1954 , the first ibv outbreak was reported in thailand [7] . although many ib vaccine strains such as connecticut, h120, ma5, m41 and armidale a3, have been used in thailand for many years, ibv outbreaks have been ongoing [1] . moreover, the relationships between recent thai ibv isolates and foreign ibv isolates are not known. the objective of this study was to characterize ibv field isolates in the recent outbreak of the disease in thailand by analyzing the s1 gene and compared them with those that have been published previously. between january and june, 2008, thirteen poultry farms in the eastern part of thailand had an outbreak of a mild-to-moderate respiratory disease (table 1) . all flocks had been vaccinated against ib with commercial live attenuated h120. chickens showed respiratory symptoms including gasping, coughing, sneezing, and tracheal rales. sick chickens were selected and sent to the department of veterinary medicine, faculty of veterinary science, chulalongkorn university. necropsy was performed and gross lesions were evaluated. gross lesions showed mildto moderated tracheitis and non-purulent airsacculitis. no gross lesion were found in the kidneys. the trachea and lung samples were taken as pools of chickens from the same farm. the samples were prepared as 10% w/v suspensions in phosphate-buffered saline (ph 7.4) and clarified at 1,800 × g for 10 min; the supernatants were then collected for analysis. viral rna was extracted by using viral nucleic acid extraction kit (real biotech, taiwan) following the manufacturer's instructions directly from the supernatants of 10% w/v sample suspensions and from the allantoic fluid of embryonated chicken eggs used for virus isolation. viral rna, extracted directly from the supernatants of 10% w/v sample suspensions, was screened for the presence of ibv by using a nested rt-pcr. the reaction was performed with accessquick rt-pcr system (promega, usa). the first amplification reaction was carried out with one-step rt-pcr using the primer sets of for1 (5´-ctt ttg ttt gca cta tgt ag-3´) and re3 (5´-taa taa cca ctc tga gct gt-3´). the second amplification reaction was carried out using the primer sets of for2 (5´-cag tgt ttg tca cac att gt-3´) and re2 (5´-cca tct gaa aaa ttg cca gt-3´). amplification products were analyzed in 1.5% agarose gel. the predicted size of nested rt-pcr product was about 400-bp. for virus isolation, the supernatants of ibv-positive samples determined by rt-pcr were inoculated into 10-day-old embryonated chicken eggs. for each sample to be examined, five embryonated chicken eggs were used. the eggs were inoculated with 0.2 ml of the sample into the allantoic cavity. the inoculated eggs were incubated at 37 o c and candled daily. allantoic fluids were harvested at 96 h postinoculation. a further blind serial passage was performed in a similar way. all of the allantoic fluids were harvested and stored at −70 o c. the allantoic fluids from the second passage of each sample positive for virus screening was submitted to another rt-pcr for amplification of a segment of 878-bp of the s1 gene coding region using a primer combination of for1 and re3. the amplification reaction was carried out with one-step rt-pcr and the reaction conditions were the same as the first amplification of nested rt-pcr described above. the rt-pcr products were cut from the gel and purified using the wizard sv gel and pcr clean-up system (promega, usa) according to the manufacturer's protocol. purified rt-pcr products were sequenced in a forward direction using primer for1 and in a reverse direction using primer re3. sequencing reactions were performed with the abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems, usa) as described by the manufacturer. sequencing reactions were run on an abi prism 310 genetic analyzer. to identify the thai ibv isolates, sequences of the s1 gene of the thai ibv isolates were compared with published ibv sequences deposited in the genbank database using a blast search via the national center of biotechnology information (usa). sequence identities by blast analysis were included in alignment and phylogenetic construction. a phylogenetic tree of the nucleotide sequences was constructed using mega version 3.1 [13] . the s1 gene sequences of the thirteen ibv isolates were submitted to the genbank database ( table 1 ). the other s1 gene sequences from the genbank database were used for comparison or phylogenetic analysis in this study included for virus screening, pooled trachea and lung samples from each flocks suspected of ibv infection were determined to be positive for ibv by screening with nested rt-pcr. a 400-bp fragment of the s1 gene was amplified in all 13 samples tested (data not shown). the allantoic fluid from the second passage of each sample screened positive for the virus was also determined to be positive with rt-pcr amplification and a segment of 878-bp of the s1 gene was obtained (data not shown). to assess the genetic relationship among the ibv isolates, a phylogenetic tree was constructed from the nucleotide sequences of s1 genes. the results are shown in fig.1 . the thirteen ibv isolates were separated into two distinct groups. group i consisted of five isolates including tha20151, tha40151, tha50151, tha60151, and tha90151. the isolates in group i showed evolutionary distances from each other. group ii consisted of eight isolates including tha30151, tha 70151, tha 80151, tha100151, tha110351, tha120351, tha130551, and tha140551, which had a close relationship with chinese ibv isolates (strain a2, sh and qxibv). the s1 gene of the thirteen ibv isolates was sequenced to characterize the isolates. the nucleotide and deduced amino acid sequences were determined and compared among each other and with other ibv strains published in the genbank database. group i thai ibv isolates had nucleotide and amino acid sequence identities between 99∼ 100% with each other. they had nucleotide sequence identities less than 85% and amino acid sequence identities less than 84% with ibvs published in the genbank database. group ii thai ibv isolates had nucleotide and amino acid sequence identities between 99∼100% with each other. they had nucleotide sequence identities of 97∼ 98% and amino acid sequence identities of 96∼98% with chinese ibvs (strain a2, sh and qxibv). one of the major problems of ibv is the frequent emergence of new variants [21] . different serotypes have been reported world wide and new variant serotypes continue to be recognized [9, 18] . thus, it is necessary and important to be able to diagnose these new serotypes. furthermore, determining the type as well as field isolates is important to select an appropriate vaccine against ibv infection in the next flock. therefore, several tests have been employed to identify the isolates into serotypes or genotypes [15] . typing with rt-pcr and sequencing of the s1 gene is easier and faster than the more traditional virus neutralization methods, but it is difficult to design pcr primers that can be used for detect all of ibv isolates [16] . in this study, ibv isolates in group i could not be detected with the primer set and pcr method described by gelb et al. [8] . to overcome this problem, the new primer sets were designed and the nested rt-pcr was developed in order to increase the sensitivity and specificity of the test used to detect ibv from the infected samples. moreover, the region amplified by the outer primer covering the hvr of the s1 gene (nucleotide position 31∼908) could be used for typing of ibv. genotyping of ibv on the basis of the s1 gene sequence, particularly the hvr region of the s1 gene, is the most common way to classify ibv isolates. it has been shown that the genetic typing based on hvr i (nucleotide position 114∼325) of the s1 gene could represent the grouping method based on the whole s1 gene [20] . furthermore, in a recent study using an approximately 450-bp region covering hvr i and hvr ii for ibv typing, it was found that typing by this region correlates with virus neutralization results [17] . in the present study, an approximately 878-bp region of s1 gene covering hvr i and hvr ii were amplified and used for typing the field isolates in thailand. although many different strains of live attenuated and inactivated vaccines have been widely used to control ib, the outbreaks of the disease have continued to be a problem in thailand [1] . in this study, thirteen ibv isolates from different commercial poultry farms in thailand were analyzed by sequencing of the hvr in s1 gene. the molecular data indicated that the recent ibv isolated in thailand evolved separately into two groups. the ibv isolates in group i were not related to other ibv strains published in the genbank database. this genotype likely represents strains indigenous to thailand. ibv isolates in group ii were genetically related to chinese strains. the results from both s1 gene comparison and phylogenetic analysis showed that ibv isolates in group i had a distant relation to vaccine strains used in thailand including ma5, h120, m41, and connecticut. new serotypes or variant strains can emerge as a result of only a few changes in the amino acid composition of s1 gene [5] . the rationale for these changes could be due to immunological pressure caused by the wide spread use of vaccines, recombination as a consequence of mixed infections, or the decrease of dominant serotypes as a result of vaccination, allowing other field strains to emerge [19] . group ii thai isolates and chinese strains shared more than 97% nucleotide identity and 96% amino acid identity; therefore, they were grouped into the same genotype. interestingly, group ii thai isolates were closely related to chinese qxibv which appeared to become widespread in several countries in the world including the uk [11] , russia [3] , and italy [2] . although no information is available currently for the introduction of qxibv from china to other countries, it had been hypothesized that wild birds were the source of introduction based on the evidence that ibv may replicate in anseriformes [3] . in summary, this finding indicated that the recent thai ibvs evolved separately and at least two groups of viruses are cocirculating in thailand. the distinctive dissimilarity between group i thai isolates and the widely used ibv vaccine indicated that the antigenic drift is likely to occur among some thai ibvs under the long-term immune pressure. group ii thai isolates were closely related to chinese strains, suggesting that the transfers of ibv infection occur among countries. avian infectious bronchitis in the southern part of thailand evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in italy molecular epizootiology of avian infectious bronchitis in russia completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus infectious bronchitis virus: evidence for recombination within the massachusetts serotype coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 studies on the attenuation of infectious bronchitis virus s1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens chinese qx strain of infectious bronchitis virus isolated in the uk a 'new' strain of infectious bronchitis virus infecting domestic fowl in great britain identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s-1 gene mega3: integrated software for molecular evolutionary genetics analysis and sequence detection and molecular characterization of infectious bronchitis virus 223 alignment phylogeny of antigenic variants of avian coronavirus ibv redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de072 strain of infectious bronchitis virus identification and analysis of the georgia 98 serotype, a new serotype of infectious bronchitis virus typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s1 gene a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia nephropathogenic infectious bronchitis in pennsylvania chickens this work was financially supported by the graduate school and the faculty of veterinary science of chulalongkorn university and intervet schering-plough animal health ltd., bangkok, thailand. we would like to thank all the staff of the virology unit, department of pathology, for their help throughout this work. key: cord-330260-xuw31zfn authors: chen, hui-wen; huang, yuan-pin; wang, ching-ho title: identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan date: 2009-01-20 journal: virus res doi: 10.1016/j.virusres.2008.11.012 sha: doc_id: 330260 cord_uid: xuw31zfn infectious bronchitis virus (ibv) infections in poultry cause great economic losses to the poultry industry worldwide. the emergence of viral variants complicates disease control. the ibv strains in taiwan were clustered into two groups, taiwan group i and taiwan group ii, based on the s1 gene. a variant was previously identified and showed a distinct s1 gene homology with other local strains. this study investigated the 3′ 7.3 kb genome of eight taiwan strains isolated from 1992 to 2007. the genes of interest were directly sequenced. sequence analyses were performed to detect any recombination event among ibvs. the results demonstrated that all of the examined viruses maintained the typical ibv genome organization as 5′-s-3a-3b-e-m-5a-5b-n-utr-3′. in the phylogenetic analyses, various genes from one strain were clustered into separate groups. moreover, frequent recombination events were identified in the simplot analyses among the taiwan and china ck/ch/ldl/97i-type strains. putative crossover sites were located in the s1, s2, 3b, m genes and the intergenic region between the m and 5a genes. all of the recombinants showed chimeric ibv genome arrangements originated from taiwan and china-like parental strains. field ibvs in taiwan undergo genetic recombination and evolution. avian infectious bronchitis virus (ibv), with a large infectious rna genome (27.6 kb), belongs to the family coronaviridae, the group 3 coronavirus. the rna molecule of the ibv is linear, singlestranded, positive sense and possesses a 5 cap and a 3 poly-a tail. ibvs can replicate in respiratory, alimentary and urogenital tracts in chickens of all ages, resulting in great economic losses to the poultry industry worldwide (cavanagh and naqi, 2003) . clinical signs associated with respiratory and enteric tracts and renal damage might be found in infected chickens (cavanagh, 2007) . during infection, ibv possesses a unique discontinuous transcription system, i.e. a nested set of 3 co-terminal subgenomic mrnas sharing a common leader sequence in the 5 end are transcribed in the presence of negative-stranded rna intermediates. the viral polymerase "jumping" possibly contributes to the high rna recombination frequency in coronaviruses (lai, 1992; lai and holmes, 2001) . the ibv genome encodes four structural proteins: spike glycoprotein (s), envelope protein (e; also known as sm), membrane glycoprotein (m), and nucleocapsid protein (n) (cavanagh and naqi, 2003) . the spike glycoprotein is post-translationally cleaved into s1 * corresponding author. tel.: +886 2 23690628; fax: +886 2 23631542. e-mail address: chingho@ntu.edu.tw (c.-h. wang) . and s2 subunits. the s1 subunit anchors onto the viral outer membrane using the s2 subunit to form a club-shaped projection on the mature virion (cavanagh, 1983) . the s1 glycoprotein is involved in cell attachment and carries epitopes for serotype-specific hemagglutination-inhibition and virus-neutralization antibodies (cavanagh and davis, 1986; hodgson et al., 2004; ignjatovic and sapats, 2005; koch et al., 1990) . the s2 glycoprotein, in which two antigenic determinants were identified (koch et al., 1990) , may possess different secondary structures that affect the s1 specific antibody binding (callison et al., 1999) . the membrane glycoprotein is associated with virus assembling and budding (lai and holmes, 2001) . the nucleocapsid protein interacts with genomic rna to form the viral nucleocapsid, playing a role in viral rna synthesis and cell immunity (lai and holmes, 2001) . in addition, four non-structural proteins of unknown function are expressed by the polycistronic genes, gene 3 and 5 . virus mutants carrying truncated 3b genes demonstrate increased virulence and growth advantages in vitro and in ovo (shen et al., 2003) . the open reading frames (orfs) of 3a, 3b, 5a, and 5b encode accessory proteins not essential for ibv replication (casais et al., 2005; hodgson et al., 2006; youn et al., 2005) . as a signature of avian coronaviruses, ibv strains are continuously evolving through point mutations and recombination of their genomes. those variants have better adaptation or increased virulence advantageous to ib outbreaks. to date, a large number of ibv sero-or genotypes have been identified worldwide (cavanagh, 2007 1171/92 1992 broiler 3 taoyuan nephropathogenic tw-i 2296/95 1995 broiler 2 taoyuan nephropathogenic tw-ii 2575/98 1998 broiler 4 changhua nephropathogenic tw-i 2992/ spike glycoprotein gene. (bochkov et al., 2006; dolz et al., 2006; dolz et al., 2008; jackwood et al., 2007) . it was reported that slight sequence differences in the s1 gene probably lead to poor crossprotection (cavanagh et al., 1997) . viruses of different types can co-circulate within a region (capua et al., 1999; liu et al., 2006) , raising the inter-strain rna recombination frequency (bochkov et al., 2007; jia et al., 1995; lee and jackwood, 2000) . the wide use of live virus vaccine may also critically contribute to the genetic evolution of ibvs by acting as a heterologous rna donor template (kusters et al., 1990; wang et al., 1993) . the emergence of viral variants has complicated disease control requiring persistent ibv molecular surveys. ibv strains in taiwan were previously clustered into two groups, taiwan group i (tw-i) and taiwan group ii (tw-ii), on the basis of the s1 gene (wang and tsai, 1996) . however, a variant isolated in 2002 showed an unusually high s1 gene homology with china strains, but not in the n gene, suggesting an inter-strain recombination event (huang et al., 2004) . in this study, the 3 7.3 kb genomes from taiwan ibv strains were investigated to elucidate the genetic diversity of viruses. eight ibvs isolated in taiwan from 1992 to 2007 (huang et al., 2004) and the vaccine strain h120 (abic biological laboratories teva ltd., israel) were recovered for this study. the case histories of local strains are listed in table 1 . viruses were propagated in the allantoic cavity of 9-11-day-old specific pathogen free embryonated eggs (animal health research institute, council of agriculture, tamsui, taiwan). each egg received 0.1-0.2 ml inoculation. after 48-72 h incubation, allantoic fluid was collected and frozen at −80 • c until use. viral rna was extracted from 200 l of virus-infected allantoic fluid using a viral nucleic acid extraction kit (geneaid biotech ltd., taipei, taiwan) following the manufacturer's protocol. previously published primers (huang and wang, 2007) were employed in this study to amplify the gene fragments. for sequencing the 3 untranslated region (utr) of h120, one additional forward primer was designed as 5 -ggaacaatgcacagctggaa-3 from strain h120-gd (genbank accession no. ay028296). reverse transcriptase-polymerase chain reaction (rt-pcr) was performed with one step in a reaction volume of 50 l containing 0.5 l of 5 u/l realtaq dna polymerase (real biotech, taipei, taiwan dna sequencing in both strands from separate rt-pcr products was conducted by a commercial service (mission biotech). each nucleotide was determined from at least four identical results. the obtained nucleotide sequences were compiled and the amino acid sequences were deduced using dnastar software (dnastar, madison, wi). the ibv reference strains were retrieved from the genbank database with the accession number listed in table 2 . the alignx program of the vector nti suite 8 software table 2 ibv reference strains included in this study. country accession numbers armidale australia dq490205 (s1-n) a vic australia dq490221 (s1-n) bj china ay319651 (s1-n) taiwan dq646406 (s1-n) 2296/95 taiwan dq646404 (s1-n) 2575/98 taiwan dq646405 (s1-n) beaudette usa nc 001451 (s1-n) cal99 usa ay514485 (s1-n) cu-t2 usa u49858 (s1-n) de072 usa af274435 (s1), ay024337 (s2) (informax, north bethesda, ml) was used to generate multiple sequence alignments and determine the nucleotide identity. phylogenetic analyses were performed with the neighbor-joining method using mega version 4 (tamura et al., 2007) . the bootstrap values were determined from 1000 replicates of the original data. the consecutive ibv nucleotide sequences from the s to n genes (6.8 kb) based on the multiple alignment results were introduced into similarity plots with simplot version 3.5.1 (lole et al., 1999) . the nucleotide identity was calculated using the kimura 2-parameter method with a transition-transversion ratio of 2 in each window of 500 bp. the window was successively furthered along the alignment using a 20-bp increment. at least four sequences were required to initiate an analysis. the ibv sequences resulting from this study were submitted to the genbank database. the accession numbers are eu822336 (strain 3468/07), eu822337 (strain 3374/05), eu822338 (strain 3263/04), eu822339 (strain 3071/03), eu822340 (strain 2992/02), and eu822341 (strain h120). fig. 1 . phylogenetic analyses of the taiwan strains (᭹), h120 ( ) and reference strains for structural and non-structural protein genes of ibvs. the phylogenetic trees were constructed using the mega version 4 by the neighbor-joining method (bootstrapping for 1000 replicates with its value >70%). sequences that covered the 3 7.3 kb genome were determined from the taiwan ibvs and strain h120. all of the examined viruses were found to maintain the typical ibv genome organization as 5 -s-3a-3b-e-m-5a-5b-n-3 (data not shown). the s gene size ranged from a minimum of 3471 nucleotides (strain 3071/03) to a maximum of 3501 nucleotides (strains 2992/02 and 3374/05). compared with other local strains, single base mutations in the 3 end of the s and 3b genes from the strain 3071/03 changed the genetic code from gaa to taa (glutamine → stop codon), resulting in 27-and 3-base truncated orfs, respectively. similarly, in the 3374/05, a single base t-insertion in the 3b gene created an early stop codon, leading to a 48-base truncation. however, the 3a, 5b, and n gene orf sizes were conserved among ibvs. all of the virus genomes carried an intergenic (ig) region located between the m and 5a genes with a size of 351-362 nucleotides. the ig region of the strain h120 was 55 bases longer than that of the strain beaudette. the 3 utr, downstream of the stop codon in the n gene, a region of 475 and 500 nucleotides was sequenced from local strains and h120, respectively. all of the local strains shared a high sequence identity (96-100%) in this region. however, only 52% and 73% identity were observed between h120 and its closely related m41 and beaudette strains, respectively. phylogenetic analyses were performed based on the nucleotide sequence alignment using each orf from the s to n genes among eight taiwan and reference strains (fig. 1) . in the s1 gene, the taiwan strains showed the highest identity (84%) with the beaudette reference strain and the lowest (60%) with the de072. the taiwan strains (except for 2992/02 and 3374/05) could be clustered into two groups, tw-i and tw-ii, based on the s1 gene. strains 2992/02 and 3374/05 were closely related (>95% identity) to the china genotype vii strains (ck/ch/ldl/97i, ck/ch/ldl/98i, q1, j2, and t3) (liu et al., 2006) . in the s2 gene, all of the local strains were grouped with ck/ch/ldl/97i and ck/ch/ldl/98i except for the two viruses isolated before 1995 (strains 1171/92 and 2296/95). strains 2575/98 and 2992/02 were classified with ck/ch/ldl/97i and ck/ch/ldl/98i in the analyses of the 3a and 3b genes. the e gene analysis segregated the 2992/02 into the china group. strains 2992/02 and 3468/07 were distributed with the china strains in the m gene analysis. the 5a, 5b and n genes analyses revealed that all of the local strains were in the same group. nucleotide sequences from the three orfs were conserved among the taiwan strains with >92% homology. the 3 6.8 kb genome sequence (s-n gene) of six taiwan ibvs were queried in the simplot analysis. strains 1171/92, h120 and the china strain ck/ch/ldl/97i were used as putative parental strains when strains 2575/98, 2992/02, 3071/03, 3374/05, 3468/07 (tw-i) were queried. the parental strain 1171/92 was replaced with 2296/95 when strain 3263/04 (tw-ii) was queried. the similarity plot displays the consecutive nucleotide identity (%) among the queried strain and parental strains. strains were considered as recombinants if any crossover event took place between two putative parental strains. the breakpoint in which the parental strains have equal identity to the query strain is the predicted recom-bination site. as fig. 2 demonstrates, crossover events between parental 1171/92 or 2296/95 and ck/ch/ldl/97i were detected in each plot. the recombination sites were located in the s1, s2, 3b, m genes, and the ig region between the m and 5a genes. each putative recombinant was schematically assembled using taiwan and china-like sequence fragments. the genomic positions of those crossover sites were indicated within the plot (numbers in red). in taiwan, field outbreaks are frequently reported despite routine vaccine use. there were originally two ibv genotypes (tw-i and tw-ii) circulating in the field wang and tsai, 1996) until genetic variants 2992/02 (huang et al., 2004) and 3374/05 emerged. both variants showed a high s1 gene homology with the proventriculus pathogenic strains j1, q2, t3, ck/ch/ldl/97i and ck/ch/ldl/98i, belonging to the china genotype vii (liu et al., 2006; yu et al., 2001) . the 3 6.8 kb gene of ck/ch/ldl/97i was used as a putative parental strain. mass type viruses have been widely used as vaccine strains in taiwan. our primary concern was the possible viral recombination resulting from the co-circulation of heterologous vaccine strains in flocks. the consecutive sequence of 3 7.3 kb genome in strain h120 was obtained first in this study and served as another putative parental strain. to our surprise, six local strains experienced crossover events with the strain ck/ch/ldl/97i instead of h120. thus, inter-strain recombination events had occurred between the ibvs from taiwan and china. taiwan and china are geographically separate. neither live poultry nor processed poultry products from china have been allowed to import into taiwan for years. how those recombinants arose remains unknown. a recently identified ibv isolate in korea (kr/d64/05) also revealed a close relationship to the china ck/ch/ldl/97i-type strains (lee et al., 2008) . furthermore, the appearance of the chinalike strains in taiwan is reminiscent of the spread of the china qxibv strain in european countries (beato et al., 2005; domanska-blicharz et al., 2006) . in this case, it could be speculated that migrating birds provide the genetic sources of ibv variants in taiwan. a chicken-nephropathogenic ibv strain was identified from a non-diseased teal (anas sp.) in china (liu et al., 2005) . thus, the transport of ibvs over long distances by other avian species appears to be possible (cavanagh, 2005) . however, we still cannot overlook illegal trafficking or unapproved vaccine use in the field. all six recombinants defined in this study were inter-genotypic recombination and the "china-like" sequence substitutions took place in multiple genes. in particular, partial s gene replacement was observed in every recombinant. since coronaviruses possess different host range or cell tropism through the variance in the s gene (casais et al., 2003; kuo et al., 2000) , alterations in the antigenic characteristic in those variants could be expected. in this study, however, nearly all of the strains showed nephropathogenicity (lesions of kidney) in chickens, rather than the proventricular lesions caused by the ck/ch/ldl/97i-type strains. how a variant with a chimeric genome arrangement from heterologous strains demonstrates its tissue tropism or pathogenicity in a host is not clear. in addition, it was found that chicks challenged with virulent ck/ch/ldl/97i were incompletely protected by commercial vaccines and other heterologous strains (liu et al., 2007) . to effectively control the ib disease in taiwan, the protective effect of vaccines against challenges from those recombinants awaits to be investigated. to our knowledge, this is the first use of simplot for genetic analyses of ibv strains. the similarity plot can depict the genetic distance among the aligned sequences in a graphical window. in this study ibv recombinants could be defined directly from the simplot analyses, and the crossover events and corresponding genome positions were readily observed. in phylogenetic analyses, strains were deduced as recombinants if different genes from the genomes were clustered into separate phylogenetic groups. parallel results were obtained from both analyses. the emergence of ibv variants through rna recombination was previously described (brooks et al., 2004; jia et al., 1995; lee and jackwood, 2000; mondal and cardona, 2007; wang et al., 1993) . recombination events occurred in multiple genes. the consensus ig sequences ct(t/g)aacaa or the conserved regions around were assumed as the recombination "hot spots" in ibvs (lee and jackwood, 2000) . in addition, the cttttg sequence was observed adjacent to the putative recombinant junction (brooks et al., 2004; wang et al., 1993) . in this study, t-rich motifs (a/t/g)ttttg, consensus among a recombinant and its parental strains, were located upstream of several crossover sites. for instance, the motifs were found in the s2 gene of the 3263/04, the 3b gene of the 2575/98, and the ig region of the 2992/02 and 3468/07. all of the recombinants experienced crossover events in the 3 end of the s gene, indicating a possible region for template switching in viral rna synthesis. similar results were obtained from the ibv strain cu-t2 and cal99, whose recombination sites were deduced in the 3 751 and 700 nucleotide of the s gene, respectively (jia et al., 1995; mondal and cardona, 2007) . in addition to viral recombination, point mutation is a way of virus evolution. the single base nonsense mutations resulted in truncations of the s and 3b genes in the taiwan strains 3071/03 and 3374/05. the naturally occurring ibv strains with mutations or complete deletions in non-structural orfs 3a, 3b, 5a, and 5b revealed lower virus growth rate or titer in ovo (liu et al., 2008) and in vivo (mardani et al., 2008) . this study describes the dual viral genomes incorporation in the separated areas having no known interactions between poultry. the molecular investigation in the 3 7.3 kb of the ibv genome demonstrates that inter-strain recombination events contribute to the genetic diversity of the taiwan strains. field ibvs in taiwan undergo genetic recombination and evolution, which might lead to disease control difficulty. the biological and antigenic characteristics of the ibv variants await further studies. evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in italy molecular epizootiology of avian infectious bronchitis in russia phylogenetic analysis of partial s1 and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity and recombination genes 3 and 5 of infectious bronchitis virus are accessory protein genes comparisons of envelope through 5b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes infectious bronchitis virus s2 gene sequence variability may affect s1 subunit specific antibody 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bronchitis coronavirus genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination infectious bronchitis viruses with a novel genomic organization genotypic and phenotypic characterization of the california 99 (cal99) variant of infectious bronchitis virus emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication mega4: molecular evolutionary genetics analysis (mega) software version 4.0 genetic grouping for the isolates of avian infectious bronchitis virus in taiwan evidence of natural recombination within the s1 gene of infectious bronchitis virus in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the financial support of the national science council and council of agriculture, executive yuan, taiwan, is greatly appreciated. key: cord-346516-lal35iyr authors: hughes, laura a.; savage, carol; naylor, clive; bennett, malcolm; chantrey, julian; jones, richard title: genetically diverse coronaviruses in wild bird populations of northern england date: 2009-07-17 journal: emerg infect dis doi: 10.3201/eid1507.090067 sha: doc_id: 346516 cord_uid: lal35iyr infectious bronchitis virus (ibv) causes a costly respiratory viral disease of chickens. the role of wild birds in the epidemiology of ibv is poorly understood. we detected diverse coronaviruses by pcr in wildfowl and wading birds in england. sequence analysis showed some viruses to be related to ibv. serial cross-sectional surveys of wild bird populations throughout northern england were undertaken from july 2004 through january 2007. samples were collected from 441 individual wild birds of 42 species, including both migratory and resident species (table 1) . fecal samples and oropharyngeal swabs were collected from live birds that had been caught primarily for ringing purposes (8) and from dead wildfowl and corvids provided by licensed shooters. biometric data and information concerning the location where birds had been trapped or shot were recorded (8) . samples were placed in virus transport media (eagle minimum essential medium supplemented with 10% fetal calf serum, penicillin, and streptomycin [10,000 units penicillin, 10 µg streptomycin/ml] and amphotericin b [250 µg/ ml]); samples collected from the same wild bird species or genus were pooled in groups of 5 and frozen at -80°c until required. viral rna was extracted from pooled fecal samples and pooled oropharyngeal swabs by using a qiaamp viral rna mini kit (qiagen, crawley, uk) following the manufacturer's instructions. reverse transcription-pcr was used to detect avian coronaviruses as previously described (9) . the primers utr41+ (5′-atgtctatcgccagggaaatgtc-3′) and utr11-(5′-gctctaactctatactagccta-3′) targeted the 3′ untranslated region (utr) of the coronavirus genome, which is highly conserved among all known types of ibv (9) . this procedure was followed by use of a heminested pcr with the same forward primer but the reverse primer utr hemi-(5′-cttaaactaaaatttagctcttcc-3′) under the same reaction conditions as the initial pcr, which had an expected product size of 214 bp. pcr products were purified by using a commercial purification kit (qiaquick pcr purification kit; qiagen) according to the manufacturer's instructions and were sequenced commercially (cogenics, essex, uk) as recommended by the manufacturer (abi 3730xl dna analyser; applied biosystems, warrington, uk). nucleotide sequences derived from this study have been deposited in the genbank sequence database under accession nos. fj490193-fj490199. sequences were aligned with previously published coronavirus sequences obtained from genbank by using the clustal program within the mega 4.0 package (10) (online appendix figure, available from www.cdc.gov/eid/ content/15/7/1091-appf.htm). phylogenetic analyses were conducted in mega 4.0. phylogenies were estimated by using a minimum-evolution method (11) , and evolutionary distances were computed by using the tamura-nei method (12) . phylogenetic trees were drawn to scale; branch lengths in the same units as those of the evolutionary distances were used to infer the phylogenetic tree. bootstrap analysis using 1,000 repetitions provided support for individual nodes (13) . animal-level prevalences and confidence limits, based on pooled samples, were estimated by using a pooled prevalence calculator (www.ausvet.com.au/pprev). generalized linear modeling was used to calculate maximum-likelihood estimates of prevalence and confidence limits (14) . coronavirus rna was detected in 7 fecal sample pools (table 2) , giving an individual animal-level prevalence estimate of 1.6% (95% confidence interval 0.7-3.1). of the pools with positive results for coronavirus, 4 were collected from ducks (designated anas/uk/p20/2005, anas/uk/ p33/2005, anas/uk/p42/2005 and anas/uk/p71/2005; table 2 ). another pool contained samples from whooper swans (cygnus cygnus) (whooper swan/uk/p3/2005), 1 consisted of samples from red knots (calidris canutus) (red knot/uk/p60/2006), and 1 contained samples from eurasian oystercatchers (haematopus ostralegus) (oystercatcher/uk/p17/2006). pcr-positive pools were from birds sampled in estuarine, salt marsh, or standing water habitats ( table 2 ). all birds appeared to be healthy. all pooled oropharyngeal samples were pcr negative. phylogenetic analyses were based on a final usable sequence of 146 nt after removal of primer sites (online appendix figure) . nucleotide distances between coronavirus sequences derived from this study were 0.0%-15.6%. sequences detected in 3 pooled duck samples and a sequence derived from a pool of whooper swan samples clustered with sequence from an ibv h120 (massachusetts) vaccine strain. sequences within this cluster were relatively homogenous with low within-group distance values (0.0%-2.8%). within this cluster, bootstrap support for the individual nodes was relatively low (figure) . coronavirus sequences detected in red knots clustered with a previously described goose coronavirus; divergence at the nucleotide level was 2.0%. sequences from viruses detected in samples from eurasian oystercatchers and ducks clustered with sequence from a published duck coronavirus (7) . the sequence from eurasian oystercatchers was identical to that of the published duck coronavirus; distance values within this cluster were 0.0%-1.0%. although samples were collected from wild bird populations comprising 46 species of wild birds from numerous and diverse habitats, coronavirus rna was detected only in wildfowl (anseriforms) and waders (charadriiformes). coronaviruses have been detected previously in wildfowl species, rock doves, wild peafowl, and some passerine species (4-7). all wild birds from which coronaviruses were detected in this study appeared to be healthy. although ibv is recognized primarily as a respiratory agent, it has been demonstrated that certain strains are able to replicate in the chicken intestine without obvious clinical disease (15) . phylogenetic analysis showed that coronavirus sequences detected by this study were genetically diverse. virus sequences from 3 pools of fecal samples from ducks and whooper swans shared high nucleotide sequence identity with sequence from the ibv h120 vaccine strain, which is commonly used for the vaccination of commercial chickens worldwide. coronaviruses sharing a high degree of identity with the ibv h120 vaccine strain have been detected previously in healthy, unvaccinated, domestic peafowl and as well as wild peafowl in china (4,5). these viit would be useful to determine the number and genome position of accessory genes of the coronaviruses detected in wild birds and to compare them with those of ibv. more detailed genetic characterization of the viruses detected including, for example, the s1 spike gene, is also needed. the detection of coronaviruses that appear to be related to ibv in wild migratory birds raises interesting questions as to their role in the transmission, dissemination, and evolution of ibv strains. figure. minimum-evolution tree (11) of coronaviruses based on a 146-bp fragment of the 3′ untranslated region of infectious bronchitis virus (ibv). evolutionary distances were computed by using the tamura-nei method (12) and are in the units of the number of base substitutions per site. coronaviruses detected in wild birds by this study are denoted with an asterisk. previously published coronavirus sequences from different sources were included for comparative purposes. genbank accession numbers are shown in brackets. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (13) . the tree is drawn to scale; branch lengths are in the same units as those of the evolutionary distances used to infer the phylogenetic tree. phylogenetic analyses were conducted in mega4 (10). cov, coronavirus. scale bar indicates nucleotide substitutions per site. infectious bronchitis a reverse transcriptasepolymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from molecular analysis of the 793/b serotype of infectious bronchitis virus in great britain isolation of avian infectious bronchitis coronavirus from domestic peafowl (pavo cristatus) and teal (anas) a massachusetts prototype-like coronavirus isolated from wild peafowls is pathogenic to chickens comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) ringer's manual. thetford (uk): british trust for ornithology detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens molecular evolutionary genetics analysis (mega) software version 4.0 a simple method for estimating and testing minimum evolution trees estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees confidence limits on phylogenies: an approach using the bootstrap a critique of methods of sampling and reporting pathogens in populations of fish early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus we thank the merseyside ringing group for collecting field samples, frodsham and district wildfowlers' club for providing wildfowl and corvid samples, rspca stapeley grange wildlife centre, the garden bird health initiative (british veterinary association animal welfare foundation, cj wildbird foods ltd, cranswick pet products, gardman ltd, rspb, the birdcare standards association, universities federation for animal welfare), and members of the public who submitted dead birds for postmortem examination.this work was funded through the defra veterinary teaching and research initiative program.dr hughes is a researcher in the department of veterinary pathology at the university of liverpool. her main research interest is infectious disease epidemiology. key: cord-305684-ipeup5mp authors: chen, yuqiu; jiang, lei; zhao, wenjun; liu, liangliang; zhao, yan; shao, yuhao; li, huixin; han, zongxi; liu, shengwang title: identification and molecular characterization of a novel serotype infectious bronchitis virus (gi-28) in china date: 2016-12-16 journal: vet microbiol doi: 10.1016/j.vetmic.2016.12.017 sha: doc_id: 305684 cord_uid: ipeup5mp avian infectious bronchitis coronavirus (ibv) is a major poultry pathogen. a characteristic feature of ibv is the occurrence of many different strains belonging to different serotypes, which renders complete control of the disease by vaccination a challenging task due to the poor cross-protection between different serotypes. in this study, based on the results of s1 sequence analysis and virus cross-neutralization tests, ibv strain ck/ch/lgx/111119 was found to be genetically and antigenically different from other known ibv types, representing not only a novel genotype, but also a novel serotype (designated as gi-28). viruses belonging to this novel serotype have been isolated from several regions in china in recent years, suggesting endemic circulation of the serotype in various geographic locations in china. further studies by complete genomic analysis showed that strain ck/ch/lgx/111119 may have originated from recombination events involving lx4 genotype ibvs and an as-yet-unidentified ibv donating a s1 gene, or from the result of accumulation of mutations and selections, especially in the s1 gene, from a lx4 genotype virus. ck/ch/lgx/111119 is a nephropathogenic strain, although it had broader tissue tropism (respiratory, digestive, urinary, and reproductive tracts) among chickens challenged at one day old. infection of the oviducts with ck/ch/lgx/111119 found in this study may have severe implications because the virus will likely induce the occurrence of false layers. coronaviruses, family coronaviridae in the order nidovirales, are generally responsible for mild enteric and respiratory infections in both humans and animals (masters, 2006) . they are now recognized as emerging viruses with a propensity to cross into new host species, as have been shown by previous and recent outbreaks of severe acute respiratory syndrome and middle east respiratory syndrome (coleman and frieman, 2014) . to date, coronaviruses were classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the genus gammacoronavirus is mostly composed of viruses isolated from birds. however, gammacoronaviruses have also been detected in the beluga whale (mihindukulasuriya et al., 2008) and bottlenose dolphin (woo et al., 2014) . the most economically important of the avian gammacoronaviruses are infectious bronchitis virus (ibv) and turkey coronaviruses. ibv, which was the first identified coronavirus and was isolated in the 1930s, causes highly contagious infectious bronchitis in domestic fowl, a respiratory, renal, and genital disease, which causes serious economic consequences worldwide (cavanagh, 2007) . coronaviruses are enveloped, positive-sense, and the largest known rna viruses, having a genome length of approximately 30 kb. about two-thirds of the genome consists of two large overlapping open reading frames (orfs), orf1a and orf1b, which are translated as the polyproteins pp1a and pp1ab, and then processed by virus-encoded proteinases into 15 or 16 nonstructural proteins (ziebuhr, 2005) . the remaining one-third of the genome encodes the virus structural spike (s), membrane (m), enveloped (e), and nucleocapsid (n) proteins, as well as four nonstructural accessory proteins (3a, 3b, 5a and 5b). the s glycoprotein can be cleaved into two subunits, s1 and s2, and contains the regions of both b-and t-cell epitopes that are important for virus neutralization and the reorganization of virus-infected cells (reguera et al., 2012; satoh et al., 2011) . the s1 domain occupies the amino proximal half, which contains the receptor-binding domain, and the s2 domain occupies the other half, which contains elements involved in membrane fusion. in contrast to species belonging to the alpha-and betacoronaviruses, which occur as only one or two different serotypes, there are many different serotypes/genotypes of the chicken coronavirus ibv (wickramasinghe et al., 2014) . typing of ibv strains is based on the feature of the s protein, especially s1 domain (cavanagh, 2007) . the high mutation and recombination rate of ibv, especially in the s1 subunit of the s protein gene, has led to the emergence of new variants, particularly in europe and north america, and more recently in intensive poultry farms in china. among the ibvs, two very important genotypes, lx4 (qxlike) and ck/ch/ldl/97i, are believed to have originated in china in mid-1995 and have since spread to other regions of the world (ababneh et al., 2012; marandino et al., 2015) . more recently, another novel genotype, guandong/xindadi (xdn), which is also believed to have originated in china from independent recombination events between the lx4 and 793/b ibv genotypes, have emerged in italy and spain (moreno et al., 2016) . the continuous emergence of new ibv genotypes in china that later spread to other regions of the world has not only prompted the development of appropriate control programs aimed to mitigate the occurrence of the disease caused by those new ibv genotypes, but also emphasize the importance of continuous surveillance of ibv in chicken flocks in china. meanwhile, extensive investigations, involving molecular characterization (especially the complete genome), antigenicity, and pathogenicity, are necessary to clarify the origin of the viruses and are significant for the control of diseases caused by new variants. in this study, a novel serotype (designated as gi-28) of ibv was found to be mainly circulated in south china in recent years and the complete genome, antigenicity, and pathogenicity were investigated to elucidate the origin and evolution of the novel serotype. the ibv strain ck/ch/lgx/111119 was isolated from the proventriculus of a diseased 80-day-old yellow broiler that had been vaccinated against ibv with live attenuated h120 vaccine at 7 days old and boosted at 25 days old with live attenuated 4/91 vaccine. the bird showed obvious respiratory signs at 70 days old. in this flock, morbidity was about 15% and mortality was 3%. gross lesions were mainly mild with tracheitis, proventriculitis, and nephritis in some chickens. the proventriculi of the dead chickens were enlarged and had pale or mottled pale serosa. kidneys of the birds were enlarged and pale. virus isolation was performed as described by . briefly, the proventriculi were homogenized, clarified by centrifugation, filtered, and then inoculated into specific pathogen-free (spf) eggs (harbin veterinary research institute, harbin, china) on embryonation day 9 via the allantoic cavity. the isolate were purified and propagated by inoculating and passaging three times in the allantoic cavity of 9-day-old spf embryonated chicken eggs until characteristic ibv lesions, such as dwarfing, stunting, or curling of embryos, were observed hewson et al., 2009 ). the infectious allantoic fluid was clarified by centrifugation at 5000g for 10 min at 4 c. total viral rna was extracted from the supernatant using trizol reagent (invitrogen corporation, carlsbad, ca, usa), following the manufacturer's protocol, and then stored at à80 c for further use. ibv-specific primers (liu et al., 2013) spanning the entire viral genome were used to reverse transcribe and amplify the viral rna contained in 2 ml of extracted total rna using the primescript tm one-step rt-pcr kit ver. 2 (takara bio inc., shiga, japan) as described by the manufacturer. briefly, a 25-ml reaction was set up on ice containing 2 ml of rna, 1 ml of 10 mm forward, 1 ml of 10 mm reverse primer, 12.5 ml of 2 â 1 step buffer (as supplied by the manufacturer), 1 ml of primescript 1 step enzyme mix, and water to a final volume of 25 ml. the reaction was incubated at 50 c for 30 min to allow cdna synthesis, and then increased to 94 c for 2 min, followed by 30 cycles of 94 c for 30 s, 50 c-60 c (depending on the primer set) for 30 s, and 72 c for 2 min. the reaction underwent a final extension phase at 72 c for 10 min and was then held at 4 c. the 3 0 and 5 0 ends of the viral genomes were confirmed by rapid amplification of cdna ends using a commercial kit (takara bio, inc.) as described by liu et al. (2013) . for each amplicon, 5 ml of pcr products were separated on a 1% agarose/ tris/borate/ethylenediaminetetraacetic acid gel to confirm the pcr product size and to estimate the amount of dna by comparison with standards. the pcr products were sequenced directly and/or cloned into a pmd 18-t vector (takara bio inc.) following the manufacturer's instructions. for each amplicon, three to five clones were sequenced. the s1 gene of strain ck/ch/lgx/111119 was used to search the genbank database (https://blast.ncbi.nlm.nih.gov/blast.cgi) using the blastn program for ibv sequences. six sequences sharing more than 95% nucleotide identity with ck/ch/lgx/111119 were selected for construction of a phylogenetic tree for sequence comparison. in addition, the s1 gene sequences of 93 ibv reference strains with different genotypes were also selected for comparison in this study ( fig. 1 ) (valastro et al., 2016) . the phylogenetic tree was constructed using a neighbor-joining algorithm with 1000 bootstraps based on an alignment generated using molecular evolutionary genetics analysis (mega) software version 4.0. (tamura et al., 2007) . the s1 nucleotide and amino acid sequences of selected viruses (based on the phylogenetic tree results) were also aligned and compared with those of strain ck/ch/lgx/111119 using lasergene software (dnastar, inc., madison, wi, usa). percent similarities were calculated to determine distances between nucleic acid and amino acid pairs. to determine a consensus sequence for the complete genome of strain ck/ch/lgx/111119, the sequence contigs were combined, manually edited, and assembled using lasergene software. the genomic sequences between ck/ch/lgx/111119 and other reference ibvs were analyzed using lasergene software to map each of the genes in the genome of strain ck/ch/lgx/111119. the sequence of the complete genome of strain ck/ch/lgx/111119 was submitted to the genbank database under the accession number kx640829. to identify the recombination events, the blastn program was used to search the genbank database for ibv sequences that were homologous to strain ck/ch/lgx/111119. simplot analyses were performed using a 1000-bp window with a 100-bp step. ibv strain h120 was used as the query strain. six ibv strains, including ck/ch/lgx/111119 in this study and five other strains representing five serotypes (4/91 for 793/b serotype, h120 for massachusetts serotype, ck/ch/ldl/140520 for tw i serotype, ck/ch/ldl/97i for ck/ch/ldl/97i serotype, and ck/ ch/ldl/091022 for lx4 serotype) were subjected to one additional fig. 1 . phylogenetic trees constructed with the nucleotide sequence alignments of the s1 glycoprotein genes using the nearest neighbor-joining method with 1000 bootstrap calculations (valastro et al., 2016) . the genbank accession numbers of the ibv strains are shown in parentheses. the strain ck/ch/lgx/111119 is indicated by a $. all the ibv strains were clustered into 6 genotypes (gi-gvi) and a number of inter-lineage recombinants. ninety-three ibv reference strains in gi were further grouped into 27 distinct viral lineages (gi-1-gi-27) (valastro et al., 2016) . our ck/ch/lgx/111119 strain was clustered into a novel lineage in gi with other 6 ibv reference strains and designated as gi-28. passage in embryonated chicken eggs for virus stock preparation and cross-neutralization tests. the virus titers were determined in 9-day-old spf chicken embryos by the allantoic route of inoculation as described by chen et al. (2015) . in addition to embryo microscopic changes, ibv rna in allantoic fluid was detected by rt-pcr as described by chen et al. (2015) to determine virus replication and subsequently calculate the virus titer. the median embryo infectious dose (eid 50 ) was calculated using the method of reed and muench (1938) . the antisera used for this test included anti-4/91, anti-h120, anti-ck/ch/ldl/140520, anti-ck/ ch/ldl/97i, anti-ck/ch/ldl/091022 , and anti-ck/ ch/lgx/111119, which were prepared as described by gao et al. (2016) . for virus neutralization, the b vn method with constant virus and diluted serum was employed in spf chicken embryos for serotyping. briefly, sera were serially diluted two-fold and mixed with 100 eid 50 of the ibv strains. after incubation for 1 h at 37 c, each of the virus-serum mixtures was inoculated into the allantoic cavity of five spf chicken embryos, which were observed for 7 days. similarly, ibv rna in allantoic fluid was detected by rt-pcr and used to determine virus replication and subsequently calculate the end-point titer of serum. the end-point titer of each serum sample was calculated using the method of reed and muench (1938) . thirty 1-day-old spf white leghorn layer chickens were separated into two groups of 15 chickens per group, which were housed in separate isolation units. birds in group 1 were challenged with the ck/ch/lgx/111119 strain at 1-day-old via the intranasal and ocular routes with 10 6 eid50 in 0.1 ml of diluent per bird. birds in group 2 were not challenged and served as negative controls. the chicks were examined daily for clinical signs of infection . morbidity and mortality were recorded daily. five chickens in each group were humanly killed using carbon dioxide/oxygen. the dead chickens were carefully examined, especially for lesions in the trachea, kidneys, and proventriculi. then, tissues of the tracheal, lungs, kidneys, and cecal tonsils were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at a thickness of 4 mm, and mounted on glass slides, which were stained with hematoxylin and eosin. immunohistochemical (ihc) analysis was performed to detect the ibv antigen using monoclonal antibody 6d10 against the n protein as described by han et al. (2016) and gao et al. (2016) . blood samples were collected on day 4 from 10 birds and on days 8, 12, 16, 20, and 24 post-challenge from all birds to confirm the presence of antibodies against ibv using a commercial enzymelinked immunosorbent assay kit (idexx laboratories, inc., westbrook, me, usa) in accordance with the manufacturer's instructions. the percentage of seropositive birds for each group in this study was calculated. all surviving birds were killed humanely using carbon dioxide/oxygen at 3 months of age, which was followed by exsanguination. post-mortem examinations were performed with special attention to abnormalities in the oviducts and kidneys. the kidney and oviduct samples were collected to detect the presence of ibv by ihc analysis. genotyping based on the phylogenetic analysis of the s1 gene from our ck/ch/lgx/111119 and 99 reference ibv strains assigned the viruses into different clusters (fig. 1) . the 93 ibv reference strains were clustered into 6 genotypes that together comprise 32 distinct viral lineages (gi-1-gi-27; gii-gvi) and a number of inter-lineage recombinants (valastro et al., 2016) . the isolate ck/ ch/lgx/111119 in this study was clustered into a novel lineage in genotype i (gi) with 6 ibv strains isolated in china and clearly set apart in the phylogenetic tree from the 93 reference strains; hence, we designated the novel genotype as gi-28. viruses of the gi-28 genotype possessed more than 97.7% and 96.0% nucleotide and amino acid identities, respectively, between each other in this study, supporting the classification of these viruses as a novel genotype. one ibv strain lie together with the qxibv strain, and have been described previously as lx4 genotype or qx-like viruses . comparatively, the lx4 genotype (qx-like) viruses are closely related to the gi-28 genotype, sharing approximately 87.1% and 87.3% nucleotide and amino acid identities, respectively, with the s1 protein of the gi-28 genotype viruses selected in this fig. 2 . simplot analysis of the complete genomic sequences of strains ck/ch/lgx/111119 and ck/ch/ldl/091022. analysis was performed using simplot software version 3.5.1 to identify potential recombination breakpoints (chen et al., 2015) . a 1000-bp window with a 100-bp step was used. strain h120 was used as the query strain. study. the massachusetts genotype h120 had less than 80% nucleotide and amino acid identities with the s1 protein of gi-28 genotype viruses. the tw i genotype, which has been described previously (xu et al., 2007) . the s1 protein of genotypes gi-28 and tw i shared approximately 80% nucleotide and amino acid identities. the strains ck/ch/ldl/97i and q1 formed a unique cluster that shared the lowest identities with the gi-28 genotype (nearly 75%). the 793/b genotype shared about 78% and 76% nucleotide and amino acid identities, respectively, with the s1 protein of the gi-28 genotype. the genome of ibv strain ck/ch/lgx/111119 is 27,667 nucleotides in length, excluding the 3 0 poly(a) tail, and shows typical ibv organization. the 5 0 end of the viral genome contains a 525-nt 5 0 untranslated region (utr) followed by a 19889-nt replicase gene encoding for two large polyproteins, pp1a and pp1ab, which occupied about two-thirds of the viral genome. four structural and accessory proteins were downstream of the replicase gene followed by a 504-nt utr at the 3 0 end of the genome. results of a blastn search showed that not only the s1 gene, but the complete genome of the ck/ch/lgx/111119 strain, showed the closest genetic relatedness with lx4 genotype strains. hence, the lx4 genotype strain ck/ch/ldl/091022 was used for simplot analysis with strain ck/ch/lgx/111119. as illustrated in fig. 2 , a fragment from approximately nt position 20,900 to 22,500 in the genome of strain ck/ch/lgx/111119 was obviously different from that of strain ck/ch/ldl/091022. this approximate 1600-nt fragment occupied most of the s1 gene and small parts of sequence at the 5 0 end of the s2 gene. the remaining regions in the genome were very similar, although some regions showed slight diversities between the two strains, implicating the involvement of lx4 genotype ibvs in the origin of ck/ch/lgx/ 111119-like strains. the results of the cross-neutralization tests using the ibv strain ck/ch/lgx/111119 and antisera against the five ibv strains, which represented the massachusetts, 793/b, lx4, ck/ch/ldl/97i, and tw i serotypes, showed that the strains belonged to different serotypes (table 1 ). in line with the genotyping results, strain ck/ ch/lgx/111119 was antigenically distinct from the massachusetts, 793/b, ck/ch/ldl/97i, and tw i serotypes. comparatively, the cross-neutralization results showed that strain ck/ch/lgx/111119 was antigenically close to the lx4 genotype strain ck/ch/ldl/ 091022 (table 1) with an r value of 0.306. these results demonstrated that strain ck/ch/lgx/111119 represents a new serotype (also designated as gi-28) that is antigenically distinct from other ibv serotypes. all of the chickens exhibited mild respiratory signs at 2-4 days post-challenge (dpc) with the ibv strain ck/ch/lgx/111119, and three of them died at 5, 7, and 10 dpc, respectively. the most remarkable lesions were confined in the kidneys of the five chickens killed at 5 days pdc and the three dead chickens. the affected kidneys were swollen and pale with tubules and ureters distended with urates (fig. 3a) . the most interesting observation was the dilatation and serous fluid accumulation in the oviducts of two of the three layer hens that survived 90 dpc (fig. 3b) . no clinical signs were observed and no birds died in the control group during the experimental period. no lesions were found in the kidneys or oviducts of the birds in the control group at the end of the experiment. viral antigens were detected by ihc analysis in the kidneys, secondary bronchi ( fig. 4a and b) , and enterocytes of the cecal table 1 results of reciprocal b virus neutralization tests using ck/ch/ldl/14520 and other ibv strains (serum dilution using a constant amount of virus). tonsils of the five chickens infected with strain ck/ch/lgx/111119 at 5 dpc ( fig. 4c and d) . obvious lesions (nephritis) were found in the kidneys of the five chickens killed at 5 days pdc and the three dead chickens, and ihc-positive cells were observed in the kidneys of the five chickens killed at 5 days pdc with ibv strain ck/ch/lgx/ 111119 as nephropathogenic strains ( fig. 4e and f) . most of the birds seroconverted by 8 dpc and all seroconverted by 12 dpc (table 2 ). there was no seroconversion in any chicken in the negative control group. ibv has progressively emerged as the cause of moderate to severe disease in chickens, with different variants/genotypes/ serotypes being detected in recent years in china. the ibv isolate ck/ch/lgx/111119, investigated in the present study, displayed distinct molecular features with respect to other genotypes and was clearly set apart from those that are not only used as vaccines in china like massachusetts and 793/b, but also from chinese variants of ibv, such as genotypes lx4, ck/ch/ldl/97i, and tw i. dead survival ck/ch/lgx/111119 15/15 3/10 3/3 0/7 2/3 0/10 6/8 7/7 7/7 7/7 7/7 negative control 0/15 0/10 -0/10 -0/10 0/10 0/10 0/10 0/10 0/10 a the post-mortem examinations were only conducted for chickens that died from challenge with ck/ch/lgx/111119 strain, as well as for the chickens that survived 90 days post-challenge. all of the dead chickens, but none of the surviving chickens, had nephritis. b antibody responses against ibv were examined from 4 to 24 days post-challenge. c only three of the surviving chickens were hens, which were examined for oviduct abnormalities. the closest relative genotype was ibv lx4, the most predominant variant in china , which shared approximately 87% nucleotide and amino acid identities with isolate ck/ch/lgx/ 111119. there are several publications of the ibv genotype that included ck/ch/lgx/111119, but unfortunately these reports do not agree on genotype designations, such as a2 by ji et al. (2011) and ch iii by luo et al. (2012) and feng et al. (2014) . also, the virus genotype designations were inconsistent even in reports by the same research group (ji et al., 2011; feng et al., 2014) . the ch iii and a2 genotypes have previously been used to classify other ibv genotypes in china (liu et al., 2006; xu et al., 2007) . herein, by comparison with the results from valastro et al. (2016) , the ibv reported in this study was designated as the gi-28 genotype. comparison of the s1 genes of the gi-28 genotype with those of other genotypes selected in this study revealed not more than 87.5% nucleotide and amino acid identities, which could explain the distinct antigenic relationship of ck/ch/lgx/111119, as compared with other serotypes (cavanagh, 2007) . analysis of complete genomic sequence of the ck/ch/lgx/111119 revealed that the virus exhibited high identity in the entire genome with lx4 genotype ibvs with the exception of the s1 protein gene, which is markedly diverse. we proposed that the emergence of gi-28 genotype viruses in chicken flocks in china resulted from recombination events involving lx4 genotype ibvs and an asyet-unidentified ibv donating a s1 gene that encoded a protein of low amino acid identity to those of other ibvs (approximately 87.3%). this was very similar to that of turkey coronavirus, which was thought to have arose through recombination of the s gene (jackwood et al., 2010) . alternatively, the gi-28 serotype might have originated from the accumulation of mutations and selection, especially in the s1 gene, because s1 domain showed the high sequence diversity in ibv (wickramasinghe et al., 2014) . if this is the case, strain ck/ch/lgx/111119 might have undergone extensive divergent evolution and acquired the abilities to infect and spread among chicken flocks. the higher sequence similarity in other regions in the genome outside of the s1 gene suggest a common origin of these sequences and could explain the closer antigenicity between ck/ch/lgx/111119 and ck/ch/ldl/091022, as compared with those of other serotypes, because in addition to the five conformation-dependent neutralizing antigenic sites mapped on s1, another immunodominant region was mapped in the nterminal region of s2 (wickramasinghe et al., 2014) and some antigenic epitopes that have roles in the protection have also been identified on n proteins of ibv (ignjatovic and sapats, 2005) . in this study, mild respiratory signs developed in the chicks at 2-4 dpc and severe nephritis present in the dead chickens was caused by infection with the isolate ck/ch/lgx/111119 and no obvious lesions were observed in the proventriculus. this is in contrast to the observation in the field cases, where obvious respiratory signs and proventriculitis were observed. there are at least two possible reasons for this discrepancy: first, ibv strain ck/ ch/lgx/111119 may not be the only causative pathogen responsible for the occurrence of the outbreak, and secondary infections caused by other microorganisms enhance the severity and contribute to the disease development. second, it is also possible that different genetics of the chicks in the field cases and of the chicks experimentally infected are responsible for the difference in tropism of the virus; it is believed that genetics of chickens play an important role in differences in ibv susceptibility and tropism (ignjatovic et al., 2003) . it is believed that ibv mainly infects the respiratory and urogenital tracts, but it has long been known that ibv can be isolated from digestive organs, such as the proventriculus, intestines, and cloaca, and persists in the cecal tonsils (ambali and jones, 1990; ambali, 1992; montgomery et al., 1997) . similarly, the results of this study showed that obvious positive immunohistochemical reactions developed not only in the cytoplasm of the tubular epithelial cells and in the mucous membrane of the ureters and collecting ducts in the kidney, and in the epithelial cells of the tracheal mucosa of the secondary bronchi at 5 dpc, but the viral antigens were also detected in the cecal tonsils. this result suggests a broad tissue tropism of strain ck/ch/lgx/111119, similar as that of its deduced parental lx4 genotype viruses (benyeda et al., 2009; de wit et al., 2011; mork et al., 2014) . the clinical manifestations of ibv in the oviduct are of high economic importance in the poultry industry because an infection of the oviduct may have severe implications comprising a drop in egg production, poor egg quality, and the occurrence of so-called "false layers." it has been reported that ibv genotypes lx4 (benyeda et al., 2009; de wit et al., 2011) , massachusetts serotype h52 (duff et al., 1971) , is/885/00-like virus isolated in the middleeastern region (awad et al., 2016) , and nrtw i serotype viruses isolated in china related to cystic oviducts in layer flocks and induce false layers. an interesting observation in this study was that the lesions of cystic oviducts were observed in the surviving chickens that were challenged at 1 day old with strain ck/ ch/lgx/111119. this finding is in agreement with the field cases, in which false layers were observed in the layer flock from which strain ck/ch/lgx/111119 was isolated. the gi-28 type first emerged in 2005 in guangdong province and has mainly circulated in south china in recent years (fig. 5) . meanwhile, some strains of this type were also isolated in other regions of central china, implicating the wide distribution and spread of this ibv type and emphasizing the importance of continuous surveillance of ibv in chicken flocks in china. in addition, it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control gi-28 type ibv strains in future because the gi-28 viruses related to cystic oviducts in layer flocks and might induce false layers. 201303033). presence of infectious bronchitis virus strain ck/ch/ldl/97i in the middle east early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus recent studies on the enterotropic strain of avian infectious bronchitis virus experimental infection of is/885/00-like infectious bronchitis virus in specific pathogen free and commercial broiler chicks comparison of the pathogenicity of qx-like, m41 and 793/b infectious bronchitis 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virus infection (ibv) in chickens in egypt associated with frequent co-infections with avian influenza and newcastle disease viruses date: 2017-03-21 journal: j virol methods doi: 10.1016/j.jviromet.2017.02.018 sha: doc_id: 322410 cord_uid: k23engcx in egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (ibv) are circulating with detrimental effects for poultry industry. a sensitive real-time rt-pcr assay targeting the ibv nucleocapsid gene (n) was developed to screen clinical samples for presence of ibv. conventional rt-pcrs amplifying hypervariable regions (hvrs 1–2 and 3) of the ibv s1 gene were developed and amplificates used for nucleotide sequence-based typing of ibv field strains in egyptian chickens directly from clinical samples. in total, fifty samples from poultry comprising swabs, tissues, and allantoic fluid were examined. twenty eight samples from chickens showed ibv-positive results. genetic analysis of the hvrs 1-2 of seven samples revealed closest amino acid homology of 83.3-89.7% in four viruses and 96.1-97.7% in the others to the previously described egyptian variant ii (eg/12197b/2012), while all seven samples shared >98.2% amino acid homology at the hvr3 locus with that genotype. in addition, in most of samples a high degree of co-infections with highly pathogenic aiv h5n1, low pathogenic h9n2, and newcastle disease was found. mixed infections in this study were detected in 19 out of 28 ibv positive samples. this indicates an intricate situation in egyptian poultry populations with unknown putative synergistic effects on pathogenicity and spread of these pathogens. effective control measures including vaccination may be severely compromised. avian infectious bronchitis virus (ibv) is a member of the genus gammacoronavirus in the coronaviridae family (king et al., 2011) . ibv induces an acute, highly contagious infectious disease of chicken. ibv is globally distributed and responsible for huge economic losses in the poultry industry. ibv was first reported in north dakota, usa, as a novel respiratory disease affecting chickens (schalk and hawn, 1931) . ibv infects initially the respiratory tract; for some ibv strains further virus spread may involve kidneys and oviduct causing reduction of growth rate, decreased performance and reduction of egg quality and quantity (cavanagh, 2003) . also, some strains showing a shift of tissue tropism may cause proventriculitis (yu et al., 2001) .the infection is spread by aerosols, direct contact and indirectly through contaminated fomites (ignjatovic and sapats, 2000) . ibv harbors an unsegmented rna genome of positive polarity which is approximately 27.6 kb in size and codes for four structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope (e) protein (spaan et al., 1988) . the n protein is a major structural protein, and highly conserved among different ibv serotypes the spike (s) glycoprotein, an integral membrane protein, is another major structural protein; it is cleaved post translationally into the s1 (n terminal part) and s2 fragments. mature s proteins trimerize and form the globular head (s1) and the stalk domain (s2) of the viral peplomer spikes (belouzard et al., 2012; cavanagh, 2007) . the s1 protein carries the receptor binding site and thus plays an important role in both tissue tropism and induction of protective immunity (belouzard et al., 2012; wickramasinghe et al., 2011) . numerous distinct serotypes have been described which differ by 20-25% and sometimes up to 50% in the s1 protein sequence (adzhar et al., 1997) . in particular, three hypervariable regions (hvrs; amino acid residues 38-67, 91-141 and 274-387) along the s1 gene are affected that elicit neutralizing and serotype-specific antibodies koch et al., 1990; moore et al., 1997) . variation in these epitopes has been implicated in escape from vaccine-induced immunity (belouzard et al., 2012) .genotypes of ibv are classified based on the genetic variation of the s gene encoding the spike protein, in particular its s1 fragment (belouzard et al., 2012; cavanagh, 2007; valastro et al., 2016) . consequently, cross protection between these different serotypes is limited (cavanagh, 2005; jackwood, 2012; kuo et al., 2010; wickramasinghe et al., 2014) . the s1 gene therefore is used for ibv strain differentiation (gough et al., 1992; kingham et al., 2000) . new s1 genotypes of ibv that often show antigenic variation and, hence, define new serotypes, appear to emerge frequently in different parts of the world (jackwood, 2012) . a number of mutation processes account for the emergence and evolution of multiple serotypes (cavanagh et al., 1992; jackwood, 2012) including point mutations, insertions, deletions, and also recombination between strains (adzhar et al., 1997; hewson et al., 2014) . in egypt, highly pathogenic avian influenza viruses (hpaiv) of subtype h5n1 and co-circulating low pathogenic aiv h9n2 have established endemic status (naguib et al., 2015) . various chicken flocks are suffering from respiratory disease caused, at least in part, by infection with ibv and ndv (abd el rahman et al., 2015) . over the last decades, different genotypes of ibv have been recognized in egypt which were related to the massachusetts, d3128, d274, d-08880 and 4/91 genotypes (abdel-moneim et al., 2006; jackwood, 2012; sheble et al., 1986) . in 2001, a unique egyptian variant (type isolate: egypt/beni-suef/01), closely related to an israeli variant strain ii, was identified in different chicken farms and classified as egyptian variant i (abdel-moneim et al., 2002) . in 2011, yet another genotype, egyptian variant ii (ck/eg/bsu-2,3/2011), was isolated, and representatives of these genotypes have been co-circulating since with the previously detected classical (vaccine like) and variant ibv genotypes as mentioned above (abdel-moneim et al., 2012) . this study is aimed at providing novel molecular diagnostic tools that can be used to detect and characterize ibv genotypes circulating on chicken farms in egypt. an n gene specific real time rt-pcr (rt-qpcr) specifically tailored to detect ibv circulating in egypt complements with previously published rt-qpcrs targeting another ibv orf e.g., (callison et al., 2006) . in addition, conventional rt-pcrs for nucleotide sequence analysis of the s1 gene hvrs have been developed and validated. fifty field samples including tracheal swabs, allantoic fluid (from isolation attempts), and tissues (kidney and trachea) were obtained in the frame of routine veterinary measures from commercial poultry farms showing severe respiratory problems and/or performance losses in egypt between 2012 and 2014 (table s1 ). detailed information on species, type, age and mortality is presented in table s1. the locations of the farms are depicted in table s1 at the gover-norate level. swab samples from alive birds were jointly obtained according to standard procedures by authorized veterinarians of the national laboratory for veterinary quality control on poultry production (nlqp, ministry of agriculture, giza) and the faculty of veterinary medicine, beni-suef university (bsu), egypt. tissues were collected from birds that had died spontaneously. following initial examination at these institutions samples were submitted to the friedrich-loeffler-institut, germany. viral rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. rna was eluted in 70 l nuclease-free water, aliquoted at 10 l and stored at −20 • c until use. by using a newly developed rt-qpcr (see below), presence of n-specific ibv rna was examined and positive samples were further subjected to two conventional rt-pcrs specific for hypervariable regions (hvrs) of the s1 gene of ibv (see below). in addition, all samples were screened for presence of the influenza a virus matrix (m) gene (fereidouni et al., 2012) , and positive samples were further subtyped using rt-qpcrs specific for subtypes h5 and h9 (monne et al., 2008) . also, samples were examined for ndv rna using an rt-qpcr specific for the avian paramyxovirus-1 matrix gene (wise et al., 2004) . pcr reactions for aiv and ndv were performed in 25 l volumes using superscript ® iii one-step rt-pcr kit with platinum ® taq dna polymerase (invitrogen) on a cfx96 thermocycler machine (bio-rad). pathotyping of h5-and ndv-positive samples was achieved by sequencing of the ha (naguib et al., 2015) or f gene cleavage sites, respectively (aldous et al., 2001) . primers were selected based on alignments of the n and s genes of a selection of avian gamma-coronavirus sequences available from genbank (ncbi). pre-selected primers were then screened in silico for their binding properties to ibv and shannon entropy plots using entropy one software (http://www.hiv.lanl.gov/content/ sequence/entropy/entropy one.html) were produced to confirm the specificity of the primers and probes for different ibv genotypes ( fig. 1) . shannon entropy analysis for ibv-specific primers and probes targeting a fragment of the nucleocapsid (n) orf was carried out using an alignment of 500 sequences of the n orf of representative ib viruses from different lineages. entropy values of primers used for amplification of the hvr of the s1 gene was performed on alignments of sequences representing various genotypes including egyptian variants i and ii, different israeli variants, qx viruses as well as different vaccine strains. the oligonucleotides finally designed are shown in table 1 . furthermore, the specificity of the newly developed rt-qpcr was evaluated using different ib reference viruses: m41, ma5, h120, h52, beaudette, qx, qx-like, cr88-121, 4/91, d880, d274, and the egyptian variants i (eg/ibv1) and ii (ibv-eg/1212b-2012). turkey coronavirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, equine torovirus, bovine and canine coronaviruses, in addition to aiv subtypes h5n1, h9n2 and ndv were used to further define specificity. the optimized thermal cycling conditions for the newly developed rt-qpcr specific for the ibv n gene fragment using the agpath-id one-step rt-pcr (thermofisher) kit were as follows: a reverse transcription step was carried out at 45 • c for 10 min, followed by an initial denaturation step at 95 • c for 10 min, and 40 cycles of pcr amplification at 95 • c for 30 s, 58 • c for 15 s, and 72 • c for 15 s in a 25 l reaction mixture using 12.5 pmol of each forward, 30 pmol of the reverse primer and 10 pmol probe per reaction. fig. 1 . analysis of the detection limit of the ibv n-specific rt-qpcr based on serial ten-fold dilutions of transcribed viral rna ranging from 10 6 to 10 0 copies/reaction (a). linear regression analysis was constructed revealing the correlation coefficients (r2) and the slope value (b). this new diagnostic tool was compared to a previously published rt-qpcr targeting ibv orf1a; thermal cycling conditions of this method were described before (callison et al., 2006) . for the conventional rt-pcrs targeting the s1 hvrs 1-3 primer concentrations of 12.5 pmol each were used per reaction. the superscript ® iii onestep rt-pcr kit with platinum-taq was used with the following cycling condition: 45 • c for 10 min, followed by an initial denaturation step at 95 • c for 10 min followed by 35 pcr amplification cycles were run at 95 • c for 15 s, 52 • c for 15 s, and 68 • c for 30 s with a final extension step of 68 • c for 5 min. an amplificate was produced from ibv strain m41 using the n-specific rt-qpcr primer set and cloned into the topo ta cloning dual promoter kit (invitrogen) containing t7 and sp6 promoter sequences. rna was transcribed with t7 rna polymerase (promega) from bamhi linearized plasmids according to the manufacturer's instructions. transcribed rna was further purified and quantified. the detection limit of the rt-qpcr assays was determined by testing in triplicate serial ten-fold dilutions of the in-vitro transcribed viral rna ranging from 10 6 to 10 0 rna copies/l/reaction, and standard curves were produced. a cq value <40 was considered positive. rna copy number was calculated based on the dna/rna copy number calculator online tool http:// endmemo.com/bio/dnacopynum.php for amplification of the hvr 1 and 2 of the s1 gene of ibv, forward and reverse primers were designed in the frame of this study, while those for the hvr3 were modified from adzhar et al. (1997) (table 1) . amplificates were size-separated by agarose electrophoresis, excised and purified from gels using the qiaquick gel extraction kit (qiagen, hilden, germany). purified pcr products were used directly for cycle sequencing reactions (bigdye terminator v3.1 cycle sequencing kit, applied biosystems). the reaction products were purified using nucleoseq columns (macherey-nagel gmbh ® 3100 genetic analyzer (life technologies). the obtained s1 hvr sequences were assembled and edited using geneious software, version 7.1.7 (kearse et al., 2012) . alignment and identity matrix analyses were performed using mafft (katoh and standley, 2013) and bioedit (hall, 1999) . sequences generated in this study were deposited in the genbank database, and assigned accession numbers are shown in table s1 . sequences of other viruses required for further analyses were extracted from public databases. phylogenetic analyses were based on manually edited alignments of the full-length open reading frames. for maximum likelihood analysis of phylogenetic relationship, a best fit model was chosen first on which further calculations and an ultrafast bootstrap equivalent analysis was based. the iq-tree software version 1.1.3 was used for all operations (minh et al., 2013; nguyen et al., 2015) . trees were finally viewed and edited using figtree v1.4.2 software (http://tree.bio.ed.ac.uk/software/figtree/) and inkscape 0.48. 3.1. performance characteristics of quantitative (n-specific) and conventional (s1-specific) rt-pcrs for ibv detection shannon entropy plots, used to evaluate the specificity of the designed primers, predicted a broad reactivity against different table 2 comparison of the analytical specificity of two real time rt-qpcrs targeting utr (callison et al., 2006) or n (this study) using a panel of ibv and other coronaviruses as well as other avian viral pathogens. rt-qpcr n rt-qpcr orf1a -a positive, cq values obtained with the two rt-qpcrs did not differ by more than 1-2 values indicating similar analytical sensitivity. b negative; cq values > 40. ibv genotypes circulating in the middle east (fig. s1 ). considerable sequence variability was evident despite selecting regions with pronounced overall conservation. thus, primers were degenerated at key positions as shown in fig. s1 to allow broader, yet specific, target hybridization. the specificity of rt-qpcr primer set was evaluated by examining different avian respiratory viruses circulating in poultry in egypt including aiv h5n1, h9n2 and ndv as well as different coronaviruses including a panel of ibv reference strains as listed in the materials section. this comprises all ibv genotypes reported to be circulating in egypt. no amplification was detected for h5n1-, h9n2-and ndv-specific rna ( table 2) . as predicted by shannon entropy plots, a distinctly specific amplification of ibv rna but not of other coronaviruses, with the exception of turkey coronavirus, was observed with the n-specific rt-qpcr primer set. genomic rna from ibv isolate (m41) was used as template for initial validation experiments. analytical specificity and sensitivity of the newly developed rt-qpcr compared favorably with the 5 utr −specific rt-qpcr of callison et al. (2006) (table 2 ) which also does not differentiate between ibv and turkey coronaviruses. run-off rna transcripts of the ibv n gene fragment were used to evaluate analytical sensitivity of the ibv-n-specific rt-qpcr. investigations using copy-based rna run-off transcripts showed a detection limit of that pcr close to 10 copies per assay. the cq values corresponding to 10 rna copies were 37 ± 0.5. the standard curves as shown in fig. 1 depict a dynamic linear range across at least 6 log units of rna copies. linear regression analysis revealed excellent reproducibility characteristics (fig. 1) . based on these results the n-specific rt-qpcr was deemed fit as a screening tool for ibv infections in clinical poultry samples from egypt. the two primer sets designed or modified to amplify ibv s1 hvr1-2 and 3 produced a band of 454-bp covering hvr 1 and hvr 2 (set 1), or a 382-bp band covering hvr3 (set 2) as predicted from an alignment of all different serotypes and variants detected in egypt so far (fig. s2) . the specificity of the two hvr primer sets was further confirmed using reference strains compromising different genotypes circulating in egypt as mentioned in the material section. an amplificate of 1010 bp was also generated using the forward primer for hvr1-2 and the reverse primer of hvr3; this amplificate covers all three hvrs of the s1 gene (fig. s2) . a trade-off had to be observed between a shorter pcr product which is less vulnerable to be affected by target rna degradation in clinical samples and a longer one yielding more sequence information (moss and thein, 1998) . therefore, a decision was taken to generally amplify the three s1 hvrs in two separate pcrs when examining clinical samples. generally, a sequenceable product was generated with these s1 rt-pcrs from clinical samples if the cq value obtained by use of the n-specific screening rt-qpcr was lower than 32 (equivalent to approximately 100 rna target copies). thus, this method gives maximum flexibility that can be adjusted according to the sample quality (virus isolate versus clinical sample). a total of 50 field samples were examined by ibv n-specific rt-qpcr. 28 samples yielded positive signals for presence of ib viruses (table 3 ). in the same sample set other avian respiratory viruses were detected by further rt-qpcrs specific for the hemagglutinin gene of aiv subtypes h9 (n = 24), and h5 aiv (n = 6), or specific for the m gene of ndv (n = 13). molecular pathotyping for the h5 viruses revealed presence of highly pathogenic strains of clade 2.2.1 as genetically characterized in a previous study (see also (naguib et al., 2015) ). for nd, three velogenic viruses all clustering with genotype vii were detected while the remaining eight were lentogenic vaccine strains. in two ibv-positive samples also h5 hpaiv was found; sixteen ibv positive samples also harbored h9 aiv, and seven ibv positive samples also contained ndv. only nine samples showed solely ibv infection without aiv or ndv. furthermore, aiv and/or ndv were detected in several ibv negative samples. details of the various co-infections are shown in table 3 . sequenceable hvr fragments were generated from seven ibv positive samples (shown in bold-face in table s1 ). no rt-pcr amplicates were obtained from two further samples that showed cq values <32 in ibv rt-qpcr; failure to amplify ibv s1-specific rna from these samples may be related to advanced rna degradation. the sequences obtained were added to an alignment that had also been used for the in silico analysis of the hvr primers: the sequences derived from three samples (ar2206, ar2185 and ar2211) revealed a high similarity of about 96.1-97.7% at the amino acid (aa) level with egyptian ibv variant ii (e.g., strain eg/1297b/2012, genbank accession number: kc533683.1) but only 72.1-72.9% to egyptian variant i (eg/beni-suef/01, genbank accession number: af395531) within their hvrs 1 and 2. the remaining four viruses (ar2189, ar2198, ar2211 and ar2212) showed 83.3-89.7% aa identity in those regions with egyptian variant ii viruses (table 4a ). this indicates that two groups of viruses can be distinguished within egyptian variant ii with respect to hvr 1, 2 sequences. all seven samples revealed 98.2-100% aa identity with egyptian variant ii for the hvr 3 and 88.4-89.3% identity compared to egyptian variant i (eg/beni-suef/01 genbank accession number: jx174183). all sequences obtained from seven clinical samples are remarkably distinct (more than 20% aa difference) to classical vaccine strains (ma5, m41 and h120) that are used in egypt. the seven field-type sequences were also grossly distinct from other vaccine strains currently or previously used in egypt (4/91, cr88 or d274) (table 4b) . phylogenetic analysis confirmed the clustering of the seven ibv positive samples with egyptian variant ii sequences (fig. 2) . within hvr 1, 2 sequences, however, the existence of two distinct table 3 rt-qpcrs reveal frequent co-infections in egyptian poultry samples with avian influenza (aiv), newcastle disease (ndv) and infectious bronchitis viruses (ibv). ibv aiv m aiv-h5 aiv-h9 ndv collective results 1 eg/ar2206-14/2012 30,13 ----ib 2 eg/ar2207-14/2012 ------3 eg/ar2208-14/2012 33,22 ----ib 4 eg/ar2164-14/2013 ------5 eg/ar2165-14/2013 -17, groups was confirmed ( fig. 2a) . interestingly, one group of hvr 1, 2 sequences was closely related to vaccine strain 1494 ( fig. 2a , black dot) which, in turn, belonged to egyptian variant i in terms of hvr 3 sequences. the other group was closely related to vaccine strain d274 whose hvr 3 was closely related to mass-like vaccine strains. controlling ib in egypt remains a challenging task due to the wide circulation of at least two different genotypes and emerging of novel strains which may pose risks of ibv vaccination failure. there also is a lack of consistent biosecurity levels in some intense production regions which opens the door to the possibility of mixed infection with other respiratory viruses as well as bacterial pathogens like mycoplasma spp. and e. coli. therefore, rapid molecular diagnostic tools covering the broad spectrum of ibv circulating in egypt are required as well as subsequent genotype identification. the ibv n-specific rt-qpcr and hvr1-3-specific rt-pcr assays developed and adapted in this study are shown to provide a reliable sensitive and specific approach for screening of suspect samples as well as for downstream genetic characterization of viruses. the hvr-specific pcrs allowed for genotyping directly from clinical samples omitting the need to isolate virus, provided a well preserved rna sample quality and sufficient viral loads. partial s1 gene analysis of the three hvrs of seven ib viruses detected and characterized in this study showed that they were closely related genetically and phylogenetically to the currently circulating egyptian variant ii. in particular, hvr3 of these viruses formed a monophyletic cluster (fig. 2b) . analyses of hvrs 1 and 2, however, revealed two distinct phylogenetic groups ( fig. 2a) . thus, there seem to exist two populations of egyptable 4 comparison of nucleotide and deduced amino acid sequences of the s1 hvr1-2 (a) and hvr3 (b) of ibv from egyptian field samples with reference and vaccine strains of different ibv serotypes. tian variant ii viruses which can be distinguished in their hvr1, 2 loci. the molecular mechanisms having caused this split remain to be elucidated. to confirm or rule out recombination full length s1 sequences will be required of a larger panel of egyptian ib viruses. in addition, it remains to be clarified whether antigenic differences exist between the two clusters within egyptian variant ii. in this set of samples, no ibv of egyptian variant i was detected; this seems to confirm previously published data from genbank indicating a dominance of egyptian variant ii. infectious bronchitis, avian influenza and newcastle disease are the three major causes of economic losses in the poultry industry; they are able to induce disease independently or in association with each other. avian influenza h5n1 and h9n2 subtypes continue to circulate in egypt since 2006 and 2011, respectively, causing many outbreaks in poultry farms (naguib et al., 2015) . velogenic ndv circulating among chickens in egypt resembles genotype viid (chicken/china/sdwf07/2011) and is associated with outbreaks in commercial poultry farms despite adherence to strict vaccination regimes (radwan et al., 2013) . similar co-infections of avian influenza and velogenic ndv were observed also in neighboring countries such as jordan and libya (kammon et al., 2015; roussan et al., 2008) . in addition, a recent similar single case was reported in egypt (hussein et al., 2014 ). an at least partial but not sterilizing, possibly vaccine-induced, specific immunity against these pathogens is likely to be at the basis of these observations. continuing clinically disguised virus circulation in the presence of specific immunity not only fosters spread of these agents but also drives the development of viral escape mutants. it should be noted that the mortality rates described for the holdings do not correlate with the presence of ibv, hpaiv h5n1 or velogenic ndv (table 3 , e.g., holding 34). this investigation focused on ibv, aiv and ndv and did not consider further avian viral or bacterial pathogens. it is highly likely that also various bacterial co-pathogens contribute to and complicate the overall clinical picture. the current situation of ib infections in egypt seems to be the result of a continuing evolution starting with infections caused by egyptian variant i since 2001, egyptian variant ii since 2011, and mass-like strains since 2006 (abdel-moneim et al., 2012; selim et al., 2013) . tracheal ciliostasis is one of the early and characteristic pathogenetic sequelae of ibv infection (cook et al., 1976) . cileostatic respiratory epithelium is known to be more vulnerable for infections with further viral and bacterial co-pathogens and may dispose for infection with aiv and ndv even in the presence of (suboptimal) vaccine-induced immunity. in conclusion, the continuous circulation of the egyptian variant ii ibv and co-infections with aiv and/or ndv severely complicate the epidemiology of viral respiratory infections of chicken in egypt. intensive surveillance is required for a better understanding of this situation. moreover, molecular identification of circulating viruses and experimental vaccination-challenge studies are required to provide data for eventual updating of vaccine strains and to strategically strengthen application programs. isolation and characterization of new variant strains of infectious bronchitis virus in northern egypt isolation and identification of egypt/beni-suef/01 a novel genotype of infectious bronchitis virus s1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt 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avian infectious bronchitis virus saving resources: avian influenza surveillance using pooled swab samples and reduced reaction volumes in real-time rt-pcr a 'new' strain of infectious bronchitis virus infecting domestic fowl in great britain bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains molecular characterization of newcastle disease virus genotype viid in avian influenza h5n1 infected broiler flock in egypt avian infectious bronchitis virus review of infectious bronchitis virus around the world characterization of avian influenza and newcastle disease viruses from poultry in libya mafft multiple sequence alignment software version 7: improvements in performance and usability geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s-1 gene antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions evolution of infectious bronchitis virus in taiwan: characterisation of rna recombination in the nucleocapsid gene ultrafast approximation for phylogenetic bootstrap development and validation of a one-step real-time pcr assay for simultaneous detection of subtype h5, h7, and h9 avian influenza viruses identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus sequencing of pcr products evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses h5n1 and h9n2 co-circulating in egypt iq-tree: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies isolation and molecular characterization of newcastle disease virus genotypes ii and viid in egypt between molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in jordan an apparently new respiratory disease of baby chicks molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 present status of infectious bronchitis in egypt coronaviruses: structure and genome expression s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity the avian coronavirus spike protein development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the authors thank diana wessler, cornelia illing, aline maksimov and gabriele adam, fli, germany, for excellent technical support. thanks are due to günther keil for the m41 reference strain. we are grateful to colleagues and co-workers at nlqp, cairo, egypt. m. naguib is recipient of a doctoral scholarship from the german academic exchange service (daad). all authors declare that they have no conflict of interest. m. m. naguib is funded by a grant from the german academic exchange service (daad grant number a/13/92967). sample collection by veterinarians was achieved from poultry kept as commercial livestock in farms in egypt. obtaining swab samples from the trachea of poultry is minimal invasive and does not require any anesthesia of the animal; minimal restrainment is used for a very short time. sampling, analysis and shipment of samples from egypt to germany was under the legal auspices of the national animal health and research institute, giza, egypt. three co-authors of the manuscript are employees of this governmental institution. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2017. 02.018. key: cord-319168-i23pqiwx authors: abro, shahid hussain; renström, lena h.m.; ullman, karin; isaksson, mats; zohari, siamak; jansson, désirée s.; belák, sándor; baule, claudia title: emergence of novel strains of avian infectious bronchitis virus in sweden date: 2012-03-23 journal: vet microbiol doi: 10.1016/j.vetmic.2011.09.022 sha: doc_id: 319168 cord_uid: i23pqiwx infectious bronchitis virus (ibv) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. recent ibv infections in sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. the complete spike gene of selected isolates from ibv cases was amplified and sequenced using conventional rt-pcr. nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the qx-like genotype. the phylogenetic analysis revealed that strains predominant in the nineties, which were of the massachusetts type, have been replaced by d388/qx-like strains, however the evolutionary link could not be established. the homology between the two genotypes was 79 and 81%. remarkably, a strong positive selection pressure was determined, mostly involving the s1 subunit of the s gene. this strong selective pressure resulted in recombination events, insertions and deletions in the s gene. two new isolates generated from recombination were found with nucleotide sequence diverging 1.7–2.4% from the d388/qx-like branch, indicating the emergence of a new lineage. the study demonstrates a constant evolution of ibv that might be in relation to increased poultry farming, trade and vaccine pressure. the findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario. infectious bronchitis virus (ibv) causes avian infectious bronchitis (ib), a highly contagious disease that affects poultry worldwide and produces severe economic losses. the disease is clinically manifested by respiratory distress, drop in egg production, poor egg quality in layers and some strains causes nephritis (cavanagh, 2003; cavanagh, 2005; mahmood et al., 2011) . the disease may lead to loss of weight and predisposes to secondary bacterial infections that may be fatal (cavanagh, 2003) . initially ibv infects the respiratory tract, however, infections of kidneys and oviduct may follow (cavanagh and naqi, 2003) . also a shift of tissue tropism in the virus has been reported (liu et al., 2006; zhou et al., 2004) , resulting in infection of a wide range of avian host species especially those reared close to domestic fowl. for example, ibv cases were found in chinese peafowl (pavo), guinea fowl (numida meleagris), partridge (alectoris) and teal (anas) (cavanagh, 2005) . ibv belongs to the order of nidovirales, family coronaviridae and to genera of gamma-coronavirus group 3 (gonzalez et al., 2003) . the genome is positive-sense veterinary microbiology 155 (2012) [237] [238] [239] [240] [241] [242] [243] [244] [245] [246] infectious bronchitis virus (ibv) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. recent ibv infections in sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. the complete spike gene of selected isolates from ibv cases was amplified and sequenced using conventional rt-pcr. nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the qx-like genotype. the phylogenetic analysis revealed that strains predominant in the nineties, which were of the massachusetts type, have been replaced by d388/qx-like strains, however the evolutionary link could not be established. the homology between the two genotypes was 79 and 81%. remarkably, a strong positive selection pressure was determined, mostly involving the s1 subunit of the s gene. this strong selective pressure resulted in recombination events, insertions and deletions in the s gene. two new isolates generated from recombination were found with nucleotide sequence diverging 1.7-2.4% from the d388/qx-like branch, indicating the emergence of a new lineage. the study demonstrates a constant evolution of ibv that might be in relation to increased poultry farming, trade and vaccine pressure. the findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario. ß 2011 elsevier b.v. all rights reserved. single stranded rna of about 27.6 kb and contains 5 0 and 3 0 untranslated regions (utrs). the proximal two-thirds of the genome encode two overlapping open reading frames (orfs) 1a and 1b that are translated into large polyproteins. these are then cleaved to generate 15 nonstructural proteins (nsp2-16), comprising a main protease, an rna-dependent rna-polymerase and other nonstructural proteins that carry different functions in transcription and replication. the remaining third of the genome encodes four main structural proteins: the envelope protein (e), the spike glycoprotein (s), the nucleocapsid protein (n) and the membrane protein (m). ibv has two accessory genes 3 and 5 that express accessory proteins 3a, 3b and 5a, 5b, respectively. (cavanagh, 2007; pasternak et al., 2006) . generally, the spike glycoprotein of the virus is translated as a pre-cursor protein (s o ) and later cleaved into the s1 and s2 subunits. however, this cleavage of the spike glycoprotein is not observed in all coronaviruses (holmes, 1990) . the spike glycoprotein of ibv consists of 1145 amino acids and its functions include attachment to the host cell, neutralization and induction of protective immunity (ignjatovic and galli, 1994; johnson et al., 2003) . it is the most variable of the ibv proteins, with most of the variability mapping to the s1 part (adzhar et al., 1997; farsang et al., 2002; keeler et al., 1998; kingham et al., 2000) due to mutations or recombination in this segment. the sequence variability in the s1 coding gene determines serotype specificity of ibv. more than 25 serotypes have been recognized to date, differing by 20-25% and sometimes 50% at amino acid level (adzhar et al., 1997; gelb et al., 2005) . as a result, cross protection between serotypes is poor (kuo et al., 2010) and changes as small as 5% in the amino-terminal half (s1) of the s protein have been shown to alter the protection ability of a vaccine (cavanagh, 2003) . the several different ibv variants are present around the globe some of them exist in particular region, while others are generally distributed (de wit et al., 2011) . the increasing number of new serotypes and genotypes of ibv is a major challenge for the prevention and control of the disease. ib had never been reported in sweden until the first outbreak of ib in commercial poultry farms in 1970s. from 1994 a series of outbreaks have been reported in layer flocks and later in parent flocks for broilers in the south of the country. due to this situation a decision was made in 1997 to vaccinate with a live attenuated vaccine of massachusetts (ma) type ib ma5. vaccination reduced the frequency and seriousness of the outbreaks. subsequent to introduction of vaccination, our study has found indications of outbreaks initiated by the vaccine virus (farsang et al., 2002) . investigations as to the origins of ib cases during 2006 have shown presence of antibodies reacting with the 4/91 variant. in 2007 and 2008, the d388 variant of ibv was identified in chickens. despite the immunization of a number of swedish poultry flocks with live attenuated vaccines, cases of infections were detected again from 2006 to 2010 in broilers. reports of similar situations abound also in other countries (beato et al., 2005; handberg et al., 2009; worthington et al., 2008; indicating a sub-optimal protection provided by the vaccines. the appearance of two new economically important field strains of ibv qx-like and italy-02 in commercial poultry flocks in western europe (beato et al., 2005; worthington et al., 2008) has further raised the need for a better understanding of the epidemiology of ib all over the world. clinically, the d388/qx-like and ita-02 genotypes are characterized by nephritis, production of severe disease symptoms and mortality (ammayappan and vakharia, 2009 ). such symptoms have not been observed in sweden, where signs of infection are limited to poor growth in broilers. the objective of the present study was to perform molecular characterization of ibv isolates in sweden in order to better understand the epidemiology and the factors behind the occurrence of new infections. samples from ib cases (trachea, bronchi, ceaca), collected in sweden from the different outbreaks between 1995 and 2010, were obtained from the national veterinary institute (sva) sample bank. available information of the isolates is summarized in table 1 . all the ibv strains have been propagated in specific pathogen-free (spf) embryonated chicken eggs (lohmann tierzucht, cuxhaven, germany) to maintain the stock. virus isolation was performed by inoculation of 9-11 day old embryonated hen's eggs with 200 ml of 10% tissue homogenates. the eggs were incubated at 37 8c for 72 h. at the end of the incubation the eggs were chilled at 4 8c for 18 h and the allantoic fluid was harvested and centrifuged at 2500 rpm for 15 min. the rna was extracted using trizol reagent as recommended by the manufacturer (invitrogen, carlsbad, table 1 list of selected swedish ibv strains isolated during 1995-2010 used in this study. year country ncbi accession number 242 1995 sweden jn022536 261 1995 sweden jn022537 381 1995 sweden jn022538 397 1995 sweden jn022539 423 1997 sweden jn022540 748 1995 sweden jn022541 1096 1997 sweden jn022542 1489 1999 sweden jn022543 6904 1999 sweden jn022544 09620 2010 sweden jn022545 09621 2010 sweden jn022546 09622 2010 sweden jn022547 062545 2009 sweden jn022548 062561 2009 sweden jn022549 065846 2010 sweden jn022550 079663 2010 sweden jn022551 079692 2010 sweden jn022552 082066 2010 sweden jn022553 900419 1997 sweden jn022554 a889 1999 sweden jn022555 usa). synthesis of cdna was performed with a primer complementary to the ibv spike gene and using super-script ii reverse transcriptase (invitrogen tm life technologies, carlsbad, usa) according to the manufacturer's instructions, in a total reaction volume of 20 ml. two sets of primers were used to amplify the spike gene by rt pcr using the phusion high-fidelity pcr enzyme (finnzymes, espoo, finland). the pcr mix contained 31 ml of distilled water, 10 ml of 5â phusion gc buffer, 1 ml of dntp mix, 1 ml of each primer, 1 ml of phusion tm high-fidelity dna polymerase and 5 ml of cdna. the thermoprofile was initial denaturation at 98 8c/30 s; and 35 cycles of 98 8c/10 s, 55 8c/10 s, and 72 8c/1 min. a final extension at 72 8c/7 min was included. pcr products of were run on 0.8% agarose gel containing ethidium bromide. the specific bands were excised from gels and purified using wizard sv gel and pcr clean-up system (promega, madison, wi, usa). sequencing reactions were carried out using big dye terminator sequencing kit (applied biosystems, foster city, ca) according to the manufacturer's instructions. a set of internal primers was used to generate overlapping sequences in both directions. due to sequence diversity, primers have been designed as new sequence data were generated. the final sequence data enables the selection of consensus primers for sequencing of similar strains. the sequences of the primers are available on request. the thermal profile was setup at 96 8c/4 min; and 25 cycles at 96 8c/15 s, 50 8c/10 s and 60 8c/4 min. the reactions were precipitated in sodium acetate and 95% ethanol. thereafter, they were centrifuged. the pellets were washed with 75% ethanol and dried at room temperature. sequencing was carried out on a 3100 dna analyzer (applied biosystems, foster city, ca). the sequences of s1 and s2 obtained were edited and assembled to the 3540 bp of the s gene. for sequence analysis, the sequences data set was pair wise aligned and edited using software lasergene dnastar (madison, usa). selective pressure of the spike glycoprotein was determined by using snap (korber, 2000) services available at http://hcv.lanl.gov/content/sequence/snap/snap.html. the difference between synonymous (ds) and non-synonymous (dns) substitutions in the codons was calculated to evaluate the substitution rate in the spike gene of the virus. the sequences were analysed for recombination events, as confirmed by analysis with a recombination detection program, rdp v.3.44. the window size was adjusted to 30 with the highest p value 0.05. the detection of recombination events were applied between sequences sharing 0 and 100% identity. phylogenetic analysis was performed with 20 sequences generated in the present study and 95 sequences of the partial s1 and complete spike gene downloaded from the genbank (accession numbers are presented in tables 1 and 2 ). all the gaps in the sequences created by insertions were deleted by using software mega4 version 4 (tamura et al., 2007) . phylogenic trees were constructed by the neighbour-joining method with the nucleotide substitution model of kimura-2 parameter model. the tree reliability was evaluated by bootstrap 1000 replicates. the data set was kept similar for the analysis. the tree topology was prepared in the fig-tree_v1 .3.1. between 1995 and 2010, the sva received specimens from different outbreaks of avian infectious bronchitis that had been submitted by commercial poultry farms for diagnosis. initially outbreaks were confirmed as ibv positive by real-time pcr. twenty swedish ibv isolates from different outbreaks were selected for comprehensive study of the complete spike gene of the virus. the isolates were assigned into two categories: one comprising isolates from nineties 1995 to 1999 and the other containing isolates from 2009 to 2010. the complete spike gene sequences were subject to blast searches in genbank and the results confirmed the isolates as ibv. comparative analysis of the nucleotide sequences of the complete spike gene was carried out to determine the relatedness among 20 swedish isolates ( the sequences were also analysed for the presence of nucleotide insertions or deletions in the spike gene. the deduced amino acid sequences of the spike glycoprotein of the swedish ibv isolates from 2009 to 2010 exhibited 95.30-100% homology. the isolates from 2009 to 2010 contained three amino acid insertions, aspartic acid, glycine and serine at positions 24, 121 and 122, respectively. the new isolates 079663 and 079692 had a deletion of arginine at position 411, whereas isolates 09620, 09621 and 09622 have lost threonine, asparagine, glutamine and arginine at positions 838, 845, 859 and 871, respectively, consequence to the nucleotide deletions. the swedish ibv isolates from 1995 to 1999 had shared homology of 89.20-99.10%. hypervariable regions were found encompassing positions 53-80, 270-296 and 521-547 within the s1 in all the analysed swedish isolates. positions 538 and 566 were highly subjected to amino acid substitutions. overall, high rates of amino acids substitution were observed in the sequences of spike glycoprotein of the virus. a bioinformatics and pair wise comparison approach was applied to estimate the synonymous and nonsynonymous substitution rates and selective evolutionary pressure in the spike glycoprotein. the analyses revealed that most of the s1 subunit was under strong positive selective pressure, showing positive codon specific differences and a high level of genetic diversity across the s1 gene (fig. 1) . in particular, codon regions 360-384 and 598-619 contained a high number of non-synonymous substitutions. overall, the s2 gene showed negative selective pressure, however, the codon regions encompassing positions 693-711 and 790-801 were found to singapore dq001341 3468 2007 taiwan eu822336 3382 2006 taiwan gq229232 1171 1992 taiwan dq646406 2296 1995 taiwan dq646404 3263 2004 taiwan eu822338 2992 2002 taiwan eu822340 3374 2005 taiwan eu822337 h120 2005 taiwan eu822341 taiwan gu386375 340552 2009 thailand gq885140 320352 2009 thailand gq885138 241251 2008 thailand gq885131 kku1 2008 thialand gq906706 2150 contain non-synonymous substitutions, thus contributing to overall variation of the s gene. there was evidence of recombination events in the s1 subunit of the swedish isolates from 2009 to 2010. recombination events were mapped to begin with break point position at amino acid 536 in isolates 079663 and 079692. the ending break point strong signals were observed at amino acid position 834. phylogenetic analyses of the swedish ibv isolates were carried out based on the partial s1 and complete spike codon positions in the sequence fig. 1 . the selective pressure analysis in the spike glycoprotein of ibv. the graph illustrates the s1 part of the spike glycoprotein was mostly subject to positive selective pressure. in s2, the selective pressure was mostly negative. gene. published ibv sequences of massachusetts type, m41, h120, baudette, italy02, qx-like and 4/91 available in the genbank were used to determine likely origin, genetic characteristics and molecular epidemiology of the swedish isolates. the phylogenetic analysis based on the partial s1 gene revealed the sequences distinguished into four main groups (fig. 2) . one group was composed of massachusetts-type, m41, h120 and beaudette strains, where the swedish isolates from 1995 to 1999 clustered, together with strains from spain, korea thailand, china and other vaccine strains. this group was classified as massachusetts genotype. the second group was divided into two sub-groups: one subgroup comprising sequences from sweden, france, the netherlands, spain, italy, uk, israel, korea and china known to belong qx-like genotype. this sub-group was further composed of three clusters from sweden, spain and mixed strains from france and the netherlands. three isolates from israel, italy and uk occupied a separate position near to these subgroups. the recent swedish isolates from 2009 to 2010 were found distributed in this sub-group as a separate cluster and closely related to the dutch strain nl1148. two new swedish isolates 079663 and 079692 branched out separately from the other swedish isolates. the remaining two sub-groups in this group consisted mostly of korean and chinese qx strains. the third group consisted of two isolates from uk and italy related to ita-02 and belonging to the ita-02 genotype. the fourth group has shown relation to the 4/ 91 genotype and consisted of reference strain 4/91 and two isolates from iraq. there were no complete spike gene sequences of the european d388/qx-like genotype available in the gen-bank. thus the comparison was limited to available sequences in the databases. the phylogenetic relationship based on the complete spike gene of ibv revealed that these sequences were separated into two main groups (fig. 3) . the first group contained sequences of the strains from usa, sweden, china and singapore related to baudette, h120, ark 99 and massachusetts genotype. this group was further divided into subgroups. swedish isolates from 1999 to 1995 were found in this group. the swedish isolates 1489, 900419 and a889 have fallen in the subgroup which is closely related to mass 41 isolates from usa. the swedish isolates 242, 748, 381, 397, 261, 423 and 1096 clustered together and showed identity as a separate subgroup of massachusetts type. one swedish isolate 6904 occupied a distinct place in this group. the second group consisted of two subgroups. one of the subgroup mostly contained isolates from taiwan. the second sub-group consisted of two main clusters. the swedish isolates from 2009 to 2010 were found forming a separate cluster in this sub-group of the sequences. the closest relationship of these swedish isolates shared with the ck/ita/90254/05 strain. ck/ita/90254/05 has occupied a distinct place within this subgroup. the other cluster consisted of different chinese isolates. ibv has been detected in the poultry population in sweden in the nineties and then in more recent years cases were reported. despite current immunization with live attenuated vaccines, occasional cases of ib have occurred in poultry flocks in 2007-2010. therefore, this study was designed to characterize strains of ibv associated with outbreaks in the nineties and in recent years in order to understand the molecular epidemiology of this disease in sweden. analysis of the nucleotide sequences of spike glycoprotein of swedish isolates from 1995 to 2010 showed that they comprise distinct sets of ibv variants; differing among themselves along that timeline (table 3 ). it is obvious that the outbreaks in the nineties and those in 2000s were caused by different ib viruses. the isolates 079663 and 079692, standing alone had the genetic signature of a new variant, as they have shown unique substitutions compared to the remaining sequences in this cluster. the nucleotide sequence data showed different regions of hypervariability in the s1 subunit of the spike glycoprotein. this is consistent with the notion that the s1 is prone to mutations, as demonstrated in different studies (adzhar et al., 1997; farsang et al., 2002; keeler et al., 1998; kingham et al., 2000) . previously it has been reported that ark-like strains have a deletion of three nucleotides in the s1 gene at nucleotides positions 69-71 (ammayappan and vakharia, 2009) but in this context, we have observed insertion rather than deletion in s1 unit of the sequences of d388/ qx-like strains. these insertions and deletions possibly have some significance in the properties related to s1 functions. further studies are needed to determine the role of these insertions and deletions in the spike glycoprotein for example in neutralization phenotype or virus tropism, which could explain the reported change in the clinical manifestation of infectious bronchitis. the s1 gene of the isolates showed much higher variation as compared to s2 gene that was mostly conserved, as previously reported (adzhar et al., 1997; keeler et al., 1998; kingham et al., 2000) . however, certain regions in the s2 were determined to be also variable, and, to an extent, contributing to sequence discrimination based on analysis of the complete s gene. therefore, it is plausible that the s2 subunit may have some role in development of new field variants. it has been reported that the evolutionary rates in the s1 of vaccinated birds were 1.5-2.5% per year. nucleotide point mutations, deletions, insertions, and rna recombination in the sequences of spike gene lead to generation of different ibv serotypes (wang and huang, 2000) . the deduced amino acid sequence data revealed different patterns of substitutions resulting from point mutations and insertions in the swedish ibv isolates especially in s1 subunit. the insertions of aspartic acid, glycine and serine in the sequences of the isolates may affect antigenicity of the virus, and this will be a question for further studies on serotyping of the isolates. our results are consistent to those were obtained by previous studies (ammayappan and vakharia, 2009; liu et al., 2009) . the strong positive selective pressure resulting from mutations in s1 gene is likely responsible for the genetic diversity and in the appearance of new phenotypic and antigenic variants. recombination events were found in the spike gene of recent swedish ibv isolates. in the particular case of strains 079663 and 079692 the recombination event was mapped to amino acid 536 and is believed to have determined a new lineage within swedish d388/qx-like cluster. in order to obtain more information concerning to molecular epidemiology of ib, we have carried out a comprehensive phylogenetic study based on partial s1 and complete spike gene of the selected swedish isolates. the phylogenetic studies, based on the partial s1 gene regions, showed that swedish ibv isolates from 1995 to 1999 were related to the massachusetts genotype. the findings confirmed our previous observation (farsang et al., 2002) . it is hypothesized that these genotypic variants have been reduced by vaccine and did not appear through diagnosis after 1999. the swedish isolates from 2009 to 2010 were found to be closely related to other western european isolates from comparison of sequences available in the genbank especially to one isolate from the netherlands (fig. 2) . the qx strains were first isolated in china in 1998 and were found associated mostly with proventriculitis, diarrhea and loss of body weight in 25-70 day old chickens (wang et al., 1998) . it was interesting that the chinese qxlike genotype of ibv was isolated from a backyard flock in a vladimir oblast (russia) where the prevalence of chinese qx-like genotype was common and this region is close to europe (bochkov et al., 2006; gough et al., 2008) . it is unclear how the qx viruses spread to europe. there were more cases of qx-like virus infection with presentation of nephritis rather than proventriculitis, and false layers in mature hens reported from belgium, china, france, germany, the netherlands, russia, taiwan and uk (ammayappan and vakharia, 2009; beato et al., 2005; bochkov et al., 2006; worthington et al., 2008; zhou et al., 2004) . similar type of ibv isolates has been reported in israel and korea (meir et al., 2004; park et al., 2005) . a qxlike strain in chicken associated with nephritis, rare proventriculitis and mortality was reported in poland in 2006 (domanska-blicharz et al., 2006 . these results demonstrated that qx-like strains comprise new emerging ibvs and presented evidence of its involvement in epidemics in many countries. it is noteworthy that despite a number of outbreaks in europe caused by qx-like strains, only a few sequences are available in the genbank. the lack of sufficient information in this aspect is limiting the comprehensible tracing of routes of infection in the continent. for example in denmark qx-like ibv variants have been reported (handberg et al., 2009 ) but the sequences of these viruses are not available in genbank. there is a possibility that trade and import of day old chicks and poultry products from other european countries or indirect routes may have contributed to the spread of the qx-like strains in sweden. the role of wild or migratory birds in dissemination of the ibv new variants still remained unclear. however, previous studies on other coronaviruses and ibv in wild birds have indicated a potential role of wild birds in dissemination of various coronaviruses, including ibv in europe (gough et al., 2008; muradrasoli et al., 2010) . taken together, the complete spike sequence data revealed different isolates of ibv of the massachusetts type and european d388/qx-like strains circulating over a time-line in sweden. so far, there was no evidence of presence of the ita-02 and 4/91 genotypes in sweden. the data suggest that the massachusetts type strains have been replaced recently with european d388/qx-like strains in sweden. sequence diversity and different molecular characteristics were observed among the swedish isolates from 1995 to 2010. the investigations revealed that presumably, d388/qx-like viruses were introduced from other european countries through imports. considering the number of investigated viruses, the present study provides the most comprehensive sequence data on ibv variants related to the d388/qxlike genotype, detected in europe to date. thus, the sequence data of this article will provide valuable reference for future studies on the evolution of genotypes, molecular epidemiology, improved diagnosis and on the virus-evolution in relation to vaccine development. ultimately, the information provided in these studies, will contribute to a more effective control of ib, this viral disease in poultry of global importance. molecular analysis of the 793/b serotype of infectious bronchitis virus in great britain complete nucleotide analysis of the 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characterization of an avian infectious bronchitis virus isolated in china from chickens with nephritis key: cord-317347-by8albr9 authors: van ginkel, frederik w.; padgett, justin; martinez-romero, gisela; miller, matthew s.; joiner, kellye s.; gulley, stephen l. title: age-dependent immune responses and immune protection after avian coronavirus vaccination date: 2015-05-28 journal: vaccine doi: 10.1016/j.vaccine.2015.04.026 sha: doc_id: 317347 cord_uid: by8albr9 infectious bronchitis virus (ibv) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for ibv. therefore we hypothesized that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response contributing to immune escape and persistence of ibv. to test this hypothesis the ibv-specific immune response and immune protection were measured in chicks vaccinated at different ages. this demonstrated a delayed production of igg and iga plasma antibodies in the 1, 7 and 14-day-old vaccination groups and also lower iga antibody levels were observed in plasma of the 1-day-old group. similar observations were made for antibodies in tears. in addition, igg antibodies from the 1-day-old group had lower avidity indices than day 28 vaccinated birds. the delayed and/or lower antibody response combined with lower igg avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv field strain challenge. the lack of vaccine-mediated protection was most pronounced in the 1-day-old vaccination group and to a lesser extent the 7-day-old group, while the 14-day-old and older chickens were protected. these data strongly support ibv vaccination after day 7 post hatch. ibv is endemic and currently one of the most important causes of economic losses for the poultry industry and represents a continuous threat for this industry. in the past, it was estimated that with the best possible management of flocks ibv infection will reduce income by approximately 3% when compared to an ibv-free flock [1] . there is approximately a 50% vaccine failure for arkansas (ark) serotype of ibv [2] , the most prevalent vaccine serotype used in the usa. symptoms of ibv infection include, but are not limited to, wet eyes, swollen face, tracheal and kidney lesions, respiratory disease, reduced weight gain in broilers, decreasing and poor egg quality in layers [3, 4] . the existence of various ibv serotypes as abbreviations: ark dpi, arkansas delmarva poultry industry; calt, conjunctivaassociated lymphoid tissue; elispot, enzyme-linked immunospot; hg, harderian gland; halt, head-associated lymphoid tissues; hrp, horseradish peroxidase; ibv, infectious bronchitis virus; eid50, median embryo infectious dose; tlr, toll-like receptor. * corresponding author. well as antigenic variants [4] complicates vaccination programs. since immunity induced by vaccination against a single serotype generally provides insufficient protection against other serotypes [5, 6] . mucosal immunity plays a role in the control of ibv in chickens as was demonstrated using ibv-resistant and ibv-susceptible inbred chicken lines [7] . this combined with the finding of gelb et al. [8] , in which ocular immunization with the massachusetts connaught strain of ibv only on day 1 or on day1 plus day 14 followed by challenge with massachusetts 41 provided protection of 8% and 50% of the chickens, respectively, while the same spf white leghorns only ocularly immunized on day 14 were 100% protected [8] . bsa immunization of 1, 7 and 12 day old broiler chickens obtained very similar results [9] . this raises questions pertaining the maturity of the immune system and in particular the mucosal immune system, and the ability of chicks to generate a protective immune response when vaccinated at a very young age. conjunctiva-associated lymphoid tissue (calt) and harderian glands do not fully mature as a lymphoid organ until weeks after hatching [5, 6, [10] [11] [12] . this combined with the practice of immunizing and boosting for ibv early after hatching may set up the immune http://dx.doi.org/10.1016/j.vaccine.2015.04.026 0264-410x/© 2015 elsevier ltd. all rights reserved. response for failure to protect. the second ibv immunization on day 14 of age, which by itself is fully protective, does not completely compensate for the premature priming on day 1 [8] . field studies by de wit et al. [3] demonstrated a lack of protective immunity when birds were boosted between day 8 through day 13 of age. the percentage of birds in a commercial flock positive for ibvspecific igm antibodies was correlated with vaccine protection and increased with the age of boosting. this data supports the notion that early vaccination and boosting of the ibv immune response may limit induction of protective immune responses to ibv. unlike the study by gelb et al. [8] , the de wit et al. [3] study can also be interpreted that maternal antibodies interfere with vaccine delivery during the first 2 weeks of life [13] . further evidence that the immune response may be limited during the first weeks of life comes from the observation that iga levels are undetectable in plasma the first week of life and igm levels are low [14] . this indicates that immunoglobulin class switching and production of antibodies is very limited during the first week post hatch and therefore chicks are highly dependent on maternal igy antibodies for protection against ibv, which drops ∼50% during the first week of life [14] . besides diminished b cell response after vaccination, splenic t cells from one week old chickens are also less responsive to polyclonal activation than that of older chickens. the splenic t cells from 1 day old chicks even produce inhibitory factors for proliferation of mature t cells in vitro [15] . furthermore, splenic lymphocytes of 40 day old chicken displayed better antigen specific proliferation after oral salmonella exposure than 10 day old chicken [16] . when measuring gene expression in lung and trachea in 1 and 4 week old birds after avian influenza exposure a reduced expression of immune-related genes was shown and included innate immune response genes in the younger birds [17] . additional evidence that innate immune mechanisms are diminished in young chickens was demonstrated by a lower salmonella phagocytic index of heterophils during the first few days of life [18] . thus, early exposure to pathogens or vaccines may induce suboptimal innate and adaptive immune responses. based on these observations we hypothesized that early ibv vaccination, i.e., within the first week after hatching, will generate an immature, poorly protective ibv-specific immune response contributing to ibv immune escape and persistence. therefore, the ability of spf chickens of different age to induce an ibv-specific antibody response and protect against challenge with an ibv field strain was measured. our data indicate that early vaccination is suboptimal for induction of ibv-specific immune responses and immune protection. chickens: specific-pathogen-free (spf) white leghorn eggs were obtained from sunrise farms, inc., catskill, ny, hatched and used in all experiments. all hatched chickens were used for the below outlined experiments regardless of sex. chickens were housed in cages in bsl 2 facilities for the duration of the experiment. food and water were provided ad libitum. all experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal use guidelines. auburn university college of veterinary medicine is an association for assessment and accreditation of laboratory animal care (aaalac)-accredited institution. ibv-vaccination and challenge: spf chickens were ocularly vaccinated with 3 × 10 5 50% embryo infectious doses (eid 50 ) of a live attenuated arkdpi ibv vaccine strain (zoetis, new york, ny) in 50 l pbs, which was expanded in our laboratory. chickens were vaccinated 1 day of age and 1, 2, 3 or 4 weeks of age. all groups were challenged ocularly with 7.3 × 10 5 eid 50 of the al/4614/98 ibv field strain 21 days after vaccination sample collection: tears were collected as previously described [19] . blood samples were obtained by puncturing the brachial vein with a sterile 20g needle into kendall monoject, edta containing, blood collection tubes (tyco healthcare group lp, mansfield, ma) and incubated on ice. blood samples were centrifuged at 500 × g for 30 min. plasma was collected and stored at −80 • c until tested. ibv propagation and purification for elisa: ibv was propagated in spf white leghorn embryonated chicken eggs (sunrise farms, inc., catskills, ny) by inoculation on day 10 of embryonation as previously reported [20] . supernatants were titrated for the ibv virus using the reed and muench method [21] . ibv was treated with 0.1% ␤-propriolactone for 30 min at 37 • c [22] . inactivation of the virus was confirmed by injection into embryonated eggs. the inactivated ibv was purified based on a previously published protocol [23] . the virus was then stored at −80 • c until used. in order to measure igg (igy), iga and igm antibody levels in plasma and tears of chicken, an ibv-specific enzyme-linked immunosorbent assay (elisa) was developed as previously described [20] . in brief, elisa plates were coated with ␤propriolactone killed, purified ibv at 2 g/ml in carbonate buffer. the plates were blocked with pbs-bsa (1%) after which the samples were loaded at two-fold dilutions. binding of chicken antibodies was detected using biotinylated anti-chicken-igg, -iga and -igm monoclonal antibodies (southern biotechnology associates, inc., birmingham, al) followed by streptavidin-horseradish peroxidase. the plates were developed using tmb (3,3 ,5,5-tetramethylbensidine; invitrogen corp., frederick, md) substrate. the highest sample dilution with at least an optical density of 0.100 above background level at 450 nm was defined as the endpointtiter. ten to thirteen chickens were analyzed per group. the control group consisted out 3 chicken from each age group for iga and igg, which were pooled in one group of 15 since no differences were observed between the controls. for igm levels in plasma the controls (each group containing 5 chickens except day 28 which had 3) were displayed separate for each age group. this was done because significant differences were observed between control igm levels to ibv in different age groups. the avidity index was determined as previously described [24, 25] using the above described ibv-specific elisa. plasma and tears were diluted 1:64 in elisa buffer and were loaded on ␤propiolactone killed ibv coated elisa plates (2 g/ml) [20] . after overnight incubation of these samples at 4 • c, 100 l of increasing concentrations of potassium thiocyanate (0.00, 0.094, 0.187, 0.375, 0.75, 1.50, 3.0 m kscn) were loaded into the wells and incubated for 30 min at room temperature. after washing the plates, the detection of ibv-specific antibodies was accomplished as previously reported [20] . the data were normalized to percent inhibition relative to samples not exposed to kscn. the concentration of kscn to inhibit 50% of the reactivity of the elisa was defined as the avidity index [24, 25] . the od 450 read-out for the kscn inhibition data was curve-fitted using 3-order polynomial regression analyses in microsoft office excell program. the excell provided formula for the inhibition curve was used to determine the x values of y = 0.5, which are the concentrations of kscn inhibiting 50% of the elisa reactivity, representing the avidity indices of those samples. all samples were analyzed in triplicates and 4-5 samples were analyzed per group. the cranial 1/3 of tracheae was collected 4 days after ibv challenge with 7.3 × 10 5 eid 50 of al/4614/98 ibv field strain. the tracheae were formalin-fixed and embedded in paraffin. longitudinal 5 m sections were made and were hematoxylin and eosin (h&e) stained and analyzed for mucosal thickness using aperio scan scope and the image j morphometry program (rsb.info.nih.gov/ij/download.html). to measure the mucosal thickness 5 measurements were made at regular intervals on one tracheal ring (see supplemental fig. 1 ). as stated above, histopathology was analyzed in h&e stained tracheal slides 4 days after ibv challenge. besides mucosal thickness (see supplemental fig. 1 ), deciliation (see supplemental fig. 2), goblet cells (see supplemental fig. 3 ) and lymphocytes scores (see supplemental fig. 1 ) of the tracheal mucosa were evaluated blindly and scored 1 through 5 based on severity (i.e., normal, mild, moderate, marked, severe). five chickens were used as positive and negative controls, i.e., one of each age group, and 10-13 chickens were analyzed for each age group. a visual depiction of the scoring of these parameters is provided in the supplemental data (supplemental figs. 1-3) . statistical analysis: data were analyzed using a one-way anova test with newman-keuls multi-comparison test or the t-test using graphpad prism5 software. groups were considered significantly different when p < 0.05. to determine whether age of ibv vaccination affected the humoral immune response, plasma samples were collected 14 and 21 days and tears 14 days after vaccination with 3 × 10 5 eid 50 of a live-attenuated arkdpi ibv vaccine strain on 1, 7 14, 21 or 28 days of age. the ibv-specific igg endpoint titers in plasma 14 days after vaccination are significantly lower for day 1 vaccinated birds with a mean of 7.8 ± 1.0 when compared to day 14, 21 and 28 vaccinated birds, which means vary between 10.2-11.4. the 7-day-old group does not differ significantly from day 1 or later vaccinated bird (fig. 1a) . the igg ibv-specific plasma levels 21 days after vaccination demonstrate, that the day 21 and 28 old vaccination groups chickens were vaccinated at day 1, 7, 14, 21 or 28 of age. unvaccinated chickens of the different age groups served as negative control. the data was analyzed by one way anova with the newman-keuls post-test. the control group contains 3 data points for each age group, which were pooled in one group (n = 15). all vaccinated age groups contained between 10 and 13 chickens for igg and iga. a significant difference is observed at p < 0.05 and is indicated by different letters. igm levels in ibv vaccinated birds are depicted by squares (n = 5) and the controls by circles (n = 5, d28 group n = 3). for the igm controls the different age groups are shown separately since significant differences were observed between them. a significant increase (p < 0.05) of igm levels in ibv vaccinated birds over their control group is indicated by a (*). stayed the same, while igg antibody titers in groups vaccinated on day 1, 7 and 14 still increased (fig. 1c) . this shows that early vaccination causes a delay in the ibv-specific igg antibody kinetics. the ibv-specific iga plasma antibody titers are not significantly different between groups, although the day 1 vaccinated group mean antibody titer is the lowest of all groups (fig. 1b) and at least 3-fold lower than the next lowest group. unlike the igg antibody titers only the day 7 vaccination group increases in mean iga plasma titer on day 21 post vaccination, while day 14 group stays the same and the day 21, 28 and 1 groups decline in mean iga titer. thus, only the day 7 group increases antibody titers on day 21 when compared to older birds. thus, the day 7 group displays a delayed response in antibody production compared to older birds. the day 7 and day 14 groups have plasma iga titers to ibv that are comparable to, or higher than, the day 21 and 28 groups on day 21 of the response. unlike the day 7 and day14 vaccination groups the day 21 and 28 groups are declining on day 21 of the response compared to the immune response on day 14. this indicates that they are past their peak response on day 21 and possibly even on day 14 based on previous observations [20] . these data are consistent with a delay in the iga plasma response to ibv in birds vaccinated at a younger age and a non-significant decline in mean iga titers in the 1-day-old group. ibv-specific igm antibody titers were measured in plasma. the plasma samples analyzed were collected on day 7 post vaccination. this time point was selected based on the literature in which the peak igm response was observed between 5-9 days after virus challenge or live virus vaccination [26] [27] [28] . due to the variability of ibv-reactive igm in the controls between different age groups, independent controls were included for each age group. the igm levels in the controls decreased considerably by ∼2 week of age after (7 days after the day 7 old chick vaccination) which increased one week later and stabilized in older age control groups (fig. 1e) . the day 14 through day 28 control igm levels were significantly higher than in the day 1 and day 7 age group controls. and the day 7 control was significantly lower than the day 1 control igm levels. all igm titers from the ibv vaccinated age groups were significantly higher when compared to their controls (p < 0.05) with exception of the day 21 age groups (p = 0.08). this was due to higher control values, which were ∼1.6 fold higher than in the age 14 or 28 days old groups. this may reflect an initial peak of natural antibodies induced to ibv before stabilizing. the ibv vaccinated age groups did not differ significantly in igm antibody levels to ibv with exception of the day 1 ibv vaccinated group, which mean ± se was 8.7 ± 0.6 had significantly lower ibv-specific igm levels in plasma than the day 14 (10.2 ± 0.4) day 21 (10.6 ± 0.7) and day 28 (11.2 ± 0.5) vaccinated birds but did not differ significantly from the day 7 (9.2 ± 0.5) vaccinated birds (fig. 1e) . the day 7 igm antibody titers were also significantly lower than those in the day 28 vaccination group but not compared to the other age groups. in tears the ibv-specific igg response is significantly higher in the day 21 and 28 vaccinated groups than in the day 1 and 7 vaccinated chickens for day 14 of the immune response ( fig. 2a) . the igg endpoint titer in the day 1 immunized group is even significantly lower than the day 7 immunized group, while the day 14 immunized group is intermediate between the day 7 group and chickens vaccinated at an older age. thus, a correlation between age of vaccination and the magnitude of the ibv-specific igg response in tears is observed on day 14 of the ibv-specific immune response. the day 14 and 21 groups have significantly higher iga anti-ibv responses in tears on day 14 after vaccination compared to the day 1 group. the day 1 group is not significantly different from the day 7 and 28 vaccination groups. the day 28 group is also significantly lower than the day 14 and 21 groups (fig. 2b) . this is likely due to the day 28 group displaying faster kinetics for iga antibody levels in tears after vaccination, rather than a lower response [20] . the day 14 group iga response is significantly higher than the day 1 and day 7 groups consistent with a delay or deficiency in the mucosal antibody response when vaccinated at an earlier age. our data indicate there is a delay in antibody production when vaccinated at a younger age. the day 1 vaccination group not only displays a delay but also lower levels of antibody production. to determine whether there are not only quantitative differences between antibodies produced when vaccinated on day 1 but also qualitative differences we compared avidity indices for igg and iga antibodies from plasma and tears generated in 1 day old versus fully matured 28 day old birds 14 days after ibv vaccination. as is illustrated in fig. 3a ,b a significantly higher avidity index is observed for igg plasma antibodies for the day 28 vaccination group when compared to the day 1 vaccinated birds, while no significant difference is observed for iga plasma antibodies. the same observations are also made for tear igg and iga antibodies (fig. 3c,d) . ciliated cells and goblet cells are the primary target of ibv in the respiratory tract [29] . fig. 4 displays the deciliation (fig. 4a ) and goblet cell (fig. 4b ) scores 4 days after ibv challenge. ibv challenge decreased ciliated epithelial cells and goblet cell score. ciliated cells were fully protected when vaccinated on day 7 of age or later but not when vaccinated on day 1 of age (fig. 4a) . the protection of goblet cells increases with the age of vaccination and were fully protected when vaccinated on day 21 of age or older (fig. 4b) . the lymphocytes score and mucosal thickness were also measured in tracheal samples on day 4 post al/4614/98 ibv field strain challenge as indicators of inflammation. as is illustrated in fig. 5a , a significant decrease in lymphocyte score was observed in the day 7 and 14 vaccination groups when compared to the day 1 group. the day 1 vaccination group did not significantly differ from the day 21 fig. 2 . the ibv-specific igg and iga response in tears. endpoint titers of ibv-specific igg (a) and iga (b) on day 14 of the immune response were measured by elisa. chickens were vaccinated on day 1, 7, 14, 21 or 28 of age. unvaccinated chickens of the different age groups served as negative control. depicted are the means and standard error of each age group and the control group, and different age groups are as described in fig. 1 (n = 10-14 per group) . the data was analyzed by one way anova with the newman-keuls post-test. differences were considered significant at p < 0.05 and are indicated by different letters. fig. 4 . tracheal deciliation and goblet cell depletion after ibv challenge. to measure the degree of protection against ibv challenge after vaccination tracheal deciliation and goblet cell depletion were measured. chickens were vaccinated at day 1, 7, 14, 21 or 28 of age and challenged 21 days later. unvaccinated/unchallenged chickens of all age groups served as negative control and unvaccinated/ibv challenge chickens as positive control. trachea were collected 4 days post challenge. depicted are the mean and one standard error. for the negative and positive controls n = 5 for the different age groups n = 10-13. the data were analyzed by one way anova with the newman-keuls post-test. significant difference was observed at p < 0.05 and are indicated by different letters. and 28 vaccination groups due to a small increase in lymphocyte score in the latter two groups (fig. 5a) . the day 1 group displayed the highest lymphocytes score from all vaccination groups, which is consistent with the highest inflammatory response to ibv challenge. this was also supported by a significant increase in mucosal thickness in the day 1 vaccinated group when compared with the day 14, 21 and 28 groups but not day 7 group, which was intermediate between the day 1 and groups vaccinated at an older age (fig. 5b ). based on our data, the hypothesis that early ibv vaccination will generate an immature, poorly protective ibv-specific immune response, is confirmed. ibv vaccination on day 1 of age, which is routinely performed in the poultry industry, will not be fully protective and as a consequence the chicks remain vulnerable to ibv exposure. thus, early vaccination perpetuates the ibv problems and is a factor in the estimated $48 million or more annual loss to the poultry industry due to ibv infection [30] . our measurements of mucosal and systemic antibody levels demonstrates a delayed production of igg and iga plasma antibodies in the day 1, day 7 and 14 of age vaccination groups. iga antibody levels in the day 1 group, unlike igg antibodies, do not recover later in the response. besides delayed igg kinetics, the day 1 group displays also a lower avidity index than the day 28 vaccinated group. lower avidity index is not observed in iga antibodies. the delayed and/or lower antibody response and lower igg avidity index translated in increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon ibv challenge when compared to chicks vaccinated later in life. a lack of vaccine-mediated protection is most noticeable in the 1 day of age vaccination group and to a lesser extend the day 7 vaccination group, while the day 14 and older vaccinated chickens are protected. the igm antibodies specific for ibv were significantly elevated above controls on days 7 of the ibv immune response in all age groups except the day 21 group. the day 21 group was not quite significant (p = 0.08) because of higher igm levels in the control group. this could be due to an initial surge of natural igm antibodies to ibv in this age group. a significant decline in the 7 day old group is observed when comparing the igm antibody levels to ibv in the control day 1 group. this would be consistent with a drop of presumably natural maternal ibv-specific igm antibodies in these spf chickens in the day 7 control age group. these igm antibodies rapidly increases in the day 14 group after which they stabilize in the older age groups. this indicates that a considerable portion of igm antibodies in plasma from the older vaccination groups reacts with ibv without seeing the virus, indicating these are natural antibodies to ibv, which only increase after day 14 of age. bacterial colonization of the intestinal tract of chickens is established during the first two weeks post-hatch [31] . this, combined with the observation that probiotics enhance natural antibodies in chicken [32] , indicates that ibv-specific natural igm antibodies to ibv are possibly generated following intestinal colonization presumably by stimulating b1 cells, which are the main producers of natural igm antibodies in sera of mammals [33] . in the ibv vaccinated groups we see a steadily incline of igm ibv-specific antibodies in plasma with age as has been reported by de wit et al. [3] . only the day 1 age group displays significantly lower igm antibody levels when compared with the day 14-28 age groups but not with the day 7 age group. this is consistent with an early in life deficiency or delay in the igm response. the lower avidity index for the igg antibodies in 1-day-old chicks is an important factor contributing to decreased protection to ibv challenge. increased antibody affinity maturation to virus vaccines strongly correlated with better protection [34, 35] . a lack of antibody affinity maturation observed following vaccination against respiratory syncytial virus was due to a lack of tlr stimulation [35] . this indicates that 1-day-old birds may be deficient in tlr expression. evidence that this is the case comes from a recent publication [36] demonstrating, that significantly lower levels of tlr7 expression in spleen and small intestines were observed when comparing 1-day-old chicks with 4-to 5-week-old chickens. inclusion of tlr activating adjuvants could alleviate the problems of early vaccination by boosting antibody production and affinity maturation in 1-day-old chicks. neither monomeric plasma nor dimeric tears-derived iga [37] displays this drop in avidity index for the day 1 vaccinated group (fig. 3b,d) . although there is a lower level of iga antibodies produced by the day 1 vaccinated birds compared to the older groups, which is consistent with a delay in class-switching in the day 1 old group, it is not clear why the lack of a mature mucosal immune system in the 1-day-old group [5, 6, [11] [12] [13] did not result in lower affinity maturation of mucosal iga antibodies compared to the 4-week-old group. another factor influencing early vaccination is the level of maternal antibodies, an issue not addressed in this study. there exists a linear relationship with the hens' plasma antibody levels and transfer of igy to the chicks' circulation [14] . chicks were over 95% protected against ibv challenge on day 1 if they had high levels of maternal antibodies but less than 30% protected when challenged on day 7. this protection correlated with local respiratory antibodies and not serum antibodies [38] . ibv-specific maternal antibodies decreased the induction of neutralizing antibodies following boosting [38] . despite this inhibition by maternal antibodies of the memory response, low or erratic maternal antibody titers to ibv in broiler flocks are associated with ibv-induced economic losses [39] . this further supports that protection by maternal antibodies, which are predominantly of the igg isotype, is important to prevent activation of an immature immune system that is not capable generating a fully protective immune response early in life. in several studies ibv vaccination was effective against ibv challenge in both spf chickens and commercial broilers when the initial vaccination was performed on day 1 [40] [41] [42] . however, in these studies day 1 vaccination was followed with a second vaccination two weeks later for optimal protection against challenge, which would have masked the relative poor ibv-specific immune responses after the day 1 immunization. extensive immunization on the day of hatch containing three different live attenuated viruses, which caused severe vaccine symptoms, provided similar protection as two single live attenuated ibv virus vaccines on day of hatch and day 14 of age when challenged with a heterologous virus [42] . this seems to indicate that induction of cross-protective immunity may be less impaired when vaccinated early in life. however, no direct comparison of the same vaccination protocol was analyzed between these two challenge groups, which makes this data harder to interpret in the context of age-dependent immune responses. a decreased humoral immune response to vaccines early in life as seen in chickens is also observed in humans. neonates are highly dependent upon passively acquired maternal antibodies, since their humoral immune system remains underdeveloped [43] . these passively obtained antibodies in infants can alter humoral and antibody-dependent immune responses to vaccines [43, 44] . in broiler chicks maternal antibodies to pathogens persisted only for ∼10 days [45] . in our study interference of maternal antibodies was excluded. the observation that 1-day-old and 7-day-old birds are not fully protected when vaccinated are confirming the importance of pathogen-specific maternal antibodies during this period. in summary we can say that ibv vaccination of chickens on day 1 of age contribute to the ibv problem in the poultry industry by inducing lower levels and/or slower kinetics of antibody production as well as lower avidity igg antibodies. this results in a poorly protective immune response as is demonstrated by subsequent ibv field strain challenge. therefore, it is advisable for the poultry industry based on our data to change their practice from vaccinating chicks on day 1 of age to vaccinating after day 7 of age. infectious bronchitis infectious bronchitis virus field vaccination coverage and persistence of arkansas-type viruses in commercial broilers efficacy of infectious bronchitis virus vaccinations in the field: association between the alpha-ibv igm response, protection and vaccine application parameters coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells delineation of b and t lymphoid cells in the chicken the harderian gland of the domestic fowl. i. histology, with reference to the genesis of plasma cells and russell bodies the secretory antibody response of inbred lines of chicken to avian infectious bronchitis virus infection infectious bronchitis virus antibodies in tears and their relationship to immunity development of immunocompetence of broiler chickens b cells and t-lymphocyte subsets of the headassociated lymphoid tissues of the chicken quantification of particle uptake by conjunctiva-associated lymphoid tissue (calt) in chickens immunoglobulin production in chicken harderian glands the avian antibody response maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens development of t cell immune responsiveness in the chicken a strong antigenspecific t cell response is associated with age and genetically dependent resistance to avian enteric salmonellosis early host responses to avian influenza a virus are prolonged and enhanced at transcriptional level depending on maturation of the immune system age-dependent phagocytosis and bactericidal activities of the chicken heterophil transfer of igg from serum to lachrymal fluid in chickens iga as an early humoral responder after mucosal avian coronavirus vaccination a simple method of estimating fifty percent endpoints evaluation of different methods of inactivation of newcastle disease virus and avian influenza virus in egg fluids and serum purification of infectious bronchitis virus propagated in embryonated chicken eggs and its confirmation by rt-pcr estimating the relative avidity of mucosal iga for antigen the measurement of relative antibody affinity by elisa using thiocyanate elution local and systemic antibody class responses to an infectious laryngotracheitic virus vaccine strain the immunoglobulin m response in chicken serum to infectious bursal disease virus development of monoclonal antibodies against chicken igm and its application in immunity studies comparative analysis of the sialic acid binding activity and the tropism for the respiratory epithelium of four different strains of avian infectious bronchitis virus the economic and social impact of the institute for animal health's work on avian diseases. in: dtz institute for animal health report. dtz institute microflora ecology of the chicken intestine using 16s ribosomal dna primers probiotics stimulate production of natural antibodies in chickens origins and functions of b1 cells with notes on the role of cd5 h5 n-terminal sheet promotes oligomerization of h7-ha1 that induces better antibody affinity maturation and enhanced protection against h7n7 and h7n9 viruses compared to inactivated influenza vaccine lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease chicken tlr21 acts as a functional homologue to mammalian tlr9 in the recognition of cpg oligonucleotides induction of mucosal immunity in the avian harderian gland with a replication deficient ad-5 vector expressing avian influenza h5 hemagglutinin maternal antibody to infectious bronchitis virus: its role in protection against infection and development of active immunity to vaccine infectious bronchitis serology in broilers and broiler breeders: correlations between antibody titers and performance in vaccinated flocks breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by vaccination programme based on the ma5 and 4/91 serotypes induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d388 (genotype qx) by maternally derived antibodies and by early vaccination the maturing immune system: implications for development and testing hiv-1 vaccines for children and adolescents determinants of infant responses to vaccines in presence of maternal antibodies decay of maternal antibodies in broiler chickens we thank the alabama agricultural experiment station for funding this research ala052-2-14016. supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2015.04. key: cord-321261-3lp54mmu authors: kuo, shu-ming; wang, ching-ho; hou, ming-hon; huang, yuan-pin; kao, hsiao-wei; su, hong-lin title: evolution of infectious bronchitis virus in taiwan: characterisation of rna recombination in the nucleocapsid gene date: 2010-08-26 journal: vet microbiol doi: 10.1016/j.vetmic.2010.02.027 sha: doc_id: 321261 cord_uid: 3lp54mmu avian infectious bronchitis virus (ibv) belongs to the coronaviridae family and causes significant economic loss in taiwan (tw), even in flocks that have been extensively immunised with massachusetts (mass)-serotype vaccines. phylogenetic analysis of all non-structural and most structural genes shows that tw ibv is genetically distinct from the us strain and more similar to chinese (ch) ibv. in contrast, the nucleocapsid (n) gene of tw ibv presents phylogenetic incongruence. rna recombination at the 5′ end of the n gene between tw and us ibv is shown to be responsible for this discordance. surprisingly, the recombinant n gene is found in all of tested tw ibv isolates, suggesting that a recombination event gave origin to a founder lineage. our data indicate that rna recombination in the recombinant 5′ end of the n gene may have caused the emergence of the current ibv population in taiwan. infectious bronchitis virus (ibv) is highly contagious and pathogenic in chickens. although the current massachusetts (mass)-and connecticut-serotype vaccines usually offer sufficient protection, they are less effective in certain areas, such as china (ch) and taiwan (tw) wang and khan, 2000; yu et al., 2001) . frequent point mutations and evolutionary fitness in the hypervariable regions (hvrs) of the spike 1 (s1) gene contribute to most of the antigenic determinants of ibv. therefore, the s1 gene is often referred to as the index of genetic diversity and of viral evolution, due to its high variability and close genotype/serotype correlation (dolz et al., 2008; liu et al., 2006; wang and huang, 2000; wang and tsai, 1996) . in addition to point mutation, rna recombination is also involved in the generation of new antigenic variants. rna recombination in a coronavirus was first demonstrated experimentally in mouse hepatitis virus (mhv) (lai et al., 1985; makino et al., 1986) , and a high frequency of recombination was found both in vitro and in vivo after mhv infection (keck et al., 1988; makino et al., 1986) . recombination sites were detected throughout nearly the entire genome, and many recombinants with multiple crossover sites were found (liao and lai, 1992) . the high frequency of rna recombination in coronaviruses is likely caused by the unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (cavanagh, 2007; lai et al., 1985; liao and lai, 1992; makino et al., 1986) . putative ibv genetic recombination in the s gene has been documented in different field isolates, including a japanese strain (kb8523), european avian infectious bronchitis virus (ibv) belongs to the coronaviridae family and causes significant economic loss in taiwan (tw), even in flocks that have been extensively immunised with massachusetts (mass)-serotype vaccines. phylogenetic analysis of all non-structural and most structural genes shows that tw ibv is genetically distinct from the us strain and more similar to chinese (ch) ibv. in contrast, the nucleocapsid (n) gene of tw ibv presents phylogenetic incongruence. rna recombination at the 5 0 end of the n gene between tw and us ibv is shown to be responsible for this discordance. surprisingly, the recombinant n gene is found in all of tested tw ibv isolates, suggesting that a recombination event gave origin to a founder lineage. our data indicate that rna recombination in the recombinant 5 0 end of the n gene may have caused the emergence of the current ibv population in taiwan. ß 2010 elsevier b.v. all rights reserved. isolates (dutch d207 and british 6/82), texas local strains (pp14 and se17) and a new york isolate (cu-t2) (jia et al., 1995; kusters et al., 1990; wang et al., 1993) , based on sequence comparison and phylogenetic incongruence. additionally, half of the nucleocapsid (n) gene of cu-t2 was further shown to be replaced by that of the holland 52 (h52) vaccine strain, indicating that genomic rna recombination in ibv may occur in multiple genes (jia et al., 1995) . in taiwan, phylogenetic analysis revealed that few tw ibv strains, such as 2992/02 and 3374/05, may undergo genetic recombination with ch ibv (chen et al., 2009; huang et al., 2004) . this experimental evidence and the sporadic detection of recombinant variants in nature support the notion that recombination occurs in coronaviruses (kottier et al., 1995) . however, available evidence remains insufficient to allow the conclusion that coronavirus evolution can be driven by interspecies recombination. tw ibv, first isolated in 1964 (lu et al., 1993) , can be classified into two individual serological groups, tw i and tw ii, based on the sequences of the hvrs of the s1 proteins (wang and huang, 2000) . the mass-serotype vaccine has shown a modest protective effect on the ibv outbreak in taiwan (huang and wang, 2006; lin et al., 2005) , possibly due to the considerable variation in s1 sequences between tw and us strains. in this study, we analyse tw ibvs and show that they are genetically closer to ch isolates than to us strains. the n gene of tw ibv is the only exception and shows phylogenetic incongruence. we further demonstrate that rna recombination has contributed to this genetic discordance. the recombinant n gene is detected in all the analysed tw ibvs, suggesting that rna recombination drives the evolution of this coronavirus at the population level. tw ibv strains were isolated and propagated through the allantoic sac route by inoculating 9-11-day-old specific pathogens free (spf) embryonated eggs from the animal health research institute, council of agriculture in taiwan (gelb and jackwood, 1998) . tw ibvs had been purified by three consecutive limiting dilutions (wang and huang, 2000; wang and tsai, 1996) . sequences of both us and ch ibvs were obtained from genbank maintained by ncbi. we listed the tested ibvs and their genbank numbers in supplementary table 1 , including 29 tw strains, 15 us strains and 30 ch strains. inquired from the genbank up to 2009, we found 12 tw and 15 us ibvs are decoded in both s1 and n genes and we included all these up-to-date ibvs in this study. among the 12 tw strains, 8 ibvs in the same clade of me tree (supplementary fig. s1 ) were chosen for the analysis of n gene in figs. 3 and 4. both tw2992/02 and tw3374/05 were excluded because of their phylogenetic discordance in s1 (fig. s1 ). both 2994/02 and 3382/06 was excluded due to the discordance in n (fig. s2) . the length of the tested genes is all intact and no 3 0 -utr deletion was found in these ibv strains. total rna was extracted from tw2575/98-infected allantoic fluid using trizol tm c&t (protech, taiwan), according to the manufacturer's protocol. rna (0.5 mg) was reverse transcribed into cdna using superscript tm iii reverse transcriptase (rt) (invitrogen, usa). pcr was performed with a proofreading dna polymerase (kod-plus, toyobo, japan). generally, the pcr conditions included 28 cycles of 94 8c for 30 s, 62 8c for 30 s, and 68 8c for 1 min, on a pcr machine (astec 818, japan). consecutive viral genomic fragments were cloned into the pcrii-topo vector (invitrogen, usa) by ta cloning and were subjected to sequencing. the 5 0 -untranslated regions (utr) of ibv 2575/98 was analysed using a smart tm race cdna amplification kit (clontech, usa). first, cdna was prepared from 1 mg of total rna from allantoic fluid, using the powerscript tm rt and a unique 3 0 reverse primer (5 0 -tac ttg caa gac aag ttc c-3 0 , nt 1247-1265 of the viral genome). after the rt reached the 5 0 end of the mrna template, it added 3-5 dc residues to the newly synthesised cdna. the smart ii a 5 0 oligonucleotide (5 0 -aagcagtggtat-caacgcagagtacgcggg-3 0 ) annealed to the dc tag and served as an additional extending primer for rt. consequently, the 5 0 single-stranded cdna was used as the pcr template with the nested primer (5 0 -aag-cagtggtatcaacgcagagt-3 0 ) and a new reverse primer, 5 0 -ccaacaagcctgcgcattgccatag-3 0 (nt 996-1020 of the viral genome). amplified dna products were cloned into the pgem-t easy vector (promega, usa) and sequenced. phylogenetic trees were constructed by bayesian inference (bi), maximum likelihood (ml) and minimum evolution (me) methods. the best-fit models and parameters for initial settings of the phylogenetic programs were selected by modeltest 3.7 for bi and ml algorithms on the basis of the akaike information criterion (posada and crandall, 1998) . estimates of evolutionary divergence over all sequence pairs were computed by averaging the number of base substitutions per site in mega 4.0. all positions containing gaps and missing data were eliminated from the dataset. standard error estimates were obtained by a bootstrap procedure from 500 replicates. for the s1 data set in fig. 1a , the best-fit model of substitution was a gtr model (rodriguez et al., 1990) with a gamma substitution parameter ( mrbayes 3.1.2 was used for bi analysis. random starting trees were used. a total of two million generations of markov chains were run. trees were saved every 100 generations, resulting in 20,000 trees in the initial samples. the stationary phase of log-likelihood was reached within 500,000 generations. thus the burn-in (number of initial trees that were discarded) was set to 5000. a majority-rule consensus tree was generated from the remaining samples (15,000 trees), and the percentage of samples recovering any particular clade represented the clade's posterior probability. we conducted 500 nonparametric bootstrap replicates for ml analysis using garli v0.951. we used the heuristic search option with substitution models set to the best-fit model chosen by modeltest 3.7. me analysis was performed using mega 4.0 with 500 replicates. twelve ibv strains, including tw2575/98, tw1171/92, tw2296/95, tp/64, mass 41, cal99, beaudette, cu-t2, ch-bj, ch-s14, ch-lx4 and ch-ldt3, were subjected to the recombination detection program (rdp) 3.0 (martin and rybicki, 2000) . genes s and n were aligned by clustalw and analysed by seven algorithms in rdp 3.0, including rdp, bootscan, geneconv, maxchi, chimaera, siscan, and 3seq. based on the results automatically calculated by rdp, tw2575/98, tw2296/ 95, cu-t2 and ch-lx4 were chosen as representative strains for which data is shown below. data were further subjected to the genetic algorithms for recombination detection (gard) program to confirm the putative single breakpoint position (kosakovsky pond et al., 2006) . to elucidate the phylogenetic relationships of tw ibv, 74 ibvs were analysed in this study, including 29 tw strains, 30 ch strains and 15 us strains. minimum evolution (me) test showed that 25 tw ibvs form a unique branch independent of the us and ch groups (fig. s1 ). some exceptional strains showing the phylogenetic incongruence were noticed, such as the tw2992/02 (chen et al., 2009; huang et al., 2004) , tw2994/02 (huang et al., 2004) , tw3374/05 (chen et al., 2009) , china ck/ch/ ldl97i and china ck/ch/ldl98i (chen et al., 2009) . in their respective clades, 11 tw strains, 9 ch strains, 9 us strains were chosen for the analyses of nucleotide-sequence identity, maximum likelihood (ml) and bayesian inference (bi). the nucleotide-sequence identity between tw s1 genes and those of us or ch isolates was similar (77-82%). the phylogenetic tree shown in fig. 1a , which were constructed by bi, shows that tw ibv is a branch independent of the us and ch groups. analysis of ml reproduces similar tree topology and the bootstrap values are illustrated together with the posterior probability (fig. 1a) . interestingly, the evolutionary divergence and me methods indicated that tw ibvs are more closely related to the ch cluster than to the us group (table 1 , fig. 1b and fig. s1 ), supported by less difference between tw-ch strains (table 1 ) and the high bootstrap value (98%) for the branch separating the tw-ch groups from us group (fig. 1b) , respectively. a virulent taiwanese field isolate, tw2575/98, was fully sequenced (27,710 bps; dq646405). the g/c content of the viral genome was 38.5%. orfs 1a, 1b, e and m of tw2575/98 were compared to those of other ibv genomes (fig. 2) . phylogenetic trees of the 1a and 1b genes of tw ibvs consistently show a closer relationship to the ch strains than to the us strains ( fig. 2a and b) . analysis of the 5 0 -utr and the orf 1-encoded proteins (including the papain-like proteinase, helicase, rna-dependent rna polymerase, p58 and hd2 protein) using the neighbourjoining algorithm reproduces the same topology (data not shown). similar results are also seen in the trees based on the full-length e and m genes of ibv, as shown in fig. 2c and d, further illustrating that tw ibv is genetically closer to the ch virus. in contrast to the above results, the n gene of tw ibv showed surprising phylogenetic incongruence and was more closely related to that of us isolates according to the me algorithm (fig. s2, fig. 3a and b) and the evolutionary divergence analysis (table 1) . a fragment containing nt 1-600 and its encoded proteins ( fig. 3c and d) also reflected the same phylogenetic relationship as the full-length n region. interestingly, results based on the 3 0 -terminus of the tw ibv n gene and its encoded protein ( fig. 3e and f) were inconsistent with those from the full-length n gene, and were similar to those for the genes described above and shown in figs. 1 and 2 . to further examine this phenomenon, unrooted phylogenetic trees produced by bi clearly illustrate that the tw n gene is phylogenetically closer to us ibv than to ch ibv (fig. 4a, c and e) . crucially, fig. 4c illustrates that the 5 0termini of the n genes of tw and us ibv belong to the same monophyletic group, rather than being separate isolated branches. the k a /k s ratio of tw2575/98 n versus all ibvs analysed in this study is 0.06-0.17, indicating the purifying selection of n protein during evolution. to further clarify whether the phylogenetic incongruence is caused by different evolutionary rates in different parts of the n gene, a ml test that are not biased by evolutionary rate variation was applied to recheck the phylogenetic relationships (holmes and rambaut, 2004) . no major difference was observed between the results from the bi and those from the ml test (fig. 4b , d and f), confirming that rna recombination probably occurred between tw and us ibvs in the 5 0terminal region of the n gene and that the phylogenetic incongruence was not caused by point mutation or variation in local evolutionary rates. to further test our hypothesis, genes 5 and n of 12 ibvs were subjected to the recombination detection program (rdp) (martin and rybicki, 2000) and the genetic algorithms for recombination detection (gard) program (kosakovsky pond et al., 2006) to examine the interspecies recombination. rdp analysis and three gard algorithms all consistently showed high significance (p value = 1.9 â 10 à3 to 3.5 â 10 à9 ) to support the hypothesis of interspecies recombination with the breakpoint at nt 278 of gene 5. rdp-bundled algorithms also revealed one candidate crossover region between the tw and us genes, located between nt 278 and 757 of the queried sequences, covering the gene 5 and the 5 0 -terminus of the n gene (fig. 5a ). for instance, the p values of the rdp and bootscan algorithms were 5.1 â 10 à4 and 1.0 â 10 à5 , respectively. alignment of the first 150 base pairs (bp) of n clearly indicated high identity between the tw and us strains (fig. 5b) . moreover, the deduced protein sequences also showed that the ch ibvs were significantly different from the tw and us strains ( fig. 5c and fig. 6 ). in addition, all the analysed tw ibvs shared the recombinant n gene, including one of the earliest isolates found in taipei in 1964, tp/64 (figs. 3 and 4) . this finding suggests that the recombination in the n gene produced a novel ibv founder distinct from ibv lineages present before 1964. the recombination event may have provided replication benefits, enabling the offspring of this founder to become dominant. the variation in the s1 region often represents viral genetic diversity and strain evolution (dolz et al., 2008; liu et al., 2006; wang and huang, 2000; wang and tsai, 1996) . the ratio of k a /k s can be an indicator of selective pressure acting on an open reading frame. the k a /k s of nucleotides in the s1 region was 0.35 ae 0.12% among evaluated tw ibvs. although previous studies of ibv s1 genes have argued that tw ibv is an independent branch of the us group (huang et al., 2004) , the relationship between tw ibv and ch isolates remains unclear. in this study, we have shown that taiwanese ibv features a recombinant ntd in the n locus and has evolved into a unique population. the interspecies recombination is evidenced by the incongruent phylogenetic relationship inferred for this gene region, high consensus in the n-terminus between tw and us strains, rare synonymous mutations in the conserved n protein, and wellsupported results from rdp analyses. a fast rate of mutation caused by an error-prone rnadependent rna polymerase is a major factor driving the evolution of rna viruses. nevertheless, as the restraint of error threshold of lethal mutations, the coronavirus is speculated to have a relatively low mutation rate due to its large genome size (eigen, 1987; maynard smith and szathmá ry, 1995) . to increase viral adaptability and fitness, coronavirus has evolved a unique discontinuous transcription system and 'copy-choice' genomic synthesis to achieve high efficiency of rna recombination (cavanagh, 2007; lai et al., 1985; liao and lai, 1992; makino et al., 1986) . however, only sporadic recombinant variants have been detected in nature. we here show that the recombinant n gene was detected in all the isolated tw ibvs, supporting the participation of recombination in viral evolution and showing that a recombinant rna virion can emerge as a local dominant strain. since the emergence of severe acute respiratory syndrome coronavirus (sars-cov) in humans, coronavirus has become a new focus of pathogenic research. in the present, little is known about the original strain and the natural host of the sars-cov. plausibly, the sars-cov may have originated, like tw ibv, through rna recombination in an unidentified host and subsequently emerged as a new virus in human beings by crossing the host boundary (holmes and rambaut, 2004; hon et al., 2008; magiorkinis et al., 2004; stavrinides and guttman, 2004; zhang et al., 2005) . however, present evidence is far from supporting this conclusion, and extensive sampling of the coronavirus genome from candidate hosts is required to solve this important issue. although studies of mhv have demonstrated that rna recombination can occur throughout the genome, genetic crossovers in coronaviruses are usually detected after tas sequences, referred to as putative hotspot sites (keck et al., 1987; kusters et al., 1990; liao and lai, 1992) . in this study, both the rdp/bootscan and gard analyses suggested the possibility of a putative 5 0 recombination crossover nick at nt 278 of gene 5 in the tw ibv genome. alternatively, given the remarkable discrepancy between the first 150 nt of ch n and those of tw and us n (fig. 5) , the start codon of n after the tas sequence may also serve as a breakpoint candidate for genetic recombination within tw ibv genome. it is noteworthy that one of the earliest tw ibvs, tp/64, shares high sequence identity in the first 150 nt of n with other tw ibvs and us ibvs (fig. 6 ). during the 1970s in taiwan, flocks were extensively immunised with the mass-serotype h120 vaccine (lu et al., 1993) . in this study, our finding suggests that the recombination in the n gene produced a novel ibv founder distinct from ibv lineages present before 1964. however, we did not exclude the possibility that the vaccination may also contribute to the antigenic shift of tw ibvs. the recombination event may have provided replication benefits, enabling the offspring of this founder to become dominant. to increase fitness under the pressure of h120 vaccination, the tw ibv with n recombinant might have undergone positive selection to escape from immune surveillance or to gain a replication advantage within quasi species. coronavirus avian infectious bronchitis virus identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan molecular epidemiology and evolution of avian infectious bronchitis virus in spain over a fourteen-year period new concepts for dealing with the evolution of nucleic acids a laboratory manual 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bronchitis virus recombination between nonsegmented rna genomes of murine coronaviruses rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments protective effect of vaccination in chicks with local infectious bronchitis viruses against field virus challenge genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between the incidence and virus isolation of infectious bronchitis in chickens in taiwan phylogenetic analysis of the full-length sars-cov sequences: evidence for phylogenetic discordance in three genomic regions high-frequency rna recombination of murine coronaviruses rdp: detection of recombination amongst aligned sequences the major transitions of evolution modeltest: testing the model of dna substitution the general stochastic model of nucleotide substitution mosaic evolution of the severe acute respiratory syndrome coronavirus isolation, pathogenicity, and h120 protection efficacy of infectious bronchitis viruses isolated in taiwan relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan evidence of natural recombination within the s1 gene of infectious bronchitis virus molecular characterization of an infectious bronchitis virus strain isolated from an outbreak in vaccinated layers characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens testing the hypothesis of a recombinant origin of the sars-associated coronavirus this work was supported by the bureau of animal and plant health inspection and quarantine, council of agriculture in taiwan (grant number 95as-13.2.1-bq-be). it is highly appreciated. we also thank dr. yi-ling lin at the institute of biomedical sciences in academia sinica for critical reading of this manuscript and valuable comments. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetmic. 2010.02.027. key: cord-337441-c5bxthwn authors: you, jae-hwan; reed, mark l.; dove, brian k.; hiscox, julian a. title: three-dimensional reconstruction of the nucleolus using meta-confocal microscopy in cells expressing the coronavirus nucleoprotein date: 2006 journal: the nidoviruses doi: 10.1007/978-0-387-33012-9_55 sha: doc_id: 337441 cord_uid: c5bxthwn nan the coronavirus nucleoprotein (n protein) is one of the most abundantly expressed viral proteins in an infected cell, with the principal function of binding the viral rna genome to form the ribonucleocapsid structure (rnp) and forming the viral core. n protein also has roles in viral replication, transcription, and translation as well as modulating cellular processes. we and others have shown that some coronavirus and arterivirus n proteins can localize to a dynamic subnuclear structure called the nucleolus and interact with nucleolar proteins. [1] [2] [3] the nucleolus is involved in ribosome subunit biogenesis, rna processing, cell cycle control, and acts as a sensor for cell stress. 4 morphologically the nucleolus can be divided into an inner fibrillar center (fc), a middle dense fibrillar component (dfc), and an outer granular component (gc). a directed proteomic analysis followed by subsequent bioinformatic analysis revealed that the nucleolus is composed of at least 400 proteins. coronavirus n proteins have the potential to be phosphorylated at multiple serine residues. however, mass spectroscopic analysis of both the avian infectious bronchitis virus (ibv) 5 and porcine transmissible gastroenteritis virus (tgev) 6 n proteins have shown that phosphorylation occurs at only three or four residues. in the case of ibv n protein, these map to predicted casein kinase ii sites. 5 based on amino acid sequence comparisons, three conserved regions have been identified in the murine coronavirus, mouse hepatitis virus (mhv) n protein. 7 in general, other coronavirus n proteins would appear to follow this pattern. we investigated the three-dimensional structure of the nucleolus and sub-nuclear bodies within cells expressing ibv and severe acute respiratory syndrome coronavirus (sars-cov) n protein. in many cases, viral proteins localize to discrete regions of the nucleolus and their specific localization can inform as to what effect they may be having on the host cell. for example, proteins that localize to, and disrupt the gc, can affect cellular transcription. therefore, we use coronavirus n proteins as a model to test our working hypothesis that disruption of nucleolar proteins and/or alterations in nucleolar architecture can perturb cellular functions. sars-cov n protein was n-terminally tagged with ecfp, creating pecfp-sars-cov-n. briefly, the sars-cov n gene was amplified (from a clone containing the sars-cov n gene-kindly provided by dr. maria zambon) using gene-specific primers to the 5' and 3' 20 nucleotides of the sars-cov n gene, but incorporating 5' restriction enzyme sites (in the case of the forward primer this was bspei and in the reverse primer bamhi) and then topo cloned into pcr2.1 (invitrogen). the insert was subcloned into pecfpc1 (clontech) such that sars-cov n protein would be c-terminal and in frame to ecfp. egfp was added n-terminally to ibv n protein, creating pegfp-ibv-n. the cloning strategy was identical to that described for sars-cov n gene, except the forward primer restriction site was bamhi and the reverse primer restriction site was ecori. the gene was ligated into pegfpc2 (clontech), such that ibv n protein would be c-terminal and in frame to egfp. all clones were verified by sequencing and expression of fusion proteins by western blot (data not shown). confocal sections of fixed samples were captured on an lsm510 meta microscope (carl zeiss ltd.) equipped with a 63x, na 1.4, oil immersion lens. pinholes were set to allow optical sections of 1 µm to be acquired. in singly transfected cells, ecfp was excited with the 458-nm argon laser line running at 10%, and emission was collected through a bp435-485 emission filter. egfp was excited with the 488-nm argon laser line running at 2%, and emission was collected through a lp505 filter. propidium iodide (pi) was excited with the helium:neon 543-nm laser line in all cases, and emission was collected through a lp560 filter. due to excitation of the egfp molecule by the 458-nm argon laser line, co-transfected samples were linearly unmixed using the meta detector. lambda plots of egfp and ecfp were generated from singly transfected reference samples excited with the 458-nm argon laser line and collected with the meta detector between 461 and 536-nm, in 10.7-nm increments. these lambda plots were then utilized to separate, or unmix, overlapping emission signal from co-transfected samples. zsections of cells expressing egfp, counterstained with pi, were generated by a two-step methodology. firstly, serial confocal sections of egfp were acquired with the meta detector. pi was then collected as described using the same z-settings. z-steps were collected 0.5 µm apart to allow over sampling of the data. the two sets of z-stacks were then pseudo-coloured and merged using the 'copy' facility within the lsm510 meta software. three-dimensional reconstruction and orthogonal views were also generated in the lsm510 meta software. studying the nucleolar localisation of proteins can be problematic. previous reports raised the possibility that charged proteins could migrate through cells postfixation and become localized to the nucleus and nucleolus. 8 although in this instance the specific example of vp22 has been challenged, the possibility arises that localization of coronavirus n proteins to discrete subcellular structures could be an artifact of fixation conditions. in addition, the successful detection of nucleolar proteins using antibodies can be related to the concentration of the protein within the nucleolus, in that the nucleolus, because of the high protein concentration, is not always amenable to antibody staining. 9 to address these concerns and also to investigate the subcellular localization of sars-cov n protein, we generated vectors that expressed fluorescent tagged sars-cov and ibv n proteins and determined the subcellular localization of these proteins first by live cell imagery, followed by fixation and confocal microscopy. sars-cov n gene was cloned downstream of ecfp (from vector pecfpc1, clontech), creating vector pecfp-sarscov-n, and ibv n gene was cloned downstream of egfp (from vector pegfpc2, clontech), creating pegfp-ibv-n, and when expressed in cells, resulted in fluorescent fusion proteins ecfp-sars-cov-n and egfp-ibv-n, respectively. cos-7 cells were transfected with pecfpc1, pegfpc2, pegfp-ibv-n or pecfp-sarscov-n (the former two as controls), and imaged 24 hr later by live cell imaging (fig. 1a) or co-transfected with both pegfp-ibv-n and pecfp-sarscov-n, fixed 24 hr post-transfection and analyzed by meta-confocal microscopy (which unmixes ecfp from egfp (fig. 1b) . live cell imaging data indicated that both egfp and ecfp, when expressed as individual proteins, had no distinct distribution pattern and were present in both the cytoplasm and nucleus but not nucleolus (fig. 1a) . however, egfp tagged ibv n protein localized to both the cytoplasm and nucleus while ecfp tagged sars-cov n protein localized to the cytoplasm only. as ecfp tagged sars-cov n has a molecular weight lower than the size exclusion limit for the nuclear pore complex, the lack of any sars-cov n protein within the nucleus suggests this protein contains a cytoplasmic retention signal. because these images were taken from live cells, the localization of ibv n protein to the nucleolus could not have been due to an artifact of fixation. confocal microscopy data (fig. 1b) reflected the localization patterns observed using live cell imaging. in contrast to egfp-tagged ibv n protein, ecfp-tagged sars-cov n protein localized to the cytoplasm and, in a minority of cells, to what appeared to be a nuclear body (arrowed). based upon morphology this structure cannot be identified but it does not have the appearance of the nucleolus. to investigate whether ibv n protein localized to a specific part of the nucleolus, cos-7 cells were transfected with pegfp-ibv-n, fixed 24 hr post-transfection, stained with pi to visualize the nucleus and nucleolus, then sectioned by confocal microscopy (fig. 2) . the data indicated that ibv n protein localized to a discrete area of the nucleolus. for example, compare the distribution of ibv n protein to the pi stained nucleolus in optical section 2.64 µm (fig. 2, arrows) , n protein would appear to occupy less nucleolar volume. to investigate this further, we utilized these z-sections to construct a threedimensional representation focusing specially on the nuclear region of the cell (fig. 3) . as can be observed, the volume taken up by ibv n protein in the nucleolus (fig. 3a) is less than total nucleolar volume (fig. 3b) . from this data we hypothesize that ibv n protein localizes to the dfc but not the gc. in conclusion, our data demonstrate that ibv n protein localizes to the cytoplasm and nucleolus and is not an artifact of fixation conditions. in contrast, sars-cov n protein remains localized in the cytoplasm and does appear to cross the nuclear pore complex, despite being below the size exclusion limit for entry into the nucleus. we hypothesize that sars-cov n protein contains a dominant cytoplasmic retention motif. meta-confocal analysis and three-dimensional reconstructions of cells expressing ibv n protein revealed that n protein does not localize throughout the nucleolus and may be confined to the dfc. this work was funded by the bbsrc, project grant number bbsb03416 and studentship bbssp200310434 to j.a.h. the confocal microscope facility in the astbury centre for structural molecular biology was funded by the wellcome trust and srif, and we would like to thank gareth howell for his help in using this facility. the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication the nucleolus--a gateway to viral infection? nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences bioinformatic analysis of the nucleolus mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein positively charged dna-binding proteins cause apparent cell membrane translocation a higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies key: cord-341541-3l6tjf3t authors: hajijafari anaraki, mozafar; sheikhi, nariman; haghbin nazarpak, hadi; nikbakht brujeni, gholamreza title: molecular characterization of infectious bronchitis virus based on rna‐dependent rna polymerase gene date: 2020-05-26 journal: microbiol immunol doi: 10.1111/1348-0421.12825 sha: doc_id: 341541 cord_uid: 3l6tjf3t extensive rate of variations in in spike glycoprotein subunit gene of infectious bronchitis virus (ibv) caused challenges for counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance. the study aimed at investigating the possibility use of rna‐dependent rna polymerase gene (rdrp) as a target for molecular characterization of ibv strains in iran. samples collected from commercial broiler flocks (n= 52) showing respiratory syndrome. specific polymerase chain reaction (pcr) primers were designed for a variable region locates in rdrp gene, flanked by highly conserved regions. reverse transcriptase pcr (rt‐pcr) followed by sequencing and sequence analysis could identified 8 ibv variants in an overall prevalence of 44.2%. deduced nucleotide and amino acid sequences were compared with published sequences for ibv strains. due to the long‐distance similarities, the field samples could be discriminated from vaccine strains. phylogenetic analysis of rdrp gene sequences resulted in clustering the ibv strains related to each area. using rdrp, as a genetic marker eliminates the challenges arise from the enormous variations that making difficult the discrimination between field and vaccine strains as well as affiliation of certain variants to various geographical areas. this article is protected by copyright. all rights reserved. replicase complex that codes for functional domains such as a papain-like proteinase (pl(pro)), main protease (m(pro)), rna helicase, and rna-dependent rna polymerase (rdrp) (4). the replicase is a multifunctional polyprotein which, after cleavage, produces a number of functional products those are indispensable for viral transcription and replication (5). mutation and recombination in ibv genome are the main cause of phenotypic variations. consequently, emergence of newly variant strains is mainly considered as a potential for new pathotypes, serotypes, and protectotypes. nucleotide sequencing and subsequent genetic analysis indicated a continuous spatio-temporal dynamics of ibv variations. most of all investigations on the ibv strains conducted based on the s and n protein gene sequences (6-8). it is believed that structural proteins are imperative target parts of the virus particles for immunization and reaching the protective immunity. therefore, variations in ibv s and n sequences are mostly considered for selecting the best vaccine strains for vaccination programs. however, extensive rate of variations in s1 gene among ibvs poses multiple challenges for clinicians in counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance (vus)(9). furthermore, in molecular epidemiology approaches, high polymorphism means that there is a low probability that a new variant could be classified in an identified profile or group. because of high degree of polymorphisms on s1 sequence, diverse genetic group designations for ibv strains are usually inconsistent with phylogenetic history (10). therefore, another powerful target for monitoring the virus variations seems to be necessary. as a genetic marker it also must be capable of being used for phylogenetic and epidemiological evolution of ibv variants. here we suggested the molecular characterization of infectious bronchitis virus based on rna-dependent rna polymerase gene. this article is protected by copyright. all rights reserved. direct sequencing method was performed for the analysis of the amplified rdrp, s1 and n genes cdna. the pcr products were purified using the purification kit (bioneer, seoul, korea) and each amplicon was separately sequenced in both directions by using specific forward and reverse primers. all procedures were carried out by sanger sequencing on an abi 3730 xl automatic dna sequencer (macrogen inc. seoul, korea). sequences were analyzed by blast through the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov/, ncbi). alignments and the amino acids prediction were made using the bioedit version 7.0.5.3 software package. the partial sequence of rdrp gene belonged to iranian ibv field strains, aligned with non-redundant sequences from all available strains, retrieved from the genbank database, were chosen for phylogenetic analysis. the partial sequence of s and n genes which is conventionally used for ibv genotyping (oie, 2013) was also used for phylogenetic analysis and making comparisons. phylogenetic analysis was conducted using mega6 beta (http://www.megasoftware.net/mega41.html) and the tree was constructed by the neighbor-joining method. bootstrapping over 1,000 replicates was done to assess the confidence level of the branch pattern. the rt-pcr with specific primers for rdrp gene of avian ibv resulted in the amplification of a figure 4) and n nucleotide sequences. the s1 tree consisted of this article is protected by copyright. all rights reserved. three main clusters divided from ancestor node. serotypes gray and jmk were separated by a long branch from other groups. gammacoronavirus, one chinese strain and two qia strains segregated with one vaccine strain (4/91). the third cluster contained h120, ma5, pakistan, massachusetts and chinese strains. the n tree also consisted of three main clusters divided from ancestor node. iranian strains separated in one cluster containing china, italy, canada and 4/91 vaccine strains. pakistan, jordan and h120 strains segregated to second cluster and poland, china and snu strains in third cluster. there are three groups of avian, mammals and insect circoviruses. iranian cacv strains phylogenetically clustered closely with canary circovirus. porcine and canine circoviruses segregated with human and goat in a two separate clade. mosquito, panda and hermit crab, which accounts for distinct amino acid sequences.. one branch only contained. replicase gene of ibv encodes pl (pro), m (pro), rna helicase and rdrp domains those are important for virus replication. the genotypic characterization assay developed in the current study focused on the variable regions of rdrp gene. this idea came from the observation that ibv s1 is a highly variability-prone region especially due to the lack of rna polymerase proofreading (6). it is also a shifting viewpoint from the assays targeting the incessant s1 gene variation to the characterization/detection based on the cause or origin of variation which is the rdrp gene. however, this idea must be elaborated by study the numerous variations as well as the association with pathogenicity and virulence. based on the rt-pcr detection and sequence analysis of ibv rdrp gene, an overall prevalence of field strains estimated as 44.2%. a similar prevalence has been reported for farms with clinical respiratory signs in which varies from 45 % to 54.4 % between districts (11-14). eight variants of the virus were identified in 23 farms, with the noticeable dominance of m6. the emergence of new ibv variants, that is an ongoing process, is a great challenge for identification, discrimination and characterization of the novel variants. in our experiment identifying the viruses that did not overlap the vaccine strains, stands for the capability of the test for detection of ibv and differentiating infected from vaccinated animals (diva). in addition, differentiations made by sequence analysis could be used for further characterizations such as affiliation of certain variants to certain geographical area. this article is protected by copyright. all rights reserved. comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus infectious bronchitis novel genotype of infectious accepted article 13 seroprevalence and genotyping of avian infectious bronchitis virus detected from iranian unvaccinated backyard chickens genotyping of avian infectious bronchitis virus in afghanistan molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in ahvaz, southwest iran, based on partial sequences of spike gene detection of newcastle disease, h9n2 avian influenza, and infectious bronchitis viruses in respiratory diseases in backyard chickens in ahvaz comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains phylogenetic analysis of a highly key: cord-312489-ywep0c08 authors: andoh, kiyohiko; suenaga, kiyotaka; sakaguchi, masashi; yamazaki, kenichi; honda, takashi title: decreased neutralizing antigenicity in ibv s1 protein expressed from mammalian cells date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.06.019 sha: doc_id: 312489 cord_uid: ywep0c08 we evaluated the antigenicity of recombinant infectious bronchitis virus (ibv) s1 protein expressed in mammalian cells. recombinant s1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using ni sepharose. the purified protein was analyzed by western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. six weeks after immunization, anti-ibv neutralizing titer and anti-s1 elisa titer were determined; immunized chickens then were inoculated with ibv via the trachea and ciliary activity was observed. results showed that the recombinant s1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant s1 protein was lower than that of inactivated virus. however, anti-s1 elisa indicated that the recombinant s1 protein induced antibodies against s1. these results suggest that the recombinant s1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. in conclusion, the neutralizing antigenicity of recombinant s1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies. infectious bronchitis virus (ibv) belongs to the order nidovirales, family coronaviridae, genus gammacoronavirus, and causes respiratory disease and pathology in the kidney and gonads of chickens and other birds (boltz et al., 2004; cavanagh, 2007) . ibv is an economically important disease in the poultry industry, and several vaccines have been used to prevent the spread of ibv. ibv shows extensive antigenic variation, reflecting mutation of the spike protein gene (cavanagh et al., 1988 (cavanagh et al., , 1997 wang and huang, 2000) . vaccines targeting individual ibv serotypes yield poor cross-protection; therefore, various attenuated and inactivated multivalent vaccines (derived from several different serotypes) are used (deguchi et al., 1998; sjaak de wit et al., 2011) . the spike protein (s) is an envelope glycoprotein that forms a dimer or trimer, and has been shown to play an important role in viral infection (cavanagh et al., 1986; ignjatovic and galli, 1994; wickramasinghe et al., 2011) . s is highly glycosylated, and based on its amino acid sequence, the spike protein is predicted to contain 21 to 35 n-glycosylation sites. s has two main functions: to attach the virus to the host cell receptor, and to activate fusion of the virion membrane with the host cell membrane (casais et al., 2003; wickramasinghe et al., 2011) . the s protein is the most important antigen in inducing neutralizing antibodies against ibv, and the n-terminal s1 region is especially important (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . the s1 domain forms the bulbous head of the spike protein, and several virus neutralization (vn) epitopes have been reported to reside within the first and third quarter of the s1 sequence (cavanagh et al., 1988; kant et al., 1992; koch et al., 1990; sjaak de wit et al., 2011) . thus, analysis of the antigenicity of the s1 domain is expected to be critical to the development of effective anti-ibv vaccines. to prevent ibv infection, several recombinant subunit vaccine developments have been attempted using the recombinant s1 protein or other proteins. immunization with recombinant s1 protein expressed from baculovirus has been shown to provide in chicken effective protection against ibv infection (song et al., 1998) . other groups also have reported that immunization with recombinant s1 http://dx.doi.org/10.1016/j.virusres.2015.06.019 0168-1702/© 2015 elsevier b.v. all rights reserved. epitope peptide expressed from escherichia coli (e. coli) protected chickens against ibv infection (yang et al., 2009a (yang et al., , 2009b . furthermore, viral vectored vaccines co-expressing the s1 protein and host cytokines have been reported to induce anti-s1 antibodies (chen et al., 2010; shi et al., 2011; tomley et al., 1987; wang et al., 2009; zeshan et al., 2011; zhang et al., 2012) . however, in these reports of protection, recombinant antigen did not provide perfect protection and antibody titers were evaluated only by hemagglutination inhibition (hi) or enzyme-linked immunosorbent assay (elisa); the respective authors did not indicate whether these antigens retained their native neutralizing antigenicity (song et al., 1998; yang et al., 2009a yang et al., , 2009b . therefore, it remains unknown whether recombinant s1 protein, without the s2 domain, completely retains its conformational epitopes and neutralizing antigenicity. to address these issues, we analyzed the recombinant s1 expressed from mammalian and avian cells by western blotting and analyzed the neutralizing antigenicity of recombinant s1 protein and compared titers against those of inactivated virus antigen. for the immunization experiment, recombinant s1 protein was expressed as the secreted protein with a trimerization motif because some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, retains the ability to bind the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) and it seemed to be suited to the vaccine antigen. the present work employed the tm86 ibv strain, an isolate of genotype jp-ii that originally was recovered from a field chicken; this strain subsequently has been used as a vaccine strain (ariyoshi et al., 2010; mase et al., 2004) . tm86 was propagated in specific-pathogen-free (spf) chicken embryonated eggs. ibv tm86 adapted to chicken kidney (ck) cells was used for the vn test and elisa of the antigen. ck cells and chicken embryo fibroblast (cef) cells were incubated in eagle's medium (emem) supplemented with 10% tryptose phosphate broth and 5% heat-inactivated fetal bovine serum (fbs; hyclone), along with 100 units of penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. 293t cells and 293 cells lacking nacetylglucosaminyltransferase i (293 gnti − , reeves et al., 2002) were propagated in dulbecco's modified eagle's medium (dmem) supplemented with 10% fbs along with 100 units penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. a segment of the s1-encoding gene, coding for the protein from the n-terminus to the cleavage site between s1 and s2, was amplified from the spike gene of ibv strain tm86 (accession no. ab120655) by reverse transcriptase polymerase chain reaction (rt-pcr). the primer set used for rt-pcr was 5 -caaattattggtcagagatgttgg-3 (s1.1) and 5 -gaatcattaaacagactttttaggtct-3 (s2r1). after amplification, the dna encoding the signal sequence of s1 (mlvkslflvtll-falcs) was replaced with a dna sequence encoding the signal sequence of marek's disease virus (mdv)-glycoprotein a (ga) (mltprvlralgwtglfflllspsnvl). furthermore, dna sequences encoding a 6× his-tag peptide sequence, with or without those encoding a t4 phage fibritin coiled-coil trimerization motif (gsgyi-peaprdgqayvrkdgewvllstflg), were inserted in-phase and downstream of the recombinant s1-encoding gene. the resulting fig. 1 . schematics of the ibv s1 segment expressed as a secreted protein. to construct the secreted s1 expression plasmid, the nucleotide sequence encoding the s1 domain lacking the cleavage sequence (rrfrr) was amplified from the ibv tm86 genome. to increase expression levels of the recombinant s1 protein, the nucleotide sequence encoding the signal sequence was replaced with a nucleotide sequence encoding mdv ga. to facilitate purification and multimerization, the resulting sequence was cloned upstream and in-phase with nucleotide sequences permitting expression as c-terminal fusions to a 6× his-tag peptide sequence with or without a t4 phage fibritin coiled-coil trimerization motif. loci encoded the recombinant s1 protein with c-terminal fusions to the indicated domains ( fig. 1) . the constructed sequence was cloned into the sal i site of the expression plasmid pcaggs, which contains a cag promoter (modified chickenˇ-actin promoter with cytomegalovirus immediate-early (cmv-ie) enhancer), rabbitˇglobin gene sequence including polyadenylation signal, and simian virus 40 (sv40) ori (niwa et al., 1991) . the constructed plasmids were named pcaggs-s1-t4-his and pcaggs-s1-his, respectively. for transfection, the plasmid was purified using a qiaprep spin miniprep kit (qiagen). the mouse mabs against ibv s1 or s2 region were established in our institute. it was confirmed that these mabs recognize nonconformational epitopes by western blotting under denaturing condition. it was also confirmed that these mabs do not show neutralizing activity in vn test. before examination, the mabs were purified using protein g column. 293t, 293 gnti − , and cef cells were transfected with either of the constructed plasmids using polyethylenimine (pei). transfection using pei was performed according to the protocol established by boussif et al. (1995) . briefly, 88 g of the plasmid was mixed with 440 l of pei (2 mg/ml) and then transfected into approximately 2 × 10 7 of 293t cells. transfected cells were incubated in opti-mem (life technologies) and the supernatant of transfected cells was harvested at 72-96 h post-transfection. the recombinant protein was purified using ni sepharose (ge healthcare) according to the manufacturer's protocol. sds-page was carried out under denaturing and nondenaturing conditions. under denaturing conditions, the purified protein was mixed with 2× sample buffer (100 mm tris-hcl (ph 6.8), 4% sds, 20% glycerol, 0.2% bromphenol blue) containing 200 mm dithiothreitol (dtt) and boiled for 5 min at 95 • c. in contrast, non-denaturing sds-page was performed according to the protocol reported by bender et al. (2005) . briefly, the purified protein was mixed with 2× sample buffer containing 0.4% sds (in the absence of a reducing agent) and loaded directly onto a gel (without boiling of the sample). the protein was separated by 5-20% polyacrylamide gradient gel (e-pagel, atto) and transferred to a polyvinylidene difluoride (pvdf) membrane (millipore). the membrane was incubated in 5% skim milk (wako) in t-pbs buffer (phosphate-buffered saline, ph 7.2 (pbs) containing 0.05% tween 20) for 60 min at 37 • c, and then incubated with a mab against s1 protein in 5% skim milk in t-pbs buffer. next, the membrane was incubated with a peroxidase-conjugated second antibody, goat anti-mouse igg (h + l) (jackson). the reacted protein was visualized using a tmb substrate kit (invitrogen). the immunization experiment consisted of 4 groups of 5 or 10 chickens. the spf chickens (layer-type) used for this study were maintained in our institute and 10 chickens were immunized with the purified recombinant s1 protein, which was his-tagged with trimerizaton motif, or inactivated virus, respectively. five chickens were immunized with pbs-mock vaccine (mock group) and 10 chickens were left untreated to serve as an unvaccinated control group. light liquid paraffin, sorbitan monooleate, and polysorbate 80 in a volume ratio of 9:36:4:1 was used as oil adjuvant. recombinant s1 protein was mixed with oil adjuvant to make an s1 suspension at a concentration of 20 g/dose. the ibv tm86 strain was inactivated using formaldehyde and mixed with oil adjuvant to generate a suspension harboring inactivated virus at a virus concentration of 10 7.0 eid 50 /dose. chickens were inoculated intramuscularly. at 6 weeks post-inoculation, blood was collected from each animal, and the resulting sera were used to determine antibody titers. each chicken then was challenged with 10 3.5 eid 50 ibv tm strain administered via the trachea. animals were sacrificed at 4 days post-challenge and ciliostasis was assessed by a slightly modified version of the previously reported protocol (cook et al., 1999) . briefly, tracheas were removed aseptically from euthanized chickens. cilia were observed microscopically and the cessation of ciliary movement was considered to be a sign of symptoms. sera were serially diluted two-fold with emem in a microplate and mixed with 200 tcid 50 of ck-adapted ibv tm strain. after incubation for 1 h at 37 • c, ck cells were inoculated with ibv and incubated at 37 • c in 5% co 2 incubators. vn titer was defined as the reciprocal of the highest dilution showing no cytopathic effect (cpe). the mab against ibv s1 protein was diluted to 2 g/ml with pbs and 50 l was added to each well of a 96-well microplate (maxisorp; nunc, denmark). after incubation overnight at 4 • c, plates were washed three times with t-pbs. next, 300 l of t-pbs containing 5% skim milk was added and incubated at room temperature (rt) for 1 h. after washing three times with t-pbs, 50 l of s1 antigen was added to each well and incubated at rt for 1 h. for the recombinant s1 elisa, supernatant of 293t cells transfected with the expression plasmid pcaggs-s1-his was used. for the native spike elisa, lysate of ibv-infected ck cells was used as the antigen. ck cells were infected with ibv at a moi of 0.2 and extracted 72 h post-infection by treatment with ripa buffer (containing 1% tritonx-100, 1% sodium deoxycholate, 0.05 m tris-hcl (ph 8.0), 0.1 m nacl, and 1 mm edta). antigens were diluted with dilution buffer (t-pbs containing 5% skim milk) and added to wells. the levels of recombinant and native s1 protein used for the elisa test were selected as the respective concentrations that yielded absorbances (following reaction with the positive serum) of 1.0 to 1.5. the contents of the wells were washed with t-pbs; an aliquot (50 l) of sera (diluted 1:100 with dilution buffer) was dispensed to each well, and plates were incubated at rt for 1 h. in the recombinant s1 elisa, primary sera were pre-treated with 1 g/ml of purified his-tagged ibv e protein at 4 • c overnight to remove antibodies against the his-tag peptide. the contents of the wells were washed with t-pbs; an aliquot (50 l) of peroxidaseconjugated anti-chicken immunoglobulin (donkey anti-chicken igy (h + l) (jackson)) was dispensed to each well, and plates were incubated at rt for 30 min. the contents of the wells were washed with t-pbs; an aliquot (100 l) of tmb substrate kit (dako) was dispensed to each well, and plates were incubated at rt for 15 min. after incubation, the enzymatic reaction was stopped by addition of 100 l/well of 1 m sulfuric acid. the absorbance was measured using a spectrophotometer (versamax), with a 450-nm and 650nm filter. a hyperimmune serum against ibv, which was prepared by immunization with attenuated and inactivated vaccines, was used as the positive serum. separate work (data not shown) using chicken antisera against ibv e, m and n proteins and anti-s2 mouse mab (described above) confirmed that native s1 elisa does not react with antibodies raised against ibv component proteins s2, e, m, or n. antisera against ibv e, m and n were prepared from chickens immunized with purified recombinant proteins expressed from e. coli and it was confirmed that these antisera reacted to ibv component by elisa (data not shown). all statistical analyses were performed with ezr (saitama medical center, jichi medical university), which is a graphical user interface for r (the r foundation for statistical computing) (kanda, 2013) . the constructed expression plasmids, pcaggs-s1-t4-his and pcaggs-s1-his, were transfected into cells and the recombinant protein was expressed as secreted protein. the secreted s1 protein was purified using ni sepharose and analyzed by western blotting. the result of western blotting under denaturing condition showed that recombinant s1 protein expressed from 293t cells ran as a broad band of nominal molecular weight ranging from 100 to 150 kda. the size range of this band was increased compared to that of native s1 protein (derived from ibv propagated in chicken embryonated eggs), with the native protein running on the denaturing gel as a single sharp band at a nominal size of approximately 100 kda (fig. 2) . recombinant s1 protein derived from 293 gnti − cells, which lack the ability to synthesize complex n-glycans, ran on the denaturing gel as a sharp band at a nominal molecular weight of 75 to 100 kda, with a size smaller than that observed for recombinant protein expressed from 293t cells (fig. 2) . recombinant s1 protein expressed in cef cells (cells that are derived from the natural host for ibv) also ran under denaturing conditions as a slightly broader and larger band than native s1 obtained from ibv virions; notably, the protein produced in cef cells ran as a sharper band than protein produced in 293t cells (fig. 3) . these results indicated that recombinant s1 protein might have an unnatural glycosylation pattern. using western blotting under non-denaturing conditions, we examined the conformation of recombinant s1 protein produced and secreted from 293t cells. we observed that recombinant s1 protein lacking the fibritin motif existed in monomeric, dimeric, and trimeric conformations. on the other hand, recombinant s1 protein with the fibritin motif existed primarily in the trimeric conformation (fig. 4) . for the immunization experiment, the purified recombinant s1fibritin protein expressed from 293t cells was mixed with adjuvant to generate a s1 inoculum at a concentration of 20 g/dose. vn titer induced by the recombinant s1 protein was lower (p < 0.05) than that induced by the inactivated virus antigen (fig. 5) . to compare the efficacy against ibv infection, ciliostasis was evaluated via ibv challenge in animals previously immunized with s1 or with inactivated ibv virus; as a control, unvaccinated and mock-vaccinated . the asterisk indicates statistical significance (p < 0.05) and the error bar indicates standard deviation. comparison between two groups was performed using t test. protection defined by ciliostasis in the trachea. protection a recombinant s1 4/10 inactivated virus 10/10 unvaccinated control 3/10 pbs-mock 0/5 a birds showing active ciliary movement/total. chickens also were subjected to ibv challenge. following challenge, all (10/10) chickens immunized with inactivated virus antigen retained ciliary movement. in contrast, post-challenge ciliostasis was observed in 60% (6/10) of animals immunized with recombinant s1, 70% (7/10) of unvaccinated animals, and all (5/5) mock-vaccinated chickens (table 1 ). these results indicated that immunization with recombinant s1 protein was not sufficient to provide protection from subsequent ibv infection. to analyze the antigenicity of the recombinant s1 protein, sera obtained from the immunization experiment were analyzed by elisa, using recombinant or native s1 protein as antigen. recombinant s1 elisa showed that sera from chickens immunized with recombinant s1 protein had a slightly high, but not significant (p = 0.14), titer than sera from chickens immunized with inactivated virus (fig. 6a) . native s1 elisa detected similar titers in the sera of animals immunized with recombinant s1 protein compared to those in sera of chickens immunized with inactivated virus (fig. 6b ) (control experiments (data not shown) demonstrated that mock and unvaccinated control groups did not differ from each other in vn and elisa tests of anti-s1 activity.) these results indicated that while recombinant s1 protein retained antigenicity (the ability to induce antibodies against s1 protein), the resulting antibodies was decreased its neutralizing activity, in contrast to those induced by inactivated virus. 6 . indirect sandwich elisa using recombinant s1 protein (a) and the lysate from ibv-infected cells (b). primary sera were collected from immunized chickens and diluted 1:100. in recombinant s1 elisa, primary sera were pre-adsorbed with purified recombinant his-tagged protein to remove the antibodies against the histag peptide sequence. reactivities are shown as the s/p ratio of absorbance at 450 nm and 650 nm. comparisons between three groups were performed using steel-dwass test and n.s. indicated not significant. this study showed that recombinant s1 protein exhibited decreased ability to induce a neutralizing antibody response, although the recombinant s1 induced non-neutralizing antibodies. the elisa using recombinant s1 used sera that already had been adsorbed with his-tag peptides, precluding a role for the purification (his-tag) domain in the induced antibody response. in the elisa using native s1, the native protein may have contained noncleaved spike protein, thus including both s1 and s2 domains as part of the antigen. however, we found (data not shown) that inclusion ("spiking") of s2 protein did not affect the results of the native s1 elisa. this observation is consistent with other reports suggesting that the s2 region contains fewer neutralizing epitopes than does the s1 region; the literature indicates that most epitopes are located in the s1 region (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . these results excluded the possibility that the other viral components affect the titer in elisa. thus, although recombinant s1 protein induced antibodies, this recombinant protein was decreased the ability to induce neutralizing antibodies, suggesting the recombinant s1 expressed in mammalian cells may change its character. in a separate test, we observed that antigen from inactivated virus that was denatured by boiling (5 min, 95 • c) did not induce neutralizing antibodies (data not shown). this result suggested that the correct conformation is important for s1 protein to induce neutralizing antibodies. in the present study, the recombinant s1 protein used for immunization was expressed in mammalian cells, rather than in avian cells; the expression efficiency of recombinant proteins in avian cells was decreased compared to that in mammalian cells, and expression in avian cells was insufficient to obtain recombinant proteins in the quantities needed for the experiments. the present work also used recombinant s1 expressed as secreted protein because secretion permitted accumulation of recombinant protein to higher levels and made it easy to purify recombinant protein. these modifications, using mammalian cells and expressing recombinant protein as the secreting form, may affect the conformation of the s1. western blotting using denaturing conditions indicated that recombinant s1 protein expressed in 293t cells exhibited increased (and less uniform) sizes compared to the native protein. in contrast, recombinant s1 protein expressed in 293 gnti − cells (which lack n-acetyl-glucosaminyltransferase i activity and so do not generate complex n-glycans) ran as a sharper band of a size similar to that of native s1 protein. together, these data indicated that the glycosylation pattern of recombinant s1 protein might differ from that of native s1, and that this difference reflects the level and nature of n-glycosyl modifications. we hypothesize that the native spike protein is typically decorated with high-mannose or hybridtype n-glycans, and/or that the complex-type glycans decorating the native spike proteins are few in number and are homogeneous. it is known that glycosylation characteristics differ between species, and glycosylation affects the conformation of glycoproteins (helenius, 1994) . therefore, glycosylation pattern differences may affect the conformation and antigenicity of the s1 protein. furthermore, glycans attached on the surface of the envelope protein are known to mask antigenic sites, thereby permitting evasion of the host immune system (sun et al., 2011; zhang et al., 2015) . we infer that recombinant s1 protein antigenic sites also may be masked by glycans. from these hypotheses, there is the possibility that s1 expressed in other expression systems, which have different glycosylation character, also changes its antigenicity. other reports suggest that the s2 region of the ibv spike protein is important for the correct conformation of s (callison et al., 1999; promkuntod et al., 2013) . therefore, the recombinant s1 protein secreted in this study might assume a non-native conformation. we hypothesize that expression of the s1 domain without the s2 region results in the assumption of an unnatural conformation, resulting in unnatural glycosylation patterns, or that unnatural glycosylation of s1 protein causes the recombinant peptide to assume an unnatural conformation. recombinant s1 protein may expose hidden epitope(s) that are normally latent in the protein. however, some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, binds the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) . these reports suggested that the secreted s1 protein retains receptor binding ability. in addition, expression of other viral envelope proteins (specifically, human immunodeficiency virus (hiv) gp120 and influenza virus hemaglutinin (ha)) as secreted proteins have been reported to induce neutralizing antibodies (pancera et al., 2005; wei et al., 2008) . these reports indicated that secreting envelope proteins with a trimerization motif is an effective method for creating vaccine antigens. additionally, cavanagh (2007) reported that ibv s1 protein was sufficient for the induction of neutralizing antibodies (thought s1 was not sufficient for protection against ibv infection). furthermore, cytotoxic t-lymphocyte (ctl) activity induced by immunization of nucleocapsid protein (np) has been shown to be important for virus clearance (collisson et al., 2000; seo and collisson, 1997) . however, in the present study, while immunization with recombinant ibv s1 (expressed as a secreted protein fused to the fibritin motif) induced antibodies, the response mainly consisted of non-neutralizing antibodies. therefore, recombinant s1 protein (expressed as a secreted protein in mammalian cells) may be insufficient for use as a vaccine antigen. recently, virus-like particle (vlp) -based antigens have been tested as new candidates for subunit vaccines against ibv (cavanagh, 2003; lv et al., 2014) . receptor binding domain (rbd) peptides also have been tested as vaccine candidates against severe acute respiratory syndrome (sars) coronavirus (jiang et al., 2012) . these alternative approaches to antigen production may overcome the challenges observed in the present work. in conclusion, we demonstrated that recombinant s1 protein (expressed as a secreted protein in mammalian cells) was decreased the ability to induce neutralizing antibodies in chicken. we infer that the recombinant protein lacked conformational epitopes as a result of 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evaluation of recombinant fowlpox virus expressing infectious bronchitis virus s1 gene and chicken interferon-␥ gene for immune protection against heterologous strains infectious bronchitis virus variants: a review of the history, current situation and control measures induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus glycosylation site alteration in the evolution of influenza a (h1n1) viruses expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus protection of chickens against infectious bronchitis by a recombinant fowlpox virus co-expressing ibv-s1 and chicken ifngamma comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian h5n1 influenza virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity multivalent dna vaccine enhanced protection efficacy against infectious bronchitis virus in chickens the protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing spike (s1) glycoprotein of infectious bronchitis virus fused/co-administered with granulocyte-macrophage colony stimulating factor protection conferred by a recombinant marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the h5n1 avian influenza virus the authors wish to thank other members of the laboratory for technical assistance, and thank mr. mcculloch for proof reading of the manuscript. the authors declare that they have no conflict of interest. key: cord-321602-88b2h06y authors: lv, chenfei; shi, tingting; zhu, pengpeng; peng, xing; cao, shangshang; yan, yan; ojha, nishant kumar; liao, min; zhou, jiyong title: construction of an infectious bronchitis virus vaccine strain carrying chimeric s1 gene of a virulent isolate and its pathogenicity analysis date: 2020-08-19 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10834-2 sha: doc_id: 321602 cord_uid: 88b2h06y abstract: infectious bronchitis virus (ibv) is a member of genus gamma-coronavirus in the family coronaviridae, causing serious economic losses to the poultry industry. reverse genetics is a common technique to study the biological characteristics of viruses. so far, there is no bac reverse genetic system available for rescue of ibv infectious clone. in the present study, a new strategy for the construction of ibv infectious cdna clone was established. the full-length genomic cdna of ibv vaccine strain h120 was constructed in pbac vector from four ibv fragment subcloning vectors by homologous recombination, which contained the cmv promoter at the 5′ end and the hepatitis d virus ribozyme (hdvr) sequence and bovine growth hormone polyadenylation (bgh) sequence after the polya tail at the 3′ end of the full-length cdna. subsequently, using the same technique, another plasmid pbac-h120/scs1 was also constructed, in which s1 gene from ibv h120 strain was replaced with that of a virulent sc021202 strain. recombinant virus rh120 and rh120/scs1 were rescued by transfecting the plasmids into bhk cells and passaged in embryonated chicken eggs. finally, the pathogenicity of both the recombinant virus strains rh120 and rh120/scs1 was evaluated in spf chickens. the results showed that the chimeric rh120/scs1 strain was not pathogenic compared with the wild-type ibv sc021202 strain and the chickens inoculated with rh120/scs1 could resist challenge infection by ibv sc021202. taken together, our results indicate that bac reverse genetic system could be used to rescue ibv in vitro and ibv s1 protein alone might not be the key factor for ibv pathogenicity. key points: • bac vector was used to construct ibv full-length cdna by homologous recombination. • based on four subcloning vectors, a recombinant chimeric ibv h120/scs1 was constructed and rescued. • pathogenicity of h120/scs1 was similar to that of h120, but different to that of sc021202. infectious bronchitis virus (ibv) is a member of the coronaviridae family in the order nidovirales. it is a highly pathogenic virus that causes serious economic losses to the world poultry industry (cavanagh 2005; cook et al. 2012) . ibv is an enveloped virus having a single-stranded, positivesense rna genome of 27.6 kb in size (boursnell et al. 1987) . because of the high degree of genome sequence variation, ibv has many genotypes circulating at any time in poultry farms worldwide (jackwood 2012) , making it difficult to control or prevent its spread through vaccination. reverse genetics is a common in vitro method to study virus biological characteristics. the development of this technique allowed researchers to manipulate the ibv genome and rescue of virus in vitro (shan et al. 2018) . previously, the most common technique to obtain chenfei lv and tingting shi contributed equally to this work. the full-length cdna of ibv was the cdna ligation (youn et al. 2005; zhou et al. 2013) . by using this method, small pieces of ibv genomic cdnas were amplified, digested with restriction enzymes (re), and then ligated. the full-length cdna with t7 promoter attached to 5′ end of the ibv genome was transcribed and transfected into bhk cells by electroporation to rescue the virus (casais et al. 2001) . another reverse genetic system for ibv was based on targeted rna recombination. this method enabled the modification of the ibv genome at its 3′ terminal by targeted rna recombination (van beurden et al. 2017) . by using this method, a recombinant h52 strain carrying the spike glycoprotein ectodomain of mhv was constructed, which had the capability to grow in a mammalian cell lines (van beurden et al. 2017 ). however, targeted rna recombination is only useful for the modification of 3′-end of the ibv genome and is difficult to modify the 5′-end polymerase gene, because the construction of donor rna vectors entering this region is hindered (van beurden et al. 2017) . bacterial artificial chromosomes (bac) vector reverse genetic system is also a common method to rescue coronaviruses in vitro. the first coronavirus full-length genomic cdna in bac was constructed for tgev (almazan et al. 2000) . later on, several coronavirus, such as sars-cov (almazan et al. 2006) , fipv (balint et al. 2012) , and mers-cov (scobey et al. 2013) , were rescued successfully using that technique. however, there is no bac reverse genetic system currently available for the rescue of ibv. the spike protein of ibv is posttranslationally cleaved into two subunits, s1 and s2, where s2 is anchored into the viral envelop and is important for the membrane fusion. s1 comprises the head domain of spike and is responsible for host receptor binding (wickramasinghe et al. 2011) . the s1 subunit is the important immunogenic component that contains epitopes for neutralizing antibody (ignjatovic and sapats 2005) . in this study, a new strategy for construction of ibv infectious clone was established. a nephropathogenic strain sc021202 (close to ibv gi-22 genotype), which causes severe kidney damage and mortality to infected chickens (zhou et al. 2004 ) was selected as donor of ibv s1 gene. first, infectious clone of ibv vaccine strain h120 (rh120) was rescued using the bac reverse genetic system. then, a chimeric ibv virus rh120/scs1 with the h120 backbone replacing its s1 subunit with that of virulent sc021202 isolate was constructed. lastly, the rh120/scs1 was used to inoculate specific pathogen free (spf) chickens for the preliminary study of the function of s1 of ibv field isolate on virus pathogenicity and the potential of recombinant virus as a vaccine candidate was also evaluated. the strains of ibv sc021202 (genbank no.: eu714029.1) and h120 (genbank no.: fj888351.1) were stored in our laboratory. viruses were propagated and titrated in the allantoic cavity of 10-day-old embryonated spf chicken eggs. baby hamster kidney (bhk-21) cell line was stored in our laboratory and cultured at 37°c in dulbecco's modified eagle's medium (dmem; gibco, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs, biological industries, israel), penicillin (100 u/ml), and streptomycin (100 μg/ml). primary chicken embryonated egg kidney (cek) cells were prepared from 17-19-day-old spf chicken embryonated eggs and maintained in dmem supplemented with 10% fbs at 37°c in 5% co 2 atmosphere. competent cells were prepared according to the instructions provided by ultra-competent cell preps kit (sang biotech, china). pbelobac11 (called pbac in the subsequent texts) vector was a gift from professor yaowei huang of zhejiang university. construction scheme of a full-length cdna clone of ibv h120 to construct the full-length cdna of ibv h120 strain, total rna was extracted from the allantoic fluid of spf chicken embryonated eggs infected with h120 and transcribed to cdna by reverse transcriptase using the thermo scientific revertaid first-strand cdna synthesis kit (thermo fisher, usa). four cdna fragments (f1, f2, f3, and f4) covering the whole h120 genome sequence were amplified separately from the cdna template with the primer sets (f1-f and f1-r, f2-f and f2-r, f3-f and f3-r, f4-f and f4-r) shown in table 1 , which also contained sequences for homologous recombination. the procedure for the construction of full-length cdna of ibv h120 has been shown in fig. 1a . the amplified 4 fragments (f1, f2, f3, and f4) carrying the same homologous recombination sequences as the pbac vector were cloned into modified linearized pbac vector. homologous recombination reaction was carried out according to the instructions provided with clonexpress one-step cloning kit (vazyme biotechnology, china). the resulting plasmids were named as pbac-f1, pbac-f2, pbac-f3, and pbac-f4. bamhi enzyme site in the first fragment was mutated as a marker (a5472c) for identification of rescued virion through amplification of the f1 fragment with primers f1a5472c-f and f1a5472c-r containing mutation site (table 1) . for the 5′utr and 3′utr fragments, the primers (5′utr-f and 5′utr-r, 3′utr-f and 3′utr-r) containing the (table 1) , and the nucleotide sequences were separately amplified. the first and fourth subcloning vectors pbac-f1 and pbac-f4 were digested with bamhi and xhoi restriction enzymes (takara, japan), respectively, resulting in a linearization of the vectors (fig. 1a) . the 5′utr was fused to the first subcloning vector construct, and the 3′utr was fused to the fourth construct by homologous recombination to obtain the first subcloning pbac-f1 and fourth subcloning pbac-f4. the first subcloning vector construct was modified by adding the cmv (cytomegalovirus) promoter sequence in the 5′ terminal of the first fragment, and then hdvr (hepatitis delta virus ribozyme) sequence and bgh (bovine growth hormone polyadenylation signal) sequence were added to the 3′ terminal of the fourth fragment after the poly(a) by homologous recombination with the primers (cmv-f and cmv-r, hb-f and hb-r) listed in table 1 . subsequently, the f2 and f4 fragments were amplified with the primer sets (f2-f1)-f and (f2-f1)-r and primer sets (f4-f3)-f and (f4-f3)-r (table 1) , respectively. they were fused to the linearized plasmids pbac-f1 and pbac-f3 digested by xhoi to obtain two semi-subclones pbac-f12 and pbac-f34, respectively (fig. 1a) . finally, two semisubclones were fused using the same method with the primer sets (f34-f12)-f and (f34-f12)-r (table 1) to complete the full-genome cdna construction of ibv h120 (fig. 1a ). replacement strategy for chimeric recombinant h120/scs1 and generation of full-length cdna procedure used to construct chimeric h120 recombinant strain has been shown in fig. 1b . the linearized plasmid dna pbac-f4(hb)-δs1 lacking the s1 gene was reverse amplified from the plasmid pbac-f4 with primer set h120-f4-△s1-f and h120-f4-△s1-r (table 1 ) as a linearized vector. the sc021202 s1 gene containing same homology arms to those of the h120f4 was amplified from total rna of allantoic fluid of spf chicken embryonated eggs infected with ibv sc021202 by rt-pcr with the primers scs1-f and scs1-r listed in table 1 and was then inserted into pbac-f4-△s1 by the same homologous recombination process using the clonexpress one-step cloning kit (vazyme biotechnology, china). then, the third segment of ibv genome and the pbac-f4/scs1 was fused to obtain two semisubclones. subsequently, both of these semi-subclones were fused to complete the construction of full-genome cdna of ibv h120/scs1. bhk-21 cells were cultured in 6-well plates. when the cells grew to 60% confluency, 4 μg (200 μl/well) of pbac-h120 or pbac-h120/scs1 was transfected to the cells using jetprime transfection reagent (polyplus, france) according to the manufacturer's instructions. transfected cells were incubated for 6 h at 37°c. after the incubation, the culture medium was replaced with dmem with 2% fbs and cells were incubated at 37°c for 48 h. after completion of the incubation period, culture medium and cells were harvested by repeated freeze-thaw (three times) and named as rh120 and rh120/scs1 p0 generation of the rescued virus. this p0 generation was inoculated into 10-day-old spf chicken embryos. after 48 h, the allantoic fluid of chicken embryo was collected and blindly passaged for 3 generations. eid 50 of passaged virus was determined by the method of reed and muench. the p0 generation rescue virus and allantoic fluid of infected chicken embryo were used for rna extraction. reverse transcription reaction was carried out using a reverse transcription kit (thermo fisher, usa) to obtain cdna. ibv h120 strain was used as template for designing primers (rm-f and rm-r, table 1 ) before and after the introduced rescue marker (rm) to amplify a 621 bp fragment spanning between 5211 and 5831 bp of the viral genome. the pcr products were purified and ligated into pmd-18t vector (takara, japan) and then transformed into competent cells, and positive clones were picked for sequencing to identify rescue marker sites. the rescue virus was further inoculated into the spf chicken embryonated eggs and observed for development of dwarf embryo lesion. to examine the stability of rescued viruses, s1 fragment of the virus of different passages was amplified from total rna extracted from allantoic fluid of infected embryonated eggs by rt-pcr using the primers h120sc-s-f and h120sc-s-r (table 1 ). the amplified fragments were then sequenced and analyzed. cek cells prepared from 18-day-old chicken embryos were infected with h120, rh120, and rh120/scs1. the medium was removed 48 hpi, and infected cells were fixed with methanol:acetone (1:1) at − 20°c. considering that membrane protein (m protein) is one of the abundant structural proteins in coronavirus (neuman et al. 2011) , in-house mouse monoclonal antibody 2b3 to ibv m protein (1:1000) was used as primary antibody in immunofluorescence assay, which were produced by our research group by the method described previously (hu et al. 2007 ). bound primary antibody was detected with fluorescein-labeled antibody to mouse the 10-day-old spf chicken embryonated eggs were inoculated with ibv h120, rh120, and rh120/scs1. after 48 h, allantoic fluids were collected and subjected to sds-page analysis. the separated proteins were then transferred to a nitrocellulose membrane at 400 ma for 30 min using 25 mm tris-192 mm glycine buffer (ph 8.3) containing 20% methanol. the membrane was blocked with 5% skim milk in pbs (20 mm tris-hcl, ph 7.5, 150 mm nacl) for 1 h and then washed three times with wash buffer . after that, the membrane was incubated with monoclonal antibody 2b3 against ibv m protein (dilution 1:1000) for 1 h. the membrane was again washed three times with wash buffer at room temperature. after that, bound primary antibody was detected with horseradish peroxidaseconjugated goat anti-mouse secondary antibody (dilution 1:5000) (sigma-aldrich, usa). signals were analyzed by enhanced chemiluminescence using the ami600 system (ge healthcare, usa). a total of fifty 7-day-old spf chickens were divided into five groups and housed in different negative pressure isolators. ten chickens in each group were inoculated with 0.3 ml of 10 4.5 eid 50 of ibv h120, rh120, rh120/scs1, and sc021202 via intranasal route. birds in the control group were inoculated with the same volume of sterilized pbs. after every 2 days, oropharyngeal and cloacal swabs were collected and subjected to rt-pcr to check the virus shedding status. the dead chickens were examined by necropsy, and the tissues displaying gross lesions were collected. meanwhile, one chicken in the groups without mortality was randomly selected for dissection and observation of gross lesions in tissues. the tissues of trachea and kidney were sent to wuhan servicebio technology co., ltd. (hangzhou, china) for processing hematoxylin and eosin (h&e) staining, and pathogenic lesion was observed under the microscope. a total of forty 7-day-old spf chicks were divided into four groups and 10 chickens in every group were immunized with 0.3 ml of 10 4.5 eid 50 of ibv h120, rh120, and rh120/scs1 by intranasal inoculation, and chickens in the control group were inoculated with the same volume of sterilized pbs. after 7 and 14 days post-immunization, serum was collected and the antibody against ibv was detected by elisa. then, the chickens were challenged with 0.3 ml of 10 5.85 eid 50 of ibv sc021202 strain. clinical signs and mortality of chickens after the challenge were recorded every day. elisa ibv n recombinant protein (1 μg/well) in 0.01 m pbs (ph 7.4) was coated on a 96-well microtiter plates (canada jet biochemicals int'l. inc.) at 4°c overnight, followed by blocking with 200 μl blocking buffer for 2 h at 37°c. the plate was washed three times with pbst and then incubated with 100 μl chicken serum samples diluted in blocking buffer (1:500) for 1 h at 37°c. after washing three times with pbst, plates were incubated with 100 μl hrp-conjugated goat antichicken igg (kpl, usa) in blocking buffer for 1 h at 37°c. after washing three times with pbst, the colorimetric reaction was developed after incubating the plates with 100 μl chromogenic substrate for 10 min at 37°c. color development was stopped with 50 μl 2 m h 2 so 4 , and an optical density at 450 nm (od 450 nm) was recorded using elx800 universal microplate reader (bio-tek instruments, inc., usa). the full-length cdna of ibv h120 strain was constructed according to the procedure shown in fig. 1a . four fragments (f1, f2, f3, and f4) covering the whole genome were amplified by rt-pcr with the total rna of ibv h120 as template (fig. 2a) . after the addition of cmv promoter to the 5′-terminal of f1 fragment and hb sequence to the 3′-terminal of f4 fragment, all four fragments were individually cloned into pbac vectors resulting into the generation of four fragment subcloning vectors. afterwards, those fragments were fused together by homologous recombination. finally, the fulllength cdna of ibv h120 in pbac vector (pbac-ibv-h120fl) was obtained and confirmed by re digestion, which showed that the full length of pbac-ibv-h120fl was digested into two major bands with expected molecular size of around 24,741 and 7507 bp by xhoi and bamhi (fig. 2b) . the full-length cdna was further confirmed by sequencing (data no shown). additionally, we made another full genome construct in which s1 gene of ibv h120 was replaced with that of strain sc020102 and termed as pbac-ibv-h120/scs1. strategy for the clone construction has been shown in fig. 1b . the recombinant plasmid pbac-ibv-h120/scs1 was also confirmed by re digestion with similar results to that of pbac-ibv-h120fl (fig. 2b) . the full-length cdna was also further confirmed by sequencing (data no shown). generation and characterization of rescued virus rh120 and rh120/scs1 pbac-ibv-h120 and pbac-ibv-h120/scs1 plasmids were first transfected into bhk-21 cells to obtain the p0 generation of rescued virus rh120 and rh120/scs1. this p0 virus was passaged in spf chicken embryonated eggs for three times. the p3 generation of the rescued viruses caused typical dwarf embryo lesions (data not shown), and ibv-specific sequences were also amplified using the primers rm-f and rm-r (table 1) with the cdna of the p3 generation of the rescued viruses (fig. 3a) . the mutant oligonucleotide site that served as rescue virus marker (ggatcc → ggctcc) was also confirmed to be in the correct position (data not shown). the s1 gene of rh120/scs1 was also amplified and sequenced, which was confirmed to be the s1 gene of sc021202 (data not shown), indicating that the s1 gene of h120 was successfully replaced with that of a heterogeneous ibv strain in a chimeric rescued virus. as results shown in fig. 3b and fig. 3c , ibv viral protein could be detected in rh120 and rh120/scs1 infected cek cells by western blot analysis and ifa. these results indicated that recombinant virus rh120 and rh120/ scs1 were successfully rescued. subsequently, the rescued rh120 and rh120/scs1 were continuously passaged in chicken embryonated eggs for 8 generations. s1 gene fragment of each passage was sequenced and analyzed. changes in the s1 gene sequence were not found in the rescued viruses, which indicated that the s1 genes of rh120 and rh120/scs1 were stable during the repeated passages. the mutant oligonucleotide site (ggatcc → ggctcc) which served as a rescue virus marker was also present in all generations (data not shown). pathogenicity and immunoprotection of chimeric vaccine strain rh120/scs1 to examine the pathogenicity of ibv h120 strain carrying s1 gene of virulent isolate, same doses of h120, rh120, rh120/ scs1, and sc0201202 were inoculated into the 7-day-old spf chickens. chickens inoculated with sterilized pbs were used as negative control. results of rt-pcr indicated that the chickens infected with h120, rh120, and rh120/scs1 showed persistent shedding of virus until 13 dpi (fig. 4a , table 2 ). clinical signs, such as respiratory distress, were only observed in sc021202 group at 3-7 dpi, and not in h120, rh120, and rh120/scs1 groups. fatality occurred only in the sc021202 infection group with mortality rate of 30% (fig. 4b) . kidneys of dead chickens showed pale discoloration and were swollen with white urate deposits, showing a typical "spotted kidney"-like lesion (fig. 4c) . mortality and severe gross pathology in kidneys were not observed in h120, rh120, and rh120/scs1 infection groups as well as the negative control group ( fig. 4b and 4c ). severe histopathological changes were observed only in the kidneys and tracheas of chickens infected with strain sc021202 (fig. 4d) . the kidney section from dead chickens in the sc021202 group showed enlarged renal tubules with epithelial cell exfoliation inside the tubular lumen, necrosis of some epithelial cells, and increased exudate with inflammatory cell infiltration (fig. 4d) . in the tracheal section from dead chicken in the sc021202 group, we observed that the epithelial cells of the mucous layer were exfoliated, degenerated, and necrosed (fig. 4d) . no obvious histopathological changes were observed in h120, rh120, and rh120/scs1 groups (fig. 4d) . in order to find out whether the chimeric virus could provide immunoprotection against the virulent ibv isolate, the chickens infected with h120, rh120, and rh120/scs1 14 dpi were challenged with ibv strain sc021202. the results showed that all the chickens survived except those in the control group (fig. 4e) . the antisera collected from h120, rh120, and rh120/scs1 immunized chickens at 7 and 14 dpi were analyzed by elisa. as result showed in fig. 4f , chickens immunized by h120, rh120, and rh120/scs1 could produce antibody against ibv at 7 and 14 dpi but not in pbs control group (fig. 4f ). compared with other avian respiratory viruses, such as avian influenza virus and newcastle disease virus, it is difficult to manipulate ibv genome by reverse genetics due to its largesized genomic rna (lai 2000) and also the availability of very few permissible cell lines for ibv infection (ferreira et al. 2003) . ligation of restriction enzyme (re)-digested fragments is a method to obtain the full-length cdna of ibv genome for rescue of ibv (casais et al. 2001 ). however, the procedure for ligation of large dna fragments to form the full-length genomic cdna is complicated, timeconsuming, and inefficient. on the other hand, the full length of ibv genome is long and selection of the suitable re for ligation of the full-length cdna of ibv genome is difficult. hence, there is a need to establish an efficient reverse genetic system to rescue ibv in vitro. bac reverse genetic system is a common in vitro method to rescue coronavirus, such as tgev (almazan et al. 2000) , sars-cov (almazan et al. 2006) , fipv (balint et al. 2012) , and mers-cov (scobey et al. 2013) . ligation of dna fragments by homologous recombination can be avoided to select many re enzyme sites. but, so far, no bac vector combined with homologous recombination has been used for the construction of ibv full-length cdna. in this study, four dna fragments of around 7000-8000 bp covering full-length genome of ibv were separately amplified and cloned into the low-copy vector pbelobac11 by homologous recombination. the necessary component sample type 1 dpi 3 dpi 5 dpi 7 dpi 9 dpi 11 dpi 13 dpi h120 oropharyngeal 4/10 7/10 9/10 6/7 6/7 6/7 5/7 cloacal 5/10 5/10 3/10 6/7 6/7 4/7 3/7 rh120 oropharyngeal 10/10 6/10 6/10 4/7 4/7 7/7 6/7 cloacal 3/10 2/10 3/10 3/7 5/7 5/7 4/7 rh120/scs1 oropharyngeal 8/10 9/10 10/10 4/7 4/7 7/7 5/7 cloacal 3/10 4/10 7/10 2/7 5/7 3/7 6/7 sc021202 oropharyngeal 4/10 10/10 a 10/10 a 7/7 a 7/7 5/7 6/7 cloacal 2/10 10/10 a 7/10 a 5/8 a 3/7 4/7 4/7 negative control oropharyngeal 0/10 0/10 0/10 0/7 0/7 0/7 0/7 cloacal 0/10 0/10 0/10 0/7 0/7 0/7 0/7 a obvious breathing distress was observed sequences of 5′-utr, 3′-utr, promoter sequence from cmv as well as hdvr and bgh were added to the 5′ terminal of the first fragment and the 3′ terminal of the last fragment by the same way. added hdvr at the end of ibv genome will increase the efficiency of rescue of ibv infectious clones by maintaining the integrity of the 3′ terminal of ibv genome. using the same strategy, two fragments, each were again fused together into the subcloning vector, and finally, both (f) detection of antibodies to ibv n protein of antisera collected one week and two weeks after immunization from ibv h120, rh120, and rh120/scs1 immunized chicken by elisa vectors each carrying two fragments were fused into a fulllength cloning vector. construction of subcloning vector by homologous recombination simplifies the operation and improves the efficiency of the ligation. in this reverse genetic system, site mutation, sequence deletion, and insertion can easily be performed based on certain sequence sites in the backbone of subcloning vector. with the reverse genetics established in this study, recombinant h120 (rh120) strain and its chimeric virus rh120/ scs1 carrying s1 gene of a virulent ibv sc021202 strain were rescued successfully. the pathogenicity of rh120/ scs1 was found to be similar to those of h120 and rh120 and was not able to cause pathological changes as shown in the case of strain sc021202-infected chickens. previous reports showed that s1 protein of ibv was responsible for pathogenicity, tropism, and antigenicity (hulswit et al. 2016; wickramasinghe et al. 2011) . however, our result indicates that only the s1 gene of sc021202 isolate is not a determinant of the pathogenicity of ibv since rh120/scs1 with h120 as the backbone and chimeric sc021202 s1 gene did not cause obvious sickness to the sensitive spf chickens as caused by the sc021202 strain. the results of this study are in agreement with previous studies showing that the s1 gene has little effect on pathogenicity (armesto et al. 2011; hodgson et al. 2004; jiang et al. 2020) . whether the s1 gene is the determinant of the pathogenicity of ibv might depend on individual isolate, which needs further study to confirm. finally, the immunization of h120, rh120, and rh120/ scs1 could protect the chicken against the challenge by ibv strain sc021202, which indicated that the recombinant clones constructed for ibv in this study may be useful for the development of novel ibv vaccine. in summary, in this study, with homologous recombination technology, an efficient reverse genetic system based on bac vectors for rescue of ibv infectious clone was established. this will provide a useful tool for further study of the pathogenicity-related genes of ibv and the development of vaccine against the virus. author contributions jyz, ml, and tts designed the experiments. cfl, tts, and ppz performed the experiments. cfl and tts analyzed the data. xp, ssc, and yy prepared the reagents. cfl, ml, and nko drafted the manuscript. all authors read and approved the final manuscript. conflict of interest the authors declare that they have no competing interests. ethical statement the animal experiment was approved by the committee on the ethics of animal of zhejiang university (zju20170667) and implemented in accordance with the animal care and ethics guidelines. this article does not contain any studies with human participants performed by any of the authors. engineering the largest rna virus genome as an infectious bacterial artificial chromosome construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype molecular characterization of feline infectious peritonitis virus strain df-2 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nephropathogenic ibyz strain remains attenuated but induces protective immunity the making of infectious viral rna-no size limit in sight reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus a reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted rna recombination binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication characterization of an avian infectious bronchitis virus isolated in china from chickens with nephritis establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-306976-p2521bl4 authors: gao, mengying; wang, qiuling; zhao, wenjun; chen, yuqiu; zhang, tingting; han, zongxi; xu, qianqian; kong, xiangang; liu, shengwang title: serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china date: 2016-08-15 journal: vet microbiol doi: 10.1016/j.vetmic.2016.05.018 sha: doc_id: 306976 cord_uid: p2521bl4 since 2009, strains of the naturally recombinant tw i genotype of infectious bronchitis virus (ibv) have caused considerable damage to the chinese poultry industry. to better understand the antigenicity and pathogenesis of this genotype, the characteristics of the ck/ch/ldl/140520 strain were compared to those of four commercial ib vaccine strains that are used commonly in china, as well as four attenuated viruses that represent two types of ibv strains, which are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world. the results showed that all eight strains were genetically and serotypically different from the strain ck/ch/ldl/140520. furthermore, neither the vaccine strains nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the ck/ch/ldl/140520 strain, indicating that it is necessary to develop new live vaccines or to evaluate the use of established vaccines in combination to control naturally recombinant tw i-type ibv strains in the future. our results showed that strain ck/ch/ldl/140520 is very pathogenic, and that it is able to cause cystic oviducts in a high percentage of birds, as well as mortality due to nephritis and respiratory distress with complete tracheal ciliostasis, especially in chickens infected at 1 day of age. being a coronavirus and, therefore, a single-stranded rna virus, infectious bronchitis virus (ibv) has an enormous capacity to change by spontaneous mutation and genetic recombination. it is believed that spontaneous mutations and genetic recombination can occur randomly in the ibv genome; however, if these events occur in the spike (s) gene, especially in its hypervariable regions, these events are most likely to result in the emergence of many different antigenic or genotypic types, which are commonly referred to as variants. therefore, ibv is ubiquitous in most parts of the world where poultry are reared, and it is able to spread very rapidly in non-protected chicken flocks, leading to heavy economic losses in poultry industries (cavanagh and gelb, 2008) . in general, while many new variants are unable to replicate or survive for a long time, a few variants that are of economic importance have emerged worldwide or in restricted geographic areas. therefore, effective surveillance, which is primarily based on the isolation and identification of the virus type causing disease, is of great importance. classification or typing of ibv strains is very important for implementing control measures, research purposes, and understanding the epidemiology and evolution of ibvs. thus far, two major groups of classification systems, including functional tests that examine the biological functions of a virus (immunotypes, protectotypes, and serotypes) and non-functional tests that assess the viral genome (genotypes), are commonly used for ibv typing (de wit, 2000) . for a specific ibv strain, although evidence from some studies suggests that there is a high correlation between the genotype and serotype, other studies have presented conflicting data (de wit, 2000; zhang et al., 2015; chen et al., 2015) , which may lead to contradictory results. the disadvantages of each system are that they only analyze one or several characteristics of a virus strain. hence, data from only one system has to be interpreted with caution, while a more objective and accurate conclusion can be drawn by completely analyzing the results from different systems, although it is suggested that the preferred typing system usually depends on the goal (e.g., selection of vaccination programs or epidemiological studies), available techniques, experience, and costs (de wit, 2000) . a large number of ibv genotypes and variants have been isolated in china in recent years (han et al., 2011) , among which two genotypes, the lx4 and ck/ch/ldl/97i types (also known as qx-and q1-like, respectively), were first isolated in china and subsequently have become widespread worldwide (valastro et al., 2016) . among these ibv genotypes, the tw1 type was first isolated in 1992 in taiwan, and it was considered to have a different genotype than all of the other ibvs (wang and tsai, 1996) . the majority of tw1 ibvs isolated from taiwan are nephropathogenic, and they have mortality rates ranging from 10 to 60% in 1-day-old specific-pathogen-free (spf) chickens (wang and tsai, 1996) . however, the naturally recombinant tw i (nrtw i) genotype was first isolated in 2009 in china, and it was thought to have originated from a natural recombination between lx4 and tw1 viruses (xu et al., 2016) . despite a previous study that characterized the genetic characteristics of an nrtw i type virus and its nephropathogenicity in 7-day-old spf chickens (xu et al., 2016) , there is no further information on this important ibv variant. to better understand the nrtw i type, a series of experiments was performed to investigate its antigenicity and pathogenicity in the oviducts of spf layers, and to evaluate the protection provided by commercial vaccines and attenuated viruses. nine ibv strains, including four vaccine strains (h120, ldt3-a, 4/91, and connecticut (conn)), four attenuated strains (ck/ch/ldl/ 091022 [ldl/091022], ck/ch/lsd/120720 [lsd/120720], ck/ch/ lgx/100508 [lgx/100508], and ck/ch/ldl/97i [ldl/97i]) and the ck/ch/ldl/140520 (nrtw i) strain (xu et al., 2016) , were used in this study. the ibv strains ldl/091022 , lsd/ 120720, and lgx/100508 (chen et al., 2015) were isolated in china and belong to the lx4 type. these three strains were attenuated by serially passaging them 120 (p120), 90 (p90), and 70 (p70) times, respectively, in 10-day-old spf chicken eggs. the ldl/091022 p120, lsd/120720 p90, and lgx/100508 p70 strains were shown to be fully attenuated in spf chickens (liu et al., 2009a) . furthermore, vaccination with attenuated viruses provided complete protection against their virulent parental viruses (data not shown). the ldl/ 97i strain was also passaged 115 times (p115) in spf chicken eggs (liu et al., 2009b) . each of the virus stocks was prepared in 10-dayold embryonated spf chicken eggs by the allantoic route of inoculation, and the infectious allantoic fluid was collected 48 h post-inoculation as previously described (liu et al., 2009a) . the titers of the viruses were determined as previously described (chen et al., 2015) , and the median embryo infectious dose (eid 50 ) was calculated using the method of reed and muench (1938) . white leghorn spf layer chickens and fertile spf chicken eggs were obtained from the harbin veterinary research institute. the birds were maintained in isolators with negative pressure, and food and water were provided ad libitum. all experimental procedures were approved by the ethical and animal welfare committee of heilongjiang province, china. the s1 genes of strains lsd/120720 p90 and lgx/100508 p70 were amplified and sequenced as previously described . the sequences have been submitted to genbank, and they have been assigned accession numbers kt957547 and kt957548, respectively. the s1 gene sequences of the remaining eight viruses, including the h120, ldt3-a, 4/91, conn vaccine, ldl/091022 p120, and ldl/97i p115, nrtw i and the tw i-type tw2575/98 strains, were selected from the genbank database and used for s1 gene comparison. both the sequences of amino acid and nucleotide were assembled and the similarities were calculated using the clustal w method available in the bioedit software package (version 7.0.3.0., available at: http://www.mbio.ncsu.edu/bioedit/bioedit). in addition, 14 other reference strains in which the s1 subunit sequences were available in genbank (www.ncbi.nlm.nih.gov/genbank/) were also selected for a phylogenetic analysis. the characteristics of the reference viruses are listed in table 1 . a phylogenetic tree based on the s1 gene was constructed from aligned amino acid sequences by the neighbor-joining method with 1000 bootstraps using the mega4 program (tamura et al., 2007) . four ibv vaccine strains (ldt3-a, 4/91, h120 and conn), the attenuated ldl/97i p115 and nrtw i were used for virus crossneutralization test in this study. because strains ldl/091022 p120, lsd/120720 p90 and lgx/100508 p70 belong to the same genotype (lx4-type or qx-like), hence, only the strains ldl/091022 p120 and lsd/120720 p90 were used. in addition, the ibv strain ck/ch/lsc/ 99i, which represents another important genotype in china (han et al., 2011) , was also used for virus cross-neutralization test in this study. sera against these ibv strains were prepared as previously described (guo et al., 2014) . briefly, 20-day-old spf chickens were inoculated by a combined intraocular and intranasal route using a total dose of 10 6 eid 50 of each virus per bird, respectively. after 2 weeks, a booster dose of each virus with 10 6 eid 50 was administered by intravenous inoculation to the bird. birds were exsanguinated 1 week after the last inoculation and the separated sera from chickens inoculated with the same virus were pooled. all sera were inactivated at 56 c for 30 min and stored in 2.0 ml aliquots at à80 c until required. for virus neutralization, sera were serially diluted two-fold with sterile phosphate-buffered saline (pbs) and mixed with 200 eid 50 of the ibv strains. after incubation for 1 h at 37 c, virus-serum mixtures were inoculated into the allantoic cavity of spf chicken embryos, which were observed for 7 d. the end-point titer of each serum sample was calculated using the method of reed and muench (1938) . fifty-five 1-day-old spf layer chickens were separated into four groups, and they were provided with food and water ad libitum. groups 1-3 included 15 birds, and they were challenged with the nrtw i strain when they were 1, 15, and 30 days old, respectively. the challenge strain (10 6 eid 50 in 0.1 ml of diluent per bird) was applied by the intranasal and ocular routes. group 4, which served as a negative control, included 10 birds that were not challenged. the chicks were examined daily for clinical signs of infection, such as tracheal rales, nasal discharge, watery eyes, and wheezing. the clinical signs from all of the birds in each group were counted by three people over a 2-min period. morbidity and mortality were recorded daily. gross lesions were also carefully examined from the dead chickens, and the kidneys of the dead chickens were subjected to immunohistochemistry (ihc) using monoclonal antibody 6d10 , which is directed against the nucleoprotein, as previously described (de wit et al., 2011; xu et al., 2016) . blood samples were collected on 4, 8, 12, 16, 20, and 24 d post-challenge (dpc) from all birds, and they were examined for the presence of antibodies against ibv using a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx, portland, me, usa) according to the manufacturer's instructions. at 4 months of age, the birds were killed humanely using carbon dioxide/oxygen, followed by exsanguination. a post-mortem examination was performed, and special attention was paid to abnormalities in the oviducts and kidneys. meanwhile, samples from the kidney were collected to detect the presence of ibv by ihc. ami no acid substit uti ons (x100 1 . phylogenetic trees constructed from the nucleotide sequences of the s1 subunit gene of the four vaccine strains, four attenuated viruses, and 14 reference ibv strains. the trees were computed using the neighbor-joining method. the significance of the tree topology was assessed by 1000 bootstrapping calculations. genbank accession numbers are indicated in table 1 . one hundred 1-day-old spf chickens were separated into 10 experimental groups, each containing 10 birds. birds in groups 1-8 were vaccinated with 10 4 eid 50 of the h120, ldt3-a, 4/91, and conn vaccines strains, and the ldl/091022 p120, lsd/120720 p90, lgx/100508 p70, and ldl/97i p115 attenuated strains, respectively, in a total of 0.1 ml of pbs per bird, by the intranasal and ocular routes. birds in groups 9 and 10 received 100 ml of sterile pbs via the same routes. blood samples were collected at 4, 8, 12, 16, and 20 d post-vaccination (dpv) . at 20 dpv, birds in groups 1-9 were challenged with 10 6 eid 50 of the virulent nrtw i strain, in a total of 100 ml of pbs per bird, by the intranasal and ocular routes. birds in group 10 were not exposed to virus and served as a negative control. nasopharyngeal and blood samples were collected from all of the birds in each group at 4, 8, 12, 16, and 20 dpc. nasopharyngeal swabs were used to recover viruses from 10day-old spf chicken eggs, and viruses were subsequently detected by reverse transcription polymerase chain reaction (rt-pcr) as previously described (liu et al., 2009a) . the serum was stored at à70 c for detecting antibodies against ibv using the aforementioned elisa kit as previously described (liu et al., 2009a) . as illustrated in fig. 1 , the four commercial vaccine strains (h120, ldt3-a, 4/91, and conn), which represent different serotypes (massachusetts (mass), tl/ch/ldt3/03, 793/b, and connecticut, respectively) of ibv, were genetically different from the nrtw i strain, as indicated by an analysis of their s1 genes. of the four attenuated strains, the ldl/091022 p120, lsd/120720 p90, and lgx/100508 p70 strains belonged to the lx4 genotype, while the ldl/97i p115 strain belonged to the ck/ch/ldl/97 genotype. these two genotypes were also genetically different from those of the nrtw i strain and the four vaccine strains. in accordance with these results, strain nrtw i did not share more than 82% nucleotide and amino acid similarities with the s1 genes and s1 proteins of the vaccine and attenuated virus strains in this study (table 2 ). in addition, similar to previous results (xu et al., 2016) , strain nrtw i shared the highest nucleotide (98.5%) and amino acid (97.4%) similarities with the tw i prototype strain tw2575/98. the results of the two-way cross-neutralization tests using the ibv strain nrtw i and the antisera against the four vaccine strains, which represented the mass, tl/ch/ldt3/03, 793/b (4/91), and conn serotypes, showed that the strains belonged to different serotypes ( table 3 ). the results using the nrtw i strain and the antisera against the deduced parental virus types, the ldl/091022 p120, lsd/120720 p90, lgx/100508 p70, and ldl/97i p115 strains, also showed non-detectable levels of cross-neutralization. none of the chickens that were vaccinated with the ldt3-a vaccine strain or the attenuated ldl/091022 and ldl/97i viruses showed clinical signs or mortality when challenged with the ibv strain nrtw i, thereby demonstrating that vaccination with these three viruses provided a high degree of clinical protection. in contrast, respiratory signs (sneezing, nasal discharge, and tracheal rales) developed at 3-4 dpc and lasted until 10 dpc in some of the chickens in the groups that were vaccinated with the h120, 4/91, and conn vaccines, and the attenuated lsd/120720 and lgx/ 100508 viruses when challenged with the nrtw i ibv strain at 20 days of age. in addition, some of the chickens died in these groups, indicating that vaccination with these viruses could not provide complete protection against the nrtw i strain. no birds died during the experiment and no clinical signs were observed in the negative control group. as listed in table 4 , the nrtw i challenge virus was re-isolated from the tracheas of nearly all of the birds in the eight vaccinated groups at 4 dpc, as well as from non-vaccinated birds that were challenged. as expected, none of the chickens in the negative control group tested positive for virus isolation. the serological responses induced by the ibv vaccines and the challenge virus are presented in table 4 . all of the birds in each of the vaccinated groups seroconverted at 20 dpv, as well as at 12 dpc with the nrtw i strain. none of the birds in the negative control group seroconverted during the experiment. table 2 nucleotide and amino acid similarities of the s1 gene and protein among strain nrtw i and the eight vaccine/attenuated strains. a amino acid identity (%) -, not tested. a reciprocal titer. all of the chickens in the 1-and 15-day-old groups that were challenged with the nrtw i strain exhibited the aforementioned respiratory signs at 3-4 dpc, and nine and two chickens, respectively, died at 4-9 dpc. similar to the negative control group, no birds died and no clinical signs were observed in the 30day-old group (table 5) . obvious hyperemia of the tracheal mucosa with catarrhal exudation was observed in all of the dead chickens. the most remarkable lesions were detected in the kidneys of the dead chickens. the affected kidneys were enlarged and pale, and urate deposition was observed in the tubules and ureters ( fig. 2a) . similar to our previous results (xu et al., 2016) , viral antigen was detected by ihc in the kidneys of all of the dead birds. antigen was found in the cytoplasm of the tubular epithelial cells and in the mucous membrane of the ureters and collecting ducts (fig. 2b) . interestingly, neither gross lesions nor ihc-positive cells were observed in the kidneys of the surviving chickens at the end of the experiment. in addition, characteristic dilatation of the oviduct developed in most (4/6) of the birds in the 1-day-old group. in contrast, one and two of the birds in the 15-and 30-day-old groups, respectively, exhibited cystic oviducts ( fig. 3 ; table 5 ). surprisingly, the dilatation and serous fluid accumulation in the oviducts were observed not only in the chickens in the 1-and 15-day-old groups, but also in chickens in the 30-day-old group, although only 2/15 of the birds in the 30-day-old group showed these lesions. as expected, no such lesions were observed in the birds of the negative control group. all of the birds in the 15-and 30-day-old groups seroconverted at 12 and 8 dpc, respectively, with strain nrtw i, which was earlier than that in the 1-day-old group, in which all of the birds seroconverted at 16 days of age (table 5) . we did not observe seroconversion in any chickens of the negative control group. in this study, most ib vaccines, including two types of government-approved vaccines (h120 and ldt3-a) and two types of non-government-approved vaccines (4/91 and conn) that are used commonly in china, as well as the nrtw i type strains, were selected for genotyping, an s1 gene comparison, serotyping, and vaccination-challenge tests. in addition, we also selected four attenuated strains representing two types (the lx4 type strains ldl/091022, lsd/120720, and lgx/100508, and the ck/ch/ldl/97i type strain ldl/97i) of ibv strains that are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world (valastro et al., 2016) . a phylogenetic analysis and an s1 gene comparison showed that the aforementioned six types of viruses are genetically different from each other and from strain nrtw i. this is in accordance with the results of the cross-neutralization tests that showed that the serotype of strain nrtw i differs from those of the selected six serotypes in this study and likely represents a novel serotype. the cross-neutralization tests did not include the tw i prototype strain tw2575/98 from taiwan, china, which was unavailable for the comparison. table 4 results of the vaccination-challenge tests (groups of chickens were vaccinated with ibv vaccine or attenuated strains, and challenged with the nrtw i strain). 10/10 4/10 0/10 0/10 7/10 10/10 10/10 10/10 6/6 6/6 6/6 6/6 10/10 5/6 4/6 2/6 1/6 ldt3-a 0/10 0/10 0/10 2/10 6/10 9/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 8/10 1/10 1/10 0/10 4/91 1/10 1/10 0/10 0/10 2/10 8/10 10/10 10/10 10/10 9/9 9/9 9/9 10/10 9/10 5/9 3/9 0/9 conn 4/10 1/10 0/10 4/10 7/10 10/10 10/10 10/10 9/9 9/9 9/9 9/9 9/10 6/9 4/9 0/9 -ldl/091022 p120 0/10 0/10 0/10 4/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 8/10 10/10 2/10 0/10 -lsd/120720 p90 3/10 2/10 0/10 0/10 3/10 7/10 10/10 10/10 10/10 8/8 8/8 8/8 10/10 8/10 4/8 0/8 -lgx/100508 p70 2/10 2/10 0/10 0/10 3/10 8/10 10/10 10/10 10/10 9/9 8/8 8/8 9/10 3/10 2/9 0/8 -ldl/97i p115 0/10 0/10 0/10 2/10 6/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 8/10 1/10 1/10 0/10 nrtw i d 10/10 7/10 0/10 6/10 10/10 10/10 10/10 0/10 3/3 3/3 3/3 3/3 10/10 3/3 1/3 0/3 -negative control 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 a number of chickens that seroconverted/number inoculated. b two procedures, including virus isolation and subsequent rt-pcr, were used for virus recovery after challenge as described previously (liu et al., 2008) . the results from the two procedures were identical. number of chickens that were positive for virus recovery/number challenged. c days after challenge. d the chickens in this group were not vaccinated with the vaccine or attenuated viruses, but were challenged at 20 days of age with the ibv nrtw i strain. the nrtw i strain was isolated from chickens that had been vaccinated against ibv with the commercial live attenuated h120 vaccine at 1 day of age and then boosted at 14 days of age (xu et al., 2016) , possibly indicating that the vaccine does not provide sufficient protection against a challenge with strain nrtw i. thus, further examination of the protection conferred by commercially available ibv vaccines and attenuated viruses against challenge with the novel serotype nrtw i strain was important for evaluating the extent of protection against distinct antigenic types. comparatively, the vaccine strain ldt3-a and two attenuated viruses, ldl/ 091022 p120 and ldl/97i p115, provided good clinical protection against challenge with the nrtw i strain, although all the strains analyzed in this study have actually percentages of similarity with nrtw i that are of almost the same value (they ranged from 76.1 to 82% of similarity at a nucleotide level, and from 75.3 to 81.4% of similarity at an amino acid level). some antigenic epitopes that have roles in the protection have also been identified on the s2 and n proteins of ibv (ignjatovic and sapats, 2005) and may attribute to the difference of the cross-protection. further studies are needed to better understand the relationship between antigenic epitopes on s (both s1 and s2) and n proteins and the level of protection provided in challenge studies. as illustrated in table 4 , the currently available vaccines and attenuated viruses did not provide sufficient respiratory protection against nrtw i challenge, which is in agreement with the genotyping and serotyping results. the results showed that the challenge virus was shed by some chickens in all of the vaccinated groups for at least 12 dpc. additionally, the high rates of challenge virus isolation (less than 80% at 4 dpc) from chickens that were vaccinated with the heterologous vaccines indicated that none of these vaccines and attenuated viruses provided sufficient protection against the ibv nrtw i strain, especially if a reduction of ibv transmission is considered to be important. these results revealed that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i-type ibv strains in the future. over the past 20 years, nephropathogenic ibv strains have emerged as the most predominant ibv strains in the poultry industry, and they are responsible for many outbreaks of kidney disease on chicken farms (de wit et al., 2011) . generally, the nephropathogenic ibv strains have a tropism for the epithelial cells of the respiratory tract, which leads to clinical signs such as excessive water consumption and wet droppings, followed by a severe renal infection that leads to increased mortality, as shown in the case for the nrtw i strain in our previous study (xu et al., 2016) , which was corroborated in the present study. our results also confirmed that the morbidity and mortality caused by nrtw i infection was age-dependent (cavanagh and gelb, 2008) . it was shown that many of the infections in young chickens caused by ibv strains result in permanent damage to the oviduct (broadfoot et al., 1954 (broadfoot et al., , 1956 , which leads to a significant reduction in egg production and quality upon sexual maturity. the effect of ibv on the oviduct of chickens is extremely variable. there are at least four major factors that influence the severity of oviduct lesions following ibv infection, as well as the outcome regarding the induction of false layers: the ibv strain that is involved, the age at infection, the presence of strain-specific, virus-neutralizing, maternally-derived antibodies at the time of infection, and the early protection induced by vaccinating young chickens (crinion and hofstad, 1972; de wit et al., 2011) . infections with different strains of lx4 type (qx-like) viruses can induce cystic oviducts with water-like fluid accumulation in some chickens that survive the infection, and cystic oviducts were observed at different ages after challenge (benyeda et al., 2009; de wit et al., 2011) , whereas no lesions could be observed in the oviducts after infection with the pathogenic 4/91 strain (benyeda et al., 2009; de wit et al., 2011) . for the mass serotype viruses, it appears that the induction of cystic lesions/false layers differs among different strains (crinion, 1972; jones and jordan, 1972; benyeda et al., 2009 ). these differences are not related to virulence because it was shown that a live attenuated strain, h52, was capable of producing similar pathological changes (duff et al., 1971) . a recent study showed that cystic oviducts were also found in some spf female chicks that were infected with an is/885/00-like virus (awad et al., 2016) , which was first detected in israel (meir et al., 2004) and later found in other parts of the world (awad et al., 2016) . in this study, we found that the nrtw i strain could induce cystic oviducts in spf chickens of different ages within 1 month post-challenge. the nrtw i strain emerged recently in china, and it originated from recombination events between tw i-and lx4-like (qx) viruses (xu et al., 2016) . one of the parental viruses, the tw1 ibv strain, was first isolated in 1992 in taiwan, and it is a nephropathogenic ibv strain; however, it was unknown whether it could induce cystic oviducts (wang and tsai, 1996) . in contrast, it was clearly shown that another parental virus (the lx4 type or qx-like) could induce both nephritis and cystic oviducts in chickens that survived infection (benyeda et al., 2009; de wit et al., 2011) . the nrtw i strain acquired the 3 0 sequences of the nsp16 and s1 genes from a tw i-like virus, and the rest of its genome from an lx4-like virus, although the tissue tropism and pathogenicity determinants of ibv remain unknown (xu et al., 2016) . in addition, our results also confirmed that as they age, chickens become more resistant to oviduct damage caused by nrtw i infections (cavanagh and gelb, 2008) . similar to other reports (benyeda et al., 2009) , our results showed that viral antigens in the kidneys and oviducts of the surviving chickens became undetectable immunohistochemically by the end of the experiment; this is in agreement with our field experience, which showed that ibvs could not be isolated (in most cases) from chickens exhibiting cystic oviducts. in conclusion, these experiments confirmed previous observations that showed that the nrtw i strain has a novel genotype, and that it is a pathogenic ibv strain that can result in high mortality rates by causing nephritis in susceptible birds. our results also showed that nrtw i infection induced age-dependent cystic oviducts. in addition, a cross-neutralization test showed that the nrtw i strain of the nrtw i genotype has a novel serotype that differs from those of the other strains used in this study. a vaccination-challenge test using different heterologous live vaccines and attenuated viruses in 1-day-old chickens did not induce high levels of protection against challenge at 20 days of age, which suggests that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i type ibv strains in future. experimental infection of is/885/00-like infectious bronchitis virus in specific pathogen free and commercial broiler chicks comparison of the pathogenicity of qx-like, m41 and 793/b infectious bronchitis strains from different pathological conditions effects of infectious bronchitis on egg production effects of infectious bronchitis in baby chicks infectious bronchitis molecular and antigenic characteristics of massachusetts genotype infectious bronchitis coronavirus in china pathogenicity of four serotypes of avian infectious bronchitis virus for the oviduct of young chickens of varying ages egg quality and production following infectious bronchitis virus exposure at one day old induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d388 (genotype qx) by maternally derived antibodies and by early vaccination infection of day old chicks with infectious bronchitis (ib) virus and subsequent anatomical abnormalities a comparative study of pigeons and chickens experimentally infected with ppmv-1 to determine antigenic relationships between ppmv-1 and ndv strains a 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china fine level epitope mapping and conservation analysis of two novel linear b-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus persistence of virus in the tissues and development of the oviduct in the fowl following infection at day old with infectious bronchitis virus altered pathogenicity, immunogenicity, tissue tropism and 3 0 -7 kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/97i strain of infectious bronchitis coronavirus characterization of a recombinant coronavirus infectious bronchitis virus with distinct s1 subunits of spike and nucleocapsid genes and a 3 0 untranslated region identification of a novel nephropathogenic infectious bronchitis virus in israel a simple method of estimating fifty percent endpoints mega4: molecular evolutionary genetics analysis (mega) software version 4.0 s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification genetic grouping for the isolates of avian infectious bronchitis virus in taiwan emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks serotype shift of a 793/b genotype infectious bronchitis coronavirus by natural recombination the authors declare that they have no competing interests. key: cord-339235-8xslz4bs authors: boroomand, zahra; jafari, ramezan ali; mayahi, mansour title: molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in ahvaz, southwest iran, based on partial sequences of spike gene date: 2018-09-15 journal: vet res forum doi: 10.30466/vrf.2018.32089 sha: doc_id: 339235 cord_uid: 8xslz4bs infectious bronchitis (ib) is a highly contagious disease involving mostly upper respiratory tract in chickens, leading to significant economic losses in the poultry industry worldwide. one of the major concerns regarding to ib is the emergence of new types of infectious bronchitis viruses (ibvs). the purpose of this study was to identify the ibvs isolated from iranian broiler chickens with respiratory symptoms. twenty-five broiler flocks around ahwaz (southwest of iran) were examined for ibv. the specimens including trachea, lung, liver, kidney, and ceacal tonsil, were collected from diseased birds and inoculated into chicken embryonated eggs. harvested allantoic fluids were subjected to reverse transcription polymerase chain reaction (rt-pcr) using primers in order to amplify spike 1 (s1) gene of ibv. the rt-pcr products of four ibv isolates were sequenced. the results showed that from 25 examined flocks with respiratory disease, 12 flocks (48.00%) were positive for ibv. in phylogenetic analysis, our isolates were closely related to the qx-like viruses such as pcrlab/06/2012 (iran), qx, hc9, hc10, ck/ch/gx/nn11-1, ck/ch/js/yc11-1, ck/ch/js/2010/13, ck/ch/js/2011/2 (china), qx/sgk-21, qx/sgk-11 (iraq) with nucleotide homology up to 99.00%. this study indicates the role of ibvs in the respiratory disorders of broiler flocks located in southwest iran, and also the existence of a variant of ibv, which is distinguishable from the other iranian variants. infectious bronchitis (ib) is an acute contagious viral infection with low mortality and a significant reduction in performance in chicken. the causative agent of ib is infectious bronchitis virus (ibv) which is an enveloped, single-stranded, positive sense and rna virus belonging to the family coronaviridae, the genus gamma coronavirus. the virus consists of three important structural proteins: the nucleocapsid (n), the membrane (m), and the spike (s1 and s2) glycoproteins. the nucleotide sequence of the s1 gene is highly variable, which makes it prone to mutation and emergence of new variants of the virus. for this reason, the molecular classification of ibvs is based on the investigation of the s1 gene. 1 vaccination is one of the best ways of immunization of susceptible birds. however, vaccinated flocks may experience disease involvement because there is almost no cross-protection among serotypes. 2 consequently, the viral strains that exist in a different geographical area must first be identified and their pathogenicity should be determined in order to select an appropriate virus strain for vaccination. in iran, aghakhan et al. identified ibv by virus isolation and serological techniques. this isolate belonged to the mass serotype. 3 in previous studies conducted to identify ibv serotypes in iran, the presence of 4/91 and massachusetts serotypes have been reported. 4, 5 recently, a new isolate of ibv (irfibv32) was identified by boroomand et al. which had 95.00% similarity to 793/b strains. 5 the other ibv serotypes also exist in iran neighboring countries such as dutch strains in pakistan. 6 in ahvaz (southwest iran), ibv vaccines including h120 and 4/91 are used in the vaccination program in broiler chicken flocks. however, ib continues to be responsible for the economic losses of the poultry industry in the region. the purpose of this study was to evaluate the role of ibv in broiler chicken respiratory complexes in ahvaz, and also to study the partial s1 sequences among field isolates of ibvs. sampling. during january 2012 to december 2013, an attempt was made to reveal the role of ibvs in broiler chickens of ahvaz which suffered from severe respiratory distress, including nasal discharge, coughing, wet rale, lacrimation, gasping, and high mortality. twenty five broiler flocks aged 2 -5 weeks old, some of which had a history of live vaccination against ibv were selected from different geographical parts of ahvaz (north, east, south and west). samples including trachea, lung, kidney, liver and cecal tonsil were obtained from dead birds, transferred in cold chain system to the faculty of veterinary medicine, shahid chamran university of ahvaz and stored at -70 ˚c until used. the homogeneous tissue samples were centrifuged at 4 ˚c and 3,000 rpm for 10 min. the supernatant was taken and antibiotics (penicillin, 10,000 iu ml -1 , streptomycin, 10000 μg ml -1 and gentamycin, 50 μg ml -1 , all from sigma, st. louis, usa) were added to prevent the growth of bacteria and fungi. an amount of 0.20 ml of the suspension was inoculated into the allantoic cavity of 9-day-old embryonated chicken eggs. 7 the eggs were incubated at 37 ˚c and after 48 hr, the allantoic fluid was harvested and examined for ibv using reverse-transcriptase polymerase chain reaction (rt-pcr). no bacterial and fungal contamination was observed in specific culture media. all experiments were carried out after approval by the animal committee of shahid chamran university of ahvaz, ahvaz, iran. extraction of rna from the allantoic fluid was carried out using rnx-plus solution (cinnagen, tehran, iran) according to manufacturer's instructions. the isolated rnas were stored at -80 ˚c. the cdna was synthesized using primescript tm rt reagent kit (takara, tokyo, japan) according to manufacturer's instructions. 8 a fragment of the s1 gene (464 bp) was amplified using a pair of specific primers including xce1 (5'-cactggtaatttttcagatgg-3') and xce2 (5'-ctctataaacacccttaca-3'). 9 the pcr reaction and its thermal condition were set up as previously described. 10 the pcr products were electrophoresed on 1.00% agarose gel. characterization of isolated ibvs. the ibv positive allantoic fluids were investigated for detection of influenza and newcastle disease viruses by rt-pcr using specific primers. out of 12 positive samples, four samples from different flocks were found to be infected with ibv alone. for genotyping, pcr products (s1 gene) of these four ibv isolates were sequenced in forward direction by bioneer co. (seoul, south korea) and their nucleotide sequences of their partial s1 gene were compared with each other and with previously reported isolates from iran, neighboring countries, and reference strains of ibv (table 1) in genbank by using nblast, and clustalw2. the phylogenetic relationship was established by http://www.phylogeny.fr/simple_phylogeny.cgi. the phylogenic tree was constructed using the neighbor joining method and mega software (version 4.0; biodesign institute, tempe, usa). the topological stability of the tree was evaluated by 1000 bootstrap replications. the results showed that 12 flocks (48.00%) were positive for ibv (fig. 1) . the nucleotide sequences of four isolates were submitted to the genbank sequence database and were given the accession numbers iribvb: kp751243, iribvc: kp751244, iribve: kp751245 and iribvf: kp751246. a phylogenetic tree (fig. 2) , based on the hypervariable region of s1 gene sequences of four ibv isolates from the present study and other strains of ibv retrieved from genbank, was generated. based on the phylogenetic analysis, these four isolates were clustered with qx-like viruses. the results demonstrated the occurrence of qx-like serotype/genotype in ahvaz, iran. the ibv isolates were closely correlated to pcrlab infectious bronchitis virus is one of the main pathogens of commercial and backyard chickens with several serotypes and genotypes circulating in the world. based on our findings, 12 out of 25 examined flocks (48.00%) were found to be infected with ibv, which show the prominent role of the virus in respiratory involvement of ahvaz broiler flocks. the ibv isolates in the present study were completely different from ibv vaccine strains (h120, ma5 and 4/91) used for vaccination in ahvaz, which indicates the incidence of ib infection despite stringent vaccination. the determination of the ibv genotype is necessary not only to understand the evolution of the virus but also for developing vaccines based on circulating ibv strains in the region. 10 one of the significant features of the ib viruses is the emergence of new variants of the virus throughout the world. therefore, new serotypes and new variants of ibvs are still isolated from chickens, even from vaccinated flocks. 10, 11 the new ibv serotypes should be identified quickly in order to develop an effective vaccination strategy. several years ago, massachusetts serotype was identified in poultry flocks of iran. 4 in recent years, 793b or 4/91 serotype has been identified in iran by several researchers. 5, 4, 12 furthermore, the distribution of different ibv genotypes (different from the vaccine strain; massachusetts) was already reported in iran. [13] [14] [15] phylogenetic analysis showed that isolated ibvs in the present study clustered with qx-like viruses. for the first time, in 1996, qx strain of ibv was identified in china, after which the occurrence of the qx-liked virus was informed and it became one of the most prevalent ibv genotypes in various countries. 16 qx-like ibvs were extended from china, to europe and recently to the south of africa. 17 also, in iraq, this genotype was also identified. 18 the pcrlab/06/2012 (jx477827) strain of ibv was isolated in iran and classified as qx-like viruses. 19 the findings of the present study revealed that qx strains of ibv may be originated from other countries (probably iraq) which has been transmitted to ahwaz. there is little information about the type of ibv spread between the middle east countries. however, borderline migrations of birds is probably an important factor. 20 taken together, this is the first report indicating the circulation of qx-like viruses in ahvaz broiler chickens with respiratory signs. finally, a comprehensive study on the pathogenesis of these ibv isolates is suggested. infectious bronchitis severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus studies on avian viral infections in iran isolation and molecular characterization of infectious bronchitis virus, isolate shiraz 3. ibv, by rt-pcr and restriction enzyme analysis isolation and identification of a new isolate of avian infectious bronchitis virus irfibv32 and study of its pathogenicity detection and seroprevalence of infectious bronchitis virus strains in phylogenetic tree generated based on the hypervariable region of s1 gene sequences of four ibv isolates from the present study and other strains of ibv retrieved from genbank using the neighbor joining method. commercial poultry in pakistan a laboratory manual for the isolation and identification of avian pathogens a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china molecular analysis of the 793/b serotype of infectious bronchitis virus in great britain factors influencing the outcome of infectious bronchitis vaccination and challenge experiments molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia a survey of the prevalence of infectious bronchitis virus type 4/91 in iran epidemiology of avian infectious bronchitis virus genotypes in iran (2010-2014) genotyping of avian infectious bronchitis viruses in iran (2015-2017) reveals domination of is-1494 like virus molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in iran the pathogenesis of a new variant genotype and qx-like infectious bronchitis virus isolated from chickens in thailand circulation of qxlike infectious bronchitis virus in the middle east genotyping of infectious bronchitis viruses from broiler farms in iraq during detection of the chinese genotype of infectious bronchitis virus (qx-type) in iran isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east the authors would like to express their gratitude to the dean for research of shahid chamran university of ahvaz for providing financial support for this work. the author disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by the research grant (31400/02/3/95) provided by shahid chamran university of ahvaz. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord-346629-770qyee8 authors: mase, m.; tsukamoto, k.; imai, k.; yamaguchi, s. title: phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan date: 2004-07-15 journal: arch virol doi: 10.1007/s00705-004-0369-9 sha: doc_id: 346629 cord_uid: 770qyee8 to define the origin and evolution of recent avian infectious bronchitis virus (ibv) in japan, a genetic analysis was performed. by phylogenetic analysis based on the s1 gene including the sequence of the hypervariable regions, ibv isolates in japan were classified into five genetic groups, which included two already-known groups (mass and gray). among them, three major genetic groups were associated with the recent outbreaks of ib in japan. one group is indigenous to japan and could not be placed within the known existing groups in other countries. the remaining two groups, which have emerged recently, are related to isolates in china and taiwan. m. mase et al. post-translational cleavage of two separate polypeptide components, designated s1 and s2 [2] . of these, the s1 glycoprotein is associated with virus attachment and is a major target of the neutralizing antibodies in chickens, so serotypic evolution in ibv is associated primarily with the sequences of the s1 glycoprotein [3, 12] . hence, the molecular characterization of ibv is based mainly on analysis of the s1 gene [16, 25] . classically, ibv strains have been placed into serologically related groups on the basis of virus neutralization tests (vnt). however, vnts are very laborious and time-consuming. in addition, there is considerable evidence that serologic relationships among ibv isolates, determined by in vitro vnts, have not been reflected by the results of in vivo cross-immunity studies [8] . moreover, the antigenic variants emerging from wild-type or vaccine viruses by point mutation or rna recombination cannot be detected by vnts [11] . recently, genetic grouping of ibv on the basis of nucleotide sequences have been applied for virus classification [15, 16] . since anyone can apply the genetic information of viruses deposited into the genbank, genetic comparison with other virus strains for epidemiological analysis can be performed quickly. since the first isolation of ibv was recorded in japan in 1954 [20] , the outbreaks have been ongoing. however, the epidemiological analysis of ibv isolates in japan has not been thorough except for a few strains [17, 18] . the relationships between japanese ibv isolates and foreign ibv isolates have also remained unknown. we were particularly interested to know whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, to define the origin and evolution of recent ibv in japan, we determined the nucleotide sequences of ibv isolated in japan using the reverse transcriptase-polymerase chain reaction (rt-pcr) method coupled with direct sequencing, and analyzed the sequences phylogenetically with viruses isolated in other countries. this information is important for determining strategies to control ib. the ibv isolates employed in this study are listed in table 1 . most of the ibv isolates were obtained from regional laboratories in japan. most of the specimens of ibv were isolated by two or three passages using embryonated specific pathogen-free (spf) eggs. the materials were submitted to our laboratory, and were propagated once in spf eggs. the presence of ibv in the inoculated embryos was initially determined by immunofluorescence assay of allantoic cells using anti-ibv chicken serum and observation of characteristic embryo changes such as dwarfing, stunting or curling, according to the procedures of a previous report [7] . the following commercial attenuated live vaccine strains in japan were also used in this study: c-78, miyazaki, on/74, kita-1, ku and tm86. they were derived from field cases of ib in japan. viral rna was extracted from infected allantoic fluids using a kit (isogen-ls, nippon gene, tokyo, japan). a reverse transcriptase (rt) reaction was carried out with superscript ii (life technologies, gaithersburg, md, u.s.a.) using random 9 mers. the n-terminus of the s1 glycoprotein, which includes [5] : country/region of origin/year of isolation except jp/kh/64 strain. jp, japan important structures such as hypervariable regions (hvr) associated with antigenic properties [3, 12, 13] , was selected for this analysis. the following primers were used: ibv-s1 (forward), 5 agg-aat-ggt-aag-ttr-ctr-gtw-aga-g 3 , and ibv-s2 (reverse), 5 gcg-cag-tac-crt-tra-yaa-aat-aag-c 3 . (the expected product was about 670 base pairs). these were designed based on a comparison and alignment of the genbank sequences of several known ibv strains. pcr products were purified using the qiaquick pcr purification kit (qiagen, hilden, germany) according to the manufacturer's instructions. purified pcr products were used as a template for sequencing on an applied biosystems 373s automated dna sequencer using dye terminator cycle sequencing chemistry (perkin-elmer/applied biosystems, foster city, ca, u.s.a.). purified pcr products were sequenced from both directions. the derived nucleotide sequences were analyzed using genetyx-mac ver. 10 .0 (software development corp., tokyo, japan), and through genbank searches. the phylogenetic analysis with available sequences from genbank was conducted using the clustal [23] , and the tree was constructed by the neighbor-joining (nj) method [21] . the expected sizes of dna fragments were successfully amplified by rt-pcr from all the employed ibv samples with our designed primers. the determination of nucleotide sequences of obtained pcr products revealed the diversity of those lengths (665-692 bp) ( table 2) . by phylogenetic analysis, the ibv isolates in japan used in this study were divided into five genetic groups (fig. 1 ). in particular, recent isolates in japan were assigned to three major genetic groups. among them, one group (jp-i) is indigenous to japan and is not present in other countries. the two other groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. some isolates that were mainly isolated in the 1950s, such as jp/ishida/51 or jp/nerima/53, and two local attenuated vaccine strains, kita-1 and ku, were classified into the mass group. another local attenuated vaccine strain, c-78, was classified into the jp-i group, and miyazaki and tm86 strains were classified into the jp-ii group. only one strain (on/74), which was also a local attenuated vaccine strain, was classified into the gray group. the deduced amino acids from obtained nucleotide sequences of pcr products were aligned with various ibv strains isolated in different geographic regions. the results are shown in fig. 2 . many insertions or deletions of amino acids among ibv strains including the japanese isolates were found in the amplified region. the generated pcr products were employed in restriction endonuclease analysis for the development of a simple and rapid classification of genetic groups. after comparing the obtained sequences in this study, we selected two endonucleases, hae ii and ecor i (takara, tokyo, japan), for this analysis. the restriction profiles fig. 1 . dendrogram of the s1 glycoprotein of infectious bronchitis viruses. amino acids 1-207 (in genbank accession number p11223) of the beaudette strain s1 glycoprotein were used for phylogenetic analysis at the amino acid level. the dendrogram is rooted to murine hepatitis virus ml-10 strain. horizontal distances are proportional to the minimum number of amino acid differences required to join nodes and sequences of the pcr products of the five genetic groups in this study, as revealed by two restriction endonucleases are shown in table 2 and fig. 3 . each japanese genetic group has a specific profile pattern. the other genetic groups represented, such as connecticut, d207, uk/4/91, and ark99, do not have sites for these two enzymes in their reported sequences. hence, the three major genetic groups in japan were easily differentiated from other genetic groups. recently, novel ibv strains have been proliferating abroad, and genetic analysis of these strains have been mainly based on the s1 gene [25] . ibv strains in japan have been considered variants different from foreign ibvs such as massachusetts or connecticut types by vnt or pcr-restriction enzyme fragment length polymorphism based on the s2 gene [9, 17, 18] . still, it remains unknown whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, it was revealed that five genetic groups, which could be differentiated by simple restriction endonuclease analysis, were present in japan. although sequencing is recommended to obtain precise genetic information, restriction endonuclease analysis is simple, easy and convenient for primary characterization in routine diagnosis. among these genetic groups, three major genetic groups were associated with recent outbreaks of ib in japan. a previously published study divided ibv strains into several distinct genetic groupings by analyzing the s1 gene [25] . however, most ibv isolates in japan formed genetic groups distinct from these. among three major genetic groups of ibv in japan, one group (jp-i) has not been found in other countries; this group may be indigenous and has been prevalent in japan for a long time, from at least the 1960s. the other two groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. recent ibv isolates in china and taiwan also form distinct genetic groups [26, 27] . it is unknown whether these genetic groups originated in japan or in these neighboring countries. interestingly, recently isolated newcastle disease viruses in japan were also closely related, genetically, to those isolated in taiwan and china [19] . furthermore, it was recently revealed that the coronavirus isolated from pheasants was genetically closely to ibv [6] . one possibility for the migration of ibv strains into this region is dissemination by some kind of wild birds close to chickens, such as pheasants. the virus neutralization antibodies that form the basis for comparison of ibv isolates are induced largely by the n-terminus in the s1 glycoprotein [3, 13] . in this study, prevalent ibv genetic groups in japan were shown to be completely distinct from other known serotypes from europe and north america by comparing amino acid sequences of the n-terminus in the s1 glycoprotein. on the basis of the relationships between genotypes and serotypes of ibv [25] , it was suggested that the serotypes of recent ibv in japan are novel. further analysis would be required to examine the antigenic property of isolates for establishment of a control strategy for ib outbreaks in japan. on the other hand, the variation of ibv may also be attributed to recombination following co-infection of a few distinct strains [11, 24] . previously, the kb8523 strain isolated in japan [22] was revealed to be genetic recombinant [14] . in japan, in addition to major massachusetts (h120 strain) and connecticut (l2 strain) type live vaccines, local attenuated vaccines isolated from outbreaks of ib in japan have been developed and used. they proved to derive from the genetic groups, mass, gray, jp-i or jp-ii, although the genetic backgrounds of these attenuated vaccines have not been clarified. so far, it has been suggested that the recombination events occur between vaccine strains and field strains [27] . it is unknown whether or not vaccine strains in japan are associated with the emergence of recombinant viruses. to detect recombinant viruses, it would be required to analyze two or more different genetic regions of isolates. further analysis of viruses would clarify whether recombinant variants have become prevalent in japan. all sequences used in this study were sent to ddbj, and their accession numbers areab120628 to ab120658. completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus ibv: structural characterization of the spike protein amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes infectious bronchitis a nomenclature for avian coronavirus isolates and the question of species status coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys use of allantoic cells for the detection of avian infectious bronchitis virus humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus serotypes of avian infectious bronchitis virus isolates from field cases in japan cross-immunity in chickens using seven isolates of avian infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s1 gene a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism typing of recent infectious bronchitis virus isolates causing nephritis in chicken phylogenetic analysis of newcastle disease virus genotypes isolated in japan a virus isolated from infectious bronchitis-like diseases of chickens the neighbor-joining method: a new method for reconstructing phylogenetic trees ibv genotypes in japan cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools evidence of natural recombination within the s1 gene of infectious bronchitis virus evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia author's address: dr. masaji mase, department of infectious diseases, national institute of animal health, 3-1-5 kannondai redaktion: sachsenplatz 4-6, 1201 wien, austria. -satz und umbruch we thank drs. t. imada and n. yuasa, national institute of animal health, japan, for valuable discussions and suggestions. we would like to thank the veterinary officials of miyazaki, akita, mie,yamanashi, nagano, shizuoka, shimane, chiba, toyama, aichi, fukui, okayama, kanagawa, osaka, and ibaraki prefectures for their cooperation in the collection of the viral samples. key: cord-329497-3jow4xbn authors: promkuntod, naruepol title: dynamics of avian coronavirus circulation in commercial and non-commercial birds in asia – a review date: 2015-12-28 journal: vet q doi: 10.1080/01652176.2015.1126868 sha: doc_id: 329497 cord_uid: 3jow4xbn it is essential to understand the latest situation regarding avian coronaviruses (acovs), commonly referred to as the well-known avian infectious bronchitis virus (ibv), given that new and diverse types of ibv are continually being identified worldwide, particularly ones that are isolated from commercial poultry and associated with a wide range of disease conditions. the existing ibvs continue to evolve in various geographic areas in asia, which results in the recombination and co-circulation between ibv types. this makes it increasingly difficult to prevent and control ibv infections, despite routine vaccination. some acovs have also been identified in other avian species and they may pose a threat of cross-transmission to commercial sectors. the present review provides an overview of ibv circulation and the dynamic emergence of new variants found throughout asia via the recombination of ibv strains. in addition to commercial poultry, backyard poultry and free-ranging birds may serve as a ‘hub’ for acov transmission within a particular area. these birds may be capable of spreading viruses, either to areas of close proximity, or to remote places via migration and trade. coronaviruses (covs) are known to be one of the major respiratory pathogens that cause a range of diseases in both human and animal communities. some have led to concerns for a threat to global public health in the twenty-first century, such as the covs causing severe acute respiratory syndrome (sars) (zhong et al. 2003) and those responsible for the recently identified middle east respiratory syndrome (mers) (zaki et al. 2012) . for covs in avian species, avian coronaviruses (acovs) are classified into the genus gammacoronavirus. the infectious bronchitis virus (ibv) is among the most important acovs affecting the poultry industry. ibv was first reported as an avian respiratory pathogen in the 1930s in massachusetts, usa. it exhibits extensive antigenic variation, and the strains present in each country are almost unique. therefore, many genotypes of ibv have been identified worldwide, and new variants keep emerging despite vaccination (reviewed in de wit et al. 2011; jackwood 2012) . vaccines against ibvs are generally effective, but new strains continue to emerge causing clinical diseases and production problems in vaccinated flocks, eventually having an economic impact on the global poultry industry liu et al. 2006a liu et al. , 2006b han et al. 2011) . the pathogenicity of ibv is very complex as it is influenced by many factors, such as the strain of the virus, the breed of chicken, environmental conditions, and concurrent infection from other pathogens. ibv alone may not cause severe and devastating disease, but ibv-infected birds can be susceptible to superinfection by bacteria (for example, escherichia coli), mycoplasma (for example, mycoplasma gallisepticum, m. synoviae) and co-infection with other viruses such as the infectious bursal disease virus (ibdv), newcastle disease virus (ndv), avian influenza virus (aiv), and marek's disease virus (mdv) (bradbury 1984; matthijs et al. 2003; cavanagh & gelb 2008; dwars et al. 2009 ). ibv consists of a single-stranded positive-sense rna genome. its genome encodes four structural proteins: phosphorylated nucleocapsid protein (n), membrane glycoprotein (m), spike glycoprotein (s), and small envelope protein (e) (cavanagh 2007) . the spike glycoprotein s, in the form of club-shaped projections present on the surface of the virus, is posttranslationally cleavable into two subunits: the s1 forming the outer spike portion of the protein and the s2, the protein anchoring it to the viral envelope. the s1 is responsible for attachment to the host cell receptors, while the s2 subunit mediates fusion of the virus and the host cell membrane, thereby entering into the host cells. moreover, the s1 subunit contains epitopes and determinants for serotypespecificity, hemagglutinin activity, and for neutralizing antibodies that provide protective immunity (masters 2006; cavanagh & gelb 2008) . all known different ibv genetic types appear to have little or no overlap to cross-protect each other, causing existing recurrent outbreaks. currently, genotypes based on the sequence of the s1 protein gene, especially in the hypervariable 5 0 region (hvr), are used for classification of ibvs. this genotyping is very useful in the continuous determination of the epidemic genotype of ibv field strains, and for predicting the effectiveness of vaccines against field isolates. in addition, the production of a new generation of vaccines, genetically related to the circulating ibv local strains, is economically beneficial for control of infectious bronchitis (ib) in global geographic regions. this review aims to provide an update on the dynamics of avian coronavirus, particularly focusing on the economically important ibv strains that are circulating in commercial farms in asian countries. the review also identifies the potential risks of ibv spreading in a traditional backyard poultry farming situation, which usually has links to rural communities and contributes to local consumption. the review goes on to discuss the ib situation occurring in species of birds other than poultry. this information draws attention to how possible diverse ibvs (and also other acovs) are persistently circulating around this continent. the collection of ibv isolates in different asian geographic regions is listed in table 1 . this includes country of origin, name and genetic types of the isolates, tissue tropism, common breeds, and age range of ibvinfected birds. genbank accession numbers are included that can document the reference citation of each representative strain. figure 1 illustrates the phylogenetic tree indicating the relationship between the s1 amino acid sequences of the selected strains obtained from the asian ibv isolates listed in table 1 , and a 4/91 strain, have been used extensively in poultry farms for many years. however, these vaccines provided very little protection against chinese isolates. this is because vaccines and field strains belong to different phylogenetic clusters that have larger evolutionary distances, indicating different genotypes (liu et al. 2006a (liu et al. , 2006b han et al. 2011; li et al. 2012; ma et al. 2012 ). the obvious signs of ib in vaccinated commercial broilers were respiratory symptoms. post-mortem findings indicated inflammation of the upper respiratory tract, the kidneys, and the proventriculus. there was a broad range of debilitating symptoms while mortality sometimes occurred when co-infection was present. the most prevalent signs in layer hens included decreased egg production, deformed eggs, and increased mortality. a nephropathogenic lx4 type, one among the predominant chinese variants, was purported to have originated in china in the mid-1980s. importantly, a predominant qx-ibv, a new ibv variant that was classified within the lx4 group, was reported (yudong et al. 1998 ) and spread extensively to other regions in china and also to other countries (reviewed in de wit et al. 2011; jackwood 2012; ma et al. 2012 ). this type of ibv caused severe nephritis, proventricultis, and atrophic oviducts, which resulted in a decrease in egg production (yudong et al. 1998) . in previous years, outbreaks of many ibv strains, such as the mass-type associated nephropathogenic strains, were reported in china. nevertheless, other strains with a partial relationship to the mass-type were also identified (li & yang 2001; yu et al. 2001; bing et al. 2007 ). in addition, virus isolation between 1995 and 2004 indicated that at least seven genotypes were detected, mainly being nephropathogenic strains. however, a number of genotypes were not only indigenous to china, but some had a genetic relationship to those isolated from neighboring countries, for example, korea and taiwan (liu et al. 2006a (liu et al. , 2006b . also the major groups of the more recently isolated ibvs belonged to the chinese types, which differed from both the vaccine and the non-chinese strains . therefore, different ibv strains have been increasingly co-circulating in the poultry population. the geographic distribution of ibvs indicated a wide variety of ibv types present in china. for instance, in southern china, the dominant signs of ib were associated with respiratory distress and nephritis in chickens of different age groups. some isolates were classified into taiwanese-type (tw-i), mass-type, and proventriculitis-type viruses (li et al. 2010; zou et al. 2010; yan et al. 2011; li et al. 2013 ). in addition, ibvs isolated from broilers and broiler breeders at various ages from eastern, southern, south-western, and central parts of china were classified into different genetic groups. these flocks exhibited typical respiratory and nephropathogenic ib symptoms, and experienced pathological changes. nephropathogenic ibvs were mainly a2like (qx-ibv) strains, and they showed evolutionary distance from vaccine strains. among others, hn08, 4/91, gray, and mass-types were also identified. moreover, recombination events were observed between a lx4 and a teal-isolate (tl/ch/ldt3/03i-type), contributing to the emergence of a new variant. interestingly, a recent taiwanese-type (tw-ii) was also detected in mainland china. corresponding investigations were presented, indicating that the chinese strains had also been isolated in taiwan (han et al. 2011; ji et al. 2011; li et al. 2012; luo et al. 2012; ma et al. 2012; li et al. 2013) . predominantly nephropathogenic lx4-type ibvs had apparently replaced the previously prevalent ibvs in china (li et al. 2013) . recently, the divergence of new ibv variants that could be further classified was also demonstrated. for instance, ibv isolates in cluster i and cluster ii had dissimilar amino acid sequences at a different position in the s1 subunit . moreover, a gx-nn09032 isolate was associated with a recombination found in four ibv strains (he et al. 2012) , while yn-type ibv was genetically similar to most of the prevalent chinese strains but displayed more severe pathogenicity than the previously reported ibvs (feng et al. 2012) . another example showed that a ck/ch/lsl/99i-type isolate was recently predominant in southern china. the s1 gene of this isolate had the greatest diversity. positive selections were detected, not only in the s1 gene but also in the m and n genes. recombination with vaccine strains, in particular a 4/91-type, was also detected (mo et al. figure 1 . phylogenetic tree of amino acid sequence of the s1 protein showing diversity of the asian ibv isolates. the tree was constructed by the maximum-likelihood method with bootstrap values calculated from 1000 replications. the amino acid sequences were aligned with clustalw in the mega 6.06 software. 2013). therefore, the trend of ibv local strains isolated in different chinese regions continually changes as novel variants are discovered. consecutive investigations over a 15-year period have so far identified at least nine genetic types and, according to sequence and phylogenetic analyses of the s1 gene (table 1) , a couple of ibv variants currently found in china. thus, it is now clear that various ibv field variants are co-circulating in china and appear to continually evolve. vaccine strains might have an important role in the appearance of new ibv variants via recombination. in addition, ibv evolution is driven by the generation of both genetic diversity and selection. for the effective control of ib, alternative vaccines rather than common mass-type vaccines are needed. the first ibv (tp/64) appeared in taiwan in 1965, and was isolated from layers that suffered from respiratory distress and a drop in egg production (tseng et al. 1996) . ib outbreaks occurred frequently even though a vaccination program was widely used. the most common vaccine used in taiwan was based on a masstype. the s1 phylogeny of the taiwanese isolates demonstrated the following groups: a taiwanese group i (tw-i), a taiwanese group ii (tw-ii), a chinese-type, and a mass-type. for the indigenous groups, the first taiwanese isolate was classified as tw-ii. however, according to the results of recent studies, most of these isolates belonged to tw-i. in addition, some of the remaining isolates appeared to be vaccine strains, including a mass-type ibv field strain and one belonging to a chinese j2 strain. the existence of chinese ibv in taiwan was unusual because of the prohibited importation of poultry products between taiwan and mainland china (huang et al. 2004 ). later, huang and wang (2006) developed attenuated vaccines derived from taiwanese ibvs by the passage of the viruses in embryonated eggs. thereafter, they demonstrated that the attenuation of these ibvs resulted in substitutions between two and six amino acids found in the s1 gene, and a few amino acid substitutions found in the s2 subunit (huang & wang 2007) . this indicated that the s1 gene had undergone a high degree of mutation. furthermore, chen et al. (2009) studied the evidence of an ibv isolated in 2002, which demonstrated that the recombinants of taiwanese ibvs had chimeric ibv genome arrangements originating from parental strains similar to those of taiwan and china. chen et al. (2010) further indicated that one isolate showed evidence of frequent recombination with the china-like strain in the s gene. another isolate demonstrated the genome organization of the china-like strain in the s2 gene, and the h120-like genome fragments within the m protein gene. this would again suggest that ibvs in taiwan undergo genetic recombination and evolution. ibvs were first isolated in japan in the 1950s. since then, outbreaks have been ongoing (nakamura et al. 1954) , although the common vaccines used in japan were mass and conn serotypes. mase et al. (2004) described five different genetic groups of the japanese isolates. these included the well-known mass and gray types, and a unique japanese type that was discovered during the outbreaks. the other two emerged groups were closely related to the chinese and taiwanese isolates. major clinical signs caused by japanese-type ibvs in poultry were associated particularly with respiratory and kidney form of ib. these types included a masstype (respiratory form), a jp-i (respiratory, kidney and reproductive form), a jp-ii (kidney form), and a jp-iii (respiratory and kidney form). later, shieh et al. (2004) demonstrated a new japanese ibv subtype, which had a s1 sequence most similar to those of australian strains, while the n sequence was closely related to those of north american strains. this new japanese variant might have recombined with australian-related and north american-related ibvs. shimazaki et al. (2008) subsequently described a new virulent 4/91 variant. these viruses caused severe respiratory symptoms and weight loss in young chicks. the s1 analysis indicated that the isolates were closely related to spanish and french isolates, suggesting that the 4/91 variants were derived from a foreign field isolate. in addition, some ibv strains had high sequence similarity with that of a 4/91 vaccine strain commonly used in japan, suggesting that vaccine-like viruses were derived from a vaccine (shimazaki et al. 2009 ). earlier, in 2002, japan had introduced the 4/91 ibv vaccine, and was trading a large amount of poultry products from european countries. the introduction of the vaccine, and the trading of poultry products, might have provided the transmission routes for those strains (mase et al. 2008 ). subsequently, mase et al. (2010) reported a novel genotype isolated in 2009, designated as jp-iv. this variant was isolated from commercial layers unable to start laying eggs. neither respiratory form of ib nor increased mortality was observed. the sequence analysis indicated that the isolate shared an ancestor with an ibv isolated in the south of china, namely the tc07-2 strain. so far, overall investigations of circulating ibvs in japan indicate cross-transmission from foreign isolates as well as continual evolution. outbreaks caused by ibvs were first reported in korea in 1986 from a laying flock that exhibited decreased egg production (rhee et al. 1986) . a nephropathogenic strain was then identified in 1990. following this, a wide variety of ibv strains were classified. three clusters were differentiated into non-korean strains, including arkansas (ark), conn, and mass types, whereas the rest of the ibv strains, such as a km91, were unique to korea ). in the beginning, a mass-type ibv was the only vaccine strain used in korea (king 1988) . but, at a later date, vaccine strains based on a mass, a h120, and a km91 were also implemented to control ib. the common korean ibv, isolated between 2001 and 2003 from broilers and broiler breeders, was classified as km91 and ark-type isolates (jang et al. 2007 ). later, lee et al. (2008) studied the genetic groups of ibvs obtained from 2003 to 2006, which indicated that the unique korean (k-i) group could be further divided into the respiratory subgroups k-ia and k-ib. furthermore, the k-ii group was closely related to nephropathogenic variants isolated from china and japan, while the k-iii group was closely related to the enteric ibv isolated from china that caused proventriculitis. from these results, k-ia and k-ii were the most prevalent strains. recently, it was shown that the new clusters of korean ibvs had recombination events of the s1 gene with putative parental strains originating from km91-like or qx-like subgroups (lim et al. 2011 ). more recently, mo et al. (2013) identified an ibv circulation in korea from the isolates from 1990 to 2011. these findings indicated that new cluster 1 was prevalent between 2009 and 2010, and was then replaced by the predominant qx-like virus in 2011. another recent study by song et al. (2013) also declared the recombination of korean ibv to be between an indigenous korean-type and mass-type vaccines currently used in korea (such as ma5, h52, and h120 vaccines). likewise, a recombination was also predicted between the following: (1) new cluster 1 and qx-like, (2) k-i and h120, and (3) km91 and qx-like. in conclusion, the currently circulating korean ibvs appear to be a respiratory strain k-i, a nephropathogenic strain k-ii with subgroups km91-like and qx-like, an enteric strain, and some other recent strains (for example, k-iii, new cluster 1, and new cluster 2). natural recombinations occur frequently between these heterologous strains and vaccines that were classified into different genetic groups, and may cause the continuous evolution of new ibv variants in korea. extensive poultry farming in thailand and malaysia is industrialized to accommodate the export of poultry products. repeated outbreaks were reported despite the use of vaccines to control ib in commercial poultry. the molecular evolution of ibv strains in malaysia is not evidently known. two malaysian ibv isolates were studied. one, with a history of high mortality and severe kidney lesions, was isolated in 1994 from a broiler farm in perak in the northern part of the malaysian peninsular. ten years later, an isolated virus, which exhibited tracheal and pulmonary congestion, was detected from a layer farm in selangor bordered by perak to the north. the s1 phylogenetic analysis of these two isolates indicated non-m41 strains, but one of them shared about 90% identity with the chinese qx-like ibv. another isolate belonged to a variant strain with lower identity to the known reference ibvs. however, these two isolates shared a common origin based on the spike s2 and n protein genes (zulperi et al. 2009 ). in thailand, the first reported ib outbreak occurred between 1953 and 1954 (chindavanig 1962 ). later, ib was shown to be a disease with important economic consequences for the thai poultry industry. although vaccines, such as conn, h120, ma5, mass, and armidale a3, appeared to be very commonly used in commercial farms, ibv spread all over the country. the most common ibv strains in thailand belonged to a mass-type, but a 4/91-type ibv was also reported (cook et al. 1996) . genetically, the recently isolated thai ibvs were characterized and found to be similar to the qx-like strain, and the indigenous strain was identified according to the hvr of the s1 gene (pohuang et al. 2009 ). most of the isolates were from h120-vaccinated commercial broiler flocks raised in central thailand. these flocks suffered from airway problems. pohuang et al. (2011) isolated and identified the viruses based on a full-length s1 gene analysis. the viruses were, therefore, classified into the following groups: a group unique to thailand group i, a qx-like group ii, and a mass-type group iii. this study also showed the recombination events of the local isolates. the viruses in group i had 5 0 -terminus of the tha001 origin isolated in thailand in the late 1990s, while the 3 0 -terminus belonged to the qx-like group ii. in addition, the viruses in group ii shared the s1 sequence of the chinese qx-ibv, but the rest of the sequence belonged to the chinese strain jx/99/01. further studies are required to determine the circulation and recombination of ibv variants in other parts of the country. in fact, the vaccination program needs to be revised. ibv was reported to be prevalent in india. the most prevalent form was primarily the respiratory form correlated to a mass-type. it is important to note that india used to be free from ibv variant forms. a novel genotype was reported only recently with the emergence of an indian nephropathogenic form of ib (bayry et al. 2005) . this ibv was isolated from the outbreak that caused diseases in one-to two-week-old commercial broilers. diseases resulted in visceral gout and nephritis. clinical signs showed respiratory disorders. grossly, distended ureters filled with uric acid, interstitial nephritis, granular degeneration, vacuolation, and desquamation of tubular epithelium were observed. eventually, by virus isolation and sequencing, sumi et al. (2012) confirmed a novel nephropathogenic ibv that belonged to a 4/91 genotype. for genotyping, this was the first occurrence of a novel ibv isolated in india. in pakistan, m41-type ibv antigens were commonly detected in the lungs and tracheas of broilers and layer hens of various ages. several ibv genotypes from commercial chickens were also tested serologically, confirming the presence of seropositive m41, d274, d1466, and 4/91 strains (ahmed et al. 2007 ). to date, no ibv classification based on genetic analysis has been performed in pakistan. in iran, ib is one of the most important respiratory diseases of broilers. the first isolation of an ibv in iranian poultry was reported in 1994 (aghakhan et al. 1994) . several iranian isolates were identified at a later date, and a 4/91-type appeared to be one of the predominant types (shoushtari et al. 2008 ). mass-type vaccines are the only officially authorized vaccines. in vaccinated flocks, failure of the mass-type vaccination often occurred, either due to the use of unrelated vaccine strains, or due to there being no partial cross protection of vaccines against field isolates (seyfi abad shapouri et al. 2004). boroomand et al. (2012) studied the iranian 4/91-ibv infection in one-day-old commercial broilers, demonstrating that this particular strain was widely distributed in tissue of the respiratory, urogenital, and digestive tracts. however, they observed only mild clinical respiratory signs and depression. the iranian 4/91 isolates were thus unlikely to cause mortality, severe clinical signs, or gross lesions in infected poultry. instead, they replicated virus in some tissues that made birds susceptible to other pathogens because of the failure of the immune system. in addition, asasi et al. (2013) studied acute-phase factors, pro-inflammatory cytokines and serum sialic acids, indicating that these variables increased after inoculation with an iranian ibv. moreover, they did not find many other ibv strains, such as the common mass-type virus (jahantigh et al. 2013). in summary, at present, ibv isolates in iran belong predominantly to the 4/91 type, and ultimately, improvements to the vaccination program will be required. ibv 4/91-type is commonly detected in iraq. three different ibv vaccines (h120, ma5, and attenuated 4/91 strains) were commonly used on poultry farms (mahmood et al. 2011). however, outbreaks have continued to occur repeatedly, which has resulted in high mortality in broiler farms, associated with renal lesions in both vaccinated and non-vaccinated flocks. recently, a new genotype in iraq, designated as the nephropathogenic sul/01/09 genotype, was discovered by sequencing the s1 gene, and was compared with the other known ibvs (mahmood et al. 2011 ). this new genotype was isolated from commercial poultry that suffered from kidney disease, despite regular immunization with 4/91 and ma5 vaccines. this kidney-type ibv was closely related to viruses isolated in israel and egypt. furthermore, amin et al. (2012) demonstrated a qx-like ibv in iraq, isolated from vaccinated broiler breeders that suffered from several clinical signs related to respiratory disease. post-mortem examination revealed purulent inflammation in the bronchi and edema in subcutaneous tissue. all of the animals were immunized with mass-type, h120, and ma5 vaccines. the s1 phylogeny revealed that the viruses showed a high nucleotide identity with those of qx-like chinese strains isolated between 2009 and 2010. however, some isolates had a nucleotide identity that correlated with the unique qxtype virus sequence from the middle east-type isolated in israel in 2004. this indicates co-circulation of two qxlike types in iraq. in israel, the only permitted vaccines are the live attenuated h120, or an inactivated mass-type serotype. since 1995, four ibv genotypes have been identified as follows: a mass-type field isolate, which differed slightly from the vaccine strain h120; two novel variants, variant i and variant ii; and a variant is/720/99 (meir et al. 1998) . later, in 2000, severe outbreaks of a novel ibv variant caused acute renal disease, high mortality, and poor weight gain in vaccinated broilers. the outbreaks resulted in huge economic losses to the israeli broiler industry. this isolated ibv was then identified as a new nephropathogenic variant. the virus shared approximately 70% similarity with the vaccine strain h120. with the h120 protection test, 91% protection to the trachea and only 25% protection to the kidney were observed. in addition to the h120 relationship, this virus was also closely related to the egyptian isolates (meir et al. 2004 ). more recently, gelb et al. (2005) studied the israeli ibv strains, mainly associated with respiratory and renal diseases, isolated between 1996 and 2000 from broilers and layers. four genetic groups were clustered by a restriction fragment length polymorphism (rflp) fragment pattern of the s1 gene as follows: a mass-type, a variant i, a variant ii, and a novel group. these isolated ibvs had a poor level of protection by the h120 vaccine. thus, using homologous viruses as vaccines would ensure the efficacy of ib control. in the past, a mass-type ibv was the only field strain present in jordan, and all commercial flocks were vaccinated against it using the corresponding vaccine. further investigations indicated the presence of ibv field serotypes other than the mass-type. by hemagglutination inhibition (hi) titers, ark, de-075-like, jmk, and mass-like serotypes were shown to be present. similarly, the mass and other serotypes were also classified in other middle-eastern countries (gharaibeh 2007; roussan, haddad, et al. 2008) . moreover, roussan, totanji, et al. (2008) studied genotyping of ibv in jordanian broiler flocks suffering from respiratory disease. they detected specific groups of a mass-type, a 4/91-type, and a d274-type ibv. roussan et al. (2009) subsequently studied commercial broilers, broiler breeders, and layers that were free from respiratory disease, indicating seropositivity for mass, 4/91, and d274 serotypes. ababneh et al. (2012) subsequently reported the ib outbreaks investigated in iraq, jordan, and saudi arabia in which diseases were associated with respiratory signs accompanied by nephritis, watery cysts in the ovary, and swollen oviducts resulting in egg production problems. phylogenetics based on the hvr of the s1 gene referred to a chinese ck/ ch/ldl/97i isolate, indicating its far eastern origins. all of these flocks were vaccinated based on mass and h120 strains, suggesting vaccination failure since less than 80% nucleotides were found to be identical to the vaccine strains. an ibv strain ck/ch/ldl/97i that caused proventriculitis in poultry was first reported in china in 1995 (liu et al. 2009 ). in recent years, this strain has been isolated from other organs of infected birds, such as the trachea. amino acid homology of the s1 gene of this strain was less than 79% compared with those vaccine strains used in jordan (ababneh et al. 2012) . therefore, only homologous protection should be considered to accomplish immunity against this strain. ultimately, the far eastern ibv variants detected in the middle east illustrate that the active spreading of ibvs within and between geographic areas in this region continues to evolve. taken overall, the published data on ibv isolation and characterization in asia suggest that the circulation of ibv strains is not only limited within each particular area, but some strains appear to spread between regions. moreover, major ibv types found in the far eastern countries consist of the following: qx-type and qx-like viruses; new ibv variants derived from recombination events between local, foreign, and vaccine strains; and some indigenous local-type viruses. however, particular qx variants seem to be the predominant asian ibv strains that may well play a crucial role in their spreading throughout asia. in addition, they have also spread to european countries (beato et al. 2005; bochkov et al. 2006; valastro et al. 2010 ). in the middle east, a 4/91-type is the prevalent type. some others (for example, iraq/is720/sul/01/09 and egypt/ is720/beni-seuf/01 isolates) are circulating in northern african countries. in addition, co-circulation of the existing ibv strains, as well as the currently used live vaccines, may also play a major role in the potential recombination of ibvs, making the incidence of new evolving ibv variants highly likely (reviewed in de wit et al. 2011; jackwood 2012). most of the newly emerged ibvs were not significantly genetically identical to vaccine strains used in a particular area, resulting in low protection or even vaccination failure. during the 2004 outbreaks of the highly pathogenic avian influenza subtype h5n1 (hpai h5n1) in southeast asia, disease control measures that covered most of the poultry operations, except for small-holder native poultry farming, were officially implemented by the governments. studies on risk analysis indicated that good sanitization in farming areas provided a lower risk of hpai, whereas backyard farms where owners traded live poultry experienced a higher risk of infections (paul et al. 2011) . in asia, the backyard poultry system is often correlated with the rural community, and greatly contributes to local consumption and agricultural activities. the purpose of backyard poultry farming in rural areas is either small-scale production or a non-commercial operation (free-ranging domesticated birds). traditional poultry trading is often conducted daily or weekly. therefore, flocks may get infected by vehicles or equipment and tools shared between farms. egg collection, transport, and trading activities among poultry farms can spread the disease to other flocks ( figure 2 ). for instance, in cambodia, poultry being moved to live bird markets via poultry traders was a potential 'hub' for the spread of hpai h5n1 (van kerkhove et al. 2009 ). in vietnam, the route from poultry traders to live bird markets was the key to hpai h5n1 transmission (soares-magalhaes 2010). furthermore, study on poultry market chains in china demonstrated that h5n1-infected live poultry markets presented a more significant problem than non-infected markets (martin 2011) . in thailand, although the number of poultry traded in traditional networks is small compared with other production systems, poultry trading continues all year round, and may thus surreptitiously contribute to the spread of any disease. live backyard poultry trading is usually based on the activities of traders who buy chickens from villages and supply urban markets with poultry meat products. in addition, trade always flows from live poultry to slaughterhouses (choprakarn & wongpichet 2007) . studied the movement and trading patterns of backyard poultry in thailand. with regard to the spread of h5n1 to the backyard farming area, they found that the pattern of disease transmission was local spreading outwardly. they also indicated a close relationship between owners' houses and fresh food markets. this relationship needs the attention of authorities to prevent future outbreaks. at the same time, poolkhet, chairatanayuth, thongratsakul, yatbantoong, et al. (2013) specified that the important factors in the spread of avian flu in backyard farming systems are the farmers themselves, neighbors with backyard poultry, and visitors to sport arenas or training fields of fighting poultry. moreover, wiratsudakul et al. (2014) studied the dynamics of backyard poultry flows in traditional trade networks. flows of backyard poultry trade indicated that traders ran businesses only near their villages. the average distance of poultry movement ranged from 4 to 25 kilometers, determining a spatial scale for the risk of h5n1 transmission to be spread through the traditional poultry market. as a result, overlapping poultry supply zones might also enable disease transmission over extensive distances through although the above studies were particularly focused on hpai h5n1 transmission in backyard poultry, this information might be of use to serve as a model for other respiratory infectious disease surveillance and control programs. it is applicable for the control and prevention of avian coronavirus-associated diseases in backyard poultry due to the same mode and route of transmission (oie 2012) . movement activities and poultry trading between farms can cause the spread of diseases throughout the neighborhood and into other areas. thus, the spread of disease could be limited by removing infected individuals, and hence breaking down the bionetwork. consequently, poor backyard farming conditions most likely play an important role in the spread of respiratory diseases. backyard poultry usually show subclinical symptoms rather than obvious clinical presentations. most of them are not vaccinated. promkuntod et al. (2014) reported ibv infection in unvaccinated backyard poultry, showing a range of clinical conditions (table 2) . poor backyard husbandry, therefore, poses a potential risk because farmers pay little attention to biosecurity, thereby generating an unconcern for disease prevention and control. therefore, backyard poultry may potentially play a role in the epidemiology of acov-associated diseases. acovs that cause diseases in poultry are mainly based on ibvs, especially those producing a range of symptoms in commercial and village poultry (cavanagh 2007; cavanagh & gelb 2008) . in recent years, covs have been isolated in other birds, for example, in pheasants and turkeys. however, the acovs found in these birds were genetically distinct from ibvs. furthermore, birds other than galliformes were also shown to be susceptible to acovs, that is, ibvs and ibv-like types (reviewed in cavanagh 2005; chu et al. 2011) . in asia, particularly in china, covs that were antigenically related to ibv (ibv-like) in terms of their genome incorporation and the sequences of the encoded genes were discovered in other galliform bird species such as peafowl, partridges, guinea fowl, and teals. ito et al. (1991) reported covs isolated from the guinea fowl. field observations and infection under laboratory conditions indicated that the virus from guinea fowl had a host range of more than one bird species. besides, it was shown that the viral protein of peafowl covs had more than 99% identity with the commercial ibv h120 vaccines (liu et al. 2005) . this indicated that the viruses recovered from peafowl had originated from vaccinated chickens in the same neighborhood. another acov isolated from peafowl was a mass-type ibv that was experimentally pathogenic to domestic chickens. this suggested that the peafowl might have acted as a natural reservoir for this ibv (sun et al. 2007) (table 2) . among non-galliform birds, the acov isolates from teal in china that were kept near domesticated chickens had a spike protein with 90% identity to a nephropathogenic ibv (liu et al. 2005 ). this isolated virus could also experimentally infect kidneys of domestic chickens, indicating that the teal virus was certainly a nephropathogenic ibv that had cross-transmitted to birds from neighboring farms. moreover, qian et al. (2006) isolated and characterized an acov from pigeons that had pancreatitis. hypothetically, this virus was identified as a novel gammacoronavirus that had a closer genetic relationship to ibv strains (table 2) . overall, these avian species would evidently act as a natural reservoir or carrier of ibv. when birds migrate, these ibv strains could be transported for long distances. in line with this, it was already shown that pathogenic avian influenza viruses could be transmitted via a variety of bird species, including wild birds, domestic captive birds, and free-range village birds (tiensin 2011) (figure 2 ). in summary, ibvs can replicate in galliform species other than chickens and in some non-galliform birds. the viruses isolated from these birds, however, are genetically related, whether they are of ibv origin or they are the common viruses found in these birds. thus, different bird species may potentially transmit viruses to other free-living birds, or to susceptible poultry populations, when they are in contact with each other under any given circumstance. ibvs still provide a major pathogen for the poultry industry in spite of routine vaccination. moreover, ibv circulation in backyard chickens and free-living birds seems to be silently endemic. the widespread distribution of ibvs in asia is due to either intra-regional or inter-regional dynamics. therefore, it is necessary to know which ibv types are circulating in particular farms and in their neighborhoods. finally, it is essential to determine whether available vaccines are expected to be protective; otherwise, new vaccines need to be developed. it is well known that two major forces drive rna virus evolution: mutations due to the high error rates of the viral rna polymerase, and the recombination of the s1 sequences in covs. this generates new ibv strains or genotypes worldwide. in line with this, it has been shown that the re-assortment and adaptation of rna viruses to the hosts that generate the diversity of viruses, for example, has led to various subtypes of pathogenic aiv (nichol et al. 2000; yu et al. 2001; jackwood 2012) . in addition to genetic variation, a complex interplay of factors influencing ibv emergence may also play an important role. these include environmental factors such as ecological change, social movement, and behavioral influences. moreover, global expansion of agriculture, rapid population growth, advances of transportation, illegal trading, inefficient biosecurity, and traditional farming systems that are unaware of ib can be associated with factors playing a role in ibv emergence (nichol et al. 2000; bayry 2013) . in asia, three major poultry production systems exist, comprising mass-produced poultry operations, small-scale poultry production, and backyard poultry farming. strict farming management provides a wellestablished biosecurity, and an adequate vaccination program for commercial poultry. in contrast, if poor husbandry continues to take place among small-holder and backyard poultry producers living in areas of geographic concentration and high housing density, dispersed throughout rural areas, this will greatly augment the spread of ib. traditional poultry farming may also facilitate continuous spreading of ibv within the area, and perhaps to flocks in the surrounding regions. thus, compartmentalization of backyard poultry farming is one of the most successful management procedures to reduce the transmission of diseases to commercial and non-commercial poultry. moreover, resident and migratory free-living birds may harbor ibvs and transmit them to other birds, or they may carry common acovs in nature that can spread to poultry and other bird populations. these birds may also play a critical role in the epidemiology of ib. in summary, this review highlights the overall dynamic emergence of ibvs in commercial and noncommercial poultry, as well as in other avian species living in the villages. factors contributing to co-circulation and generation of new emerging ibv strains are as follows: (1) genetic variation of the ibv as a result of mutations, insertions, and deletions, especially in the s1 genes; (2) recombination between local ibv strains circulating in the poultry flocks; (3) recombination between local strains and vaccine strains used in the field; and (4) interaction of environmental and social factors. therefore, future multidisciplinary approaches require further useful information, focusing on the ongoing genotype evolution, monitoring acov circulation in avian species, and providing risk analyses of small-scale native poultry production. this information could be variously obtained by means of virology, bioinformatics, and epidemiology of acov-associated diseases. presence of infectious bronchitis virus strain ck/ch/ldl/97i in the middle east. isrn vet sci studies on avian viral infections in iran detection and seroprevalence of infectious bronchitis virus strains in commercial poultry 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virus from recent outbreaks in broiler flocks reveals emergence of novel strain in india a massachusetts prototype like coronavirus isolated from wild peafowls is pathogenic to chickens epidemiology and control of avian influenza h5n1 virus in thailand [dissertation isolation and adaptation of infectious bronchitis virus in taiwan from 1993 to 1995 qx-type infectious bronchitis virus in commercial flocks in the uk poultry movement networks in cambodia: implications for surveillance and control of highly pathogenic avian influenza (hpai/h5n1) modeling the dynamics of backyard chicken flows in traditional trade networks in thailand: implications for surveillance and control of avian influenza phylogenetic analysis of s1 gene of infectious bronchitis virus isolates from china molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia isolation and identification of glandular stomach type ibv (qx ibv) in chickens isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiology and cause of severe acute respiratory syndrome people's republic of china genetic analysis revealed lx4 genotype strains of avian infectious bronchitis virus became predominant in recent years in sichuan area sequence and phylogenetic analysis of s1, s2, m, and n genes of infectious bronchitis virus isolates from malaysia all avian coronavirus and relevant documents published all over the world, especially asian contributors, are greatly acknowledged for 'up-to-date' fruitful information. no potential conflict of interest was reported by the author. key: cord-344297-qqohijqi authors: smith, jacqueline; sadeyen, jean-remy; cavanagh, david; kaiser, pete; burt, david w. title: the early immune response to infection of chickens with infectious bronchitis virus (ibv) in susceptible and resistant birds date: 2015-10-09 journal: bmc vet res doi: 10.1186/s12917-015-0575-6 sha: doc_id: 344297 cord_uid: qqohijqi background: infectious bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. this is due to both mortality (either directly provoked by ibv itself or due to subsequent bacterial infection) and lost egg production. the virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease. methods: whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with infectious bronchitis virus (ibv). tracheal tissue was examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. the host innate immune response was evaluated over these 3 days and differences between the susceptible and resistant lines examined. results: genes and biological pathways involved in the early host response to ibv infection were determined andgene expression differences between susceptible and resistant birds were identified. potential candidate genes for resistance to ibv are highlighted. conclusions: the early host response to ibv is analysed and potential candidate genes for disease resistance are identified. these putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to ibv. electronic supplementary material: the online version of this article (doi:10.1186/s12917-015-0575-6) contains supplementary material, which is available to authorized users. infectious bronchitis (ib) is a highly contagious respiratory disease of chickens first described in the usa in the 1930's [1] [2] [3] . clinical signs include: coughing, sneezing, rales and nasal discharge. the disease can also affect the reproductive organs, which leads to a decrease in egg quality and production, thus making it a major cause of economic losses within the poultry industry [4] . the causative virus, infectious bronchitis virus (ibv) is a coronavirus, which is an enveloped virus with a single positive-stranded rna genome, which replicates in the host cell cytoplasm [5] . proteins encoded by ibv include the viral rna polymerase, structural spike proteins, membrane and nucleocapsid and various other regulatory proteins. the spike glycoprotein mediates cell attachment and plays a significant role in host cell specificity [6] . the existence of many different ibv serotypes, which are not cross-protective means that control of ib, is very difficult. mortality is usually fairly low (~5 %), however some strains of the virus can also cause nephritis meaning that, depending on strain, mortality can be greater than 50 % [7, 8] or even up to 80 % with some australian isolates [9] . ibv infection leaves birds more susceptible to colibacillosis [10] and subsequent bacterial infections can also lead to a high level of mortality [11] . currently, attenuated live vaccines are used in broilers and pullets, and killed vaccines are used in layers and breeders [12] . however, virus control is very difficult, as there are only a few vaccine types and many different strains of ibv. the virus also continues to mutate rapidly, generating more virulent strains of the disease [13] [14] [15] . coronaviruses have now also been detected in other avian species such as turkey, duck, goose, pheasant, guinea fowl, teal, pigeon, peafowl and partridge [4] . the extent to which the virus affects the host is highly dependent on the chicken breed [4] and the mhc b locus is known to play a role in susceptibility to the virus [16] . in this study we attempt to identify non-mhc genes, which may be involved in resistance to ibv. no genetic analyses have thus far been undertaken in order to try and do this and no quantitative trait loci or genes associated with resistance have been determined, so far. based on differential gene expression in susceptible and resistant lines of chickens, we identify potential candidate genes for disease resistance towards ibv (virulent m41 strain). building on the previous work by dar et al. [17] and wang et al. [18] we used affymetrix wholegenome chicken microarrays to examine the tracheal gene expression profiles of a line of birds known to be susceptible to ibv infection (line 15i) and a line known to show resistance (line n). we determined the early host response to infection and propose possible candidate genes for involvement in disease resistance towards ibv. understanding how coronaviruses infect the host and identifying genes involved in resistance is important not only for the poultry industry but also has important implications for human health, as diseases such as sars are also caused by coronaviruses [19, 20] . all animal work was conducted according to uk home office guidelines and approved by the roslin institute animal welfare and ethical review body. the lines used in these experiments are an ibv susceptible lineline 15i (inbred white leghorn strain) [21] and an ibv resistant lineline n (non-inbred cornell strain). line 15i was developed at east lansing in the usa in the 1940s [22] and line n at cornell, usa in the 1960s [23] . the lines have since been maintained at the institute for animal health in compton, uk. twoweek-old chicks from each line (15i and n) were separated into two experimental rooms, with ad libitum access to food and water. in one room, 54 birds (27 from each line) were infected with 4 log 10 cid 50 (10 4 cid 50 ) of virulent ibv-m41 strain in a total of 100 μl of 0.2 % bsa in pbs equally by intra nasal and ocular routes. in the other room, 54 control birds (27 from each line) received 100ul pbs via the same route. trachea samples (upper half ) were collected at 2, 3 and 4 days postinfection (9 individual birds from each line at each time point). the trachea of infected and control birds from each line were analysed for viral load using taqman real-time quantitative rt-pcr assays. tissue samples (~30 mg) were stabilized in rnalater (ambion, life technologies, paisley, uk) and disrupted using a bead mill (retsch mm 300, retsch, haan, germany) at 20 hz for 4 min. total rna was prepared using an rneasy kit (qiagen, crawley, uk) extraction method as per the manufacturer's protocol. samples were resuspended in a final volume of 50 μl of rnasefree water. concentrations of the samples were calculated by measuring od 260 and od 280 on a spectrophotometer (nanodrop, thermo scientific, paisley, uk). quality of the rna was checked on a bioanalyser (agilent technologies, south queensferry, uk). an rna integrity number (rin) > 8 proved the integrity of the rna. biotinylated fragmented crna was hybridized to the affymetrix chicken genome array. this array contains comprehensive coverage of 32,773 transcripts corresponding to over 28,000 chicken genes. the chicken genome array also contains 689 probe sets for detecting 684 transcripts from 17 avian viruses. for each experimental group (control and infected birds in each of the two lines at each of 2, 3 and 4 dpi), three biological replicates (3 rna pools from 3 birds) were hybridized. thus, 36 arrays were used in total. hybridization was performed at 45°c for 16 hours in a hybridization oven with constant rotation (60 rpm). the microarrays were then automatically washed and stained with streptavidin-phycoerythrin conjugate (sape; invitrogen, paisley, uk) in a genechip fluidics station (affymetrix, santa clara, ca). fluorescence intensities were scanned with a genearray scanner 3000 (affymetrix, santa clara, ca). the scanned images were inspected and analyzed using established quality control measures. array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm-1128. gene expression data generated from the genechip operating software (gcos) was normalised using the plier (probe logarithmic intensity error) method [24] within the affymetrix expression console software package. this normalised data was then analysed using the limma and farms [25] packages within r in bioconductor [26] . probes with a false discovery rate (fdr) value <0.05 and a fold change ≥1.5 were deemed to be biologically significant. in order to determine which biological pathways are involved in the responses to viral infection, we analysed our differentially-expressed (de) genes using pathway express [27, 28] which uses kegg pathways [29] to pictorially display up/down regulation of genes. (nb. these diagrams are based on the human pathways and so are not completely representative of the chicken pathways). genes differentially expressed during the host response (fdr <0.05) were analysed against a reference background consisting of all genes expressed in the experiment. factors considered by pathway express include the magnitude of a gene's expression change and its position and interactions in any given pathway, thus including an 'impact factor' when calculating statistically significant pathways. anything with a p-value <0.25 is deemed significant when using this software. use of the ingenuity pathway analysis (ipa) program [30] revealed which canonical pathways are being switched on by ibv infection in the host (with benjamini-hochberg multiple testing correction) and allowed us to analyze the gene interaction networks involved in the host response. genes were clustered by similar expression pattern and analysed for enriched go-terms and transcription factor binding sites (tfbs) using expander (v5.2) [31]. normalised expression data from control samples were compared with infected samples to examine the host response to ibv infection. enrichment analysis of particular go terms or tfbs within clusters was done using the tango and prima functions, respectively, within the expander package. taqman real-time quantitative rt-pcr (qrt-pcr) was used to quantify viral rna levels and for confirmation of the microarray results for the mrna levels of selected genes. this was performed on 3 replicate pools of 3 samples (9 birds). primers (sigma) and probe (pe applied biosystems, warrington, uk) ( table 1) were designed using primer express (pe applied biosystems). briefly, the assays were performed using 2 μl of total rna and the taqman fast universal pcr master mix and one-step rt-pcr mastermix reagents (pe applied biosystems) in a 10 μl reaction. amplification and detection of specific products were performed using the applied biosystems 7500 fast real-time pcr system with the following cycle profile: one cycle at 48°c for 30 min and 95°c for 20 sec, followed by 40 cycles at 95°c for 3 sec and 60°c for 30 sec. data are expressed in terms of the cycle threshold (ct) value, normalised for each sample using the ct value of 28s rrna product for the same sample, as well described previously [32] [33] [34] . final results are shown as 40-ct using the normalised value, or as fold-change from uninfected controls. taqman real-time quantitative rt-pcr analysis was used to measure viral load in trachea samples from both control and infected birds from both lines 15i and n. tracheal tissue was chosen for examination in this study as the target of ibv is the epithelial surface of the respiratory tract. viral rna was detected in infected birds, but no significant difference in viral load was detected between lines at any of the days 2, 3 or 4 post infection (fig. 1) . this would indicate that the resistance to the virus seen in line n is due to how the birds respond to the virus once it has entered the body and is not a measure of how the birds can prevent initial infection by the virus itself. when resistance to ibv infection was originally determined in these lines, it was noted that they were equally susceptible to infection, but a variation in outcome was seen. in line n, 33 % of birds showed air sac lesions whereas 73 % of 15i birds presented lesions. mortality was 0 in line n, but 47 % within line 15i birds. it was hypothesized that the different lines were producing different immunological responses upon infection [21] . gene expression differences found in the susceptible 15i line between infected and control birds over days 2, 3 and 4 post infection were analysed, with a view to examining the innate host response to infection by ibv. genes seen to be induced during the host response to infection include c1s, irf1, stat1, mx1, tlr3 and ctss as previously recognised by guo et al. [35] . we also identified ifit5, oasl, sca2, lyg2, isg12-2, ddx60, ifih1, irf7, table s1 . to elucidate which biological pathways are being perturbed during the host response to ibv infection, we analysed our data using pathway express [36] . the resulting pathway diagrams are extremely useful in establishing which gene networks are involved in a particular experimental response. as seen in fig. 2 , genes involved in antigen presentation and the toll-like receptor (tlr) pathway are up-regulated. tlrs identify pathogen associated molecular patterns (pamps) and are crucial to the innate immune system. in this study tlr3 is shown to be induced at 3 dpi. tlr3 recognizes double-stranded rna intermediates produced during viral replication and has previously been shown to be induced in the trachea at this time after ibv infection [37] . another pathway involved is the phosphatidylinositol signalling pathway (table 2) . phosphatidylinositol kinases are known to play an important role in the viral life cycle after infection of the host and pi4kb is known to be exploited by coronaviruses for viral entry. the product of pi4kb catalysis is phosphatidylinositol 4-phosphate (pi4p) and coronavirus entry into the host is mediated by the pi4p lipid microenvironment [38] . genes involved in the complement system are also highlighted as being up-regulated in response to ibv infection. complement-mediated lysis of viruses is an important facet of the host innate immune system and its role in defence against viral infection [39] as reflected in the induction of these genes in this study. use of ingenuity pathway analysis (ipa) software also allowed us to determine which biological systems are active during the host response. up-regulated genes are seen to be part of the canonical biological pathways shown in fig. 3a . biological processes involving pattern recognition receptors and interferon signalling feature heavily. the interferon response is a powerful antiviral mechanism, which has previously been shown to be involved in the host response after ibv infection. a very early induction of ifn-γ has been reported in splenocytes [40] , and in peripheral blood mononuclear cells (pbmcs) and lung leukocytes [41] . ifnb expression has also been reported in trachea between 1 and 2 dpi [42] . we do not see this increase in expression of interferon genes (due to the absence of data earlier than 2 dpi), but we do see the downstream effects, with increased expression of many interferon-induced genes. specific physiological processes activated upon ibv infection can also be seen in fig. 3b . the stimulation of various different immune cells is seen along with the indication of reproductive abnormality, which would reflect the problems seen with egg-laying upon ibv infection. in order to cluster genes seen to be involved in the host response to infectious bronchitis into groups with similar expression profiles and probably sharing similar functions or gene regulatory pathways, we utilised the click algorithm within the expander program [43] . figure 4a shows the expression profile of genes upregulated during the response to virus. the expander program was also used to analyse the gene ontology (go) functional annotations of the genes being differentially expressed. figure 4b shows the biological process terms, which are significantly enriched in the genes responding during the host response to infection. as would be expected, these include terms like 'innate immune response' and 'antigen processing and presentation'. 'nad + adp-ribosyltransferase activity' and 'phosphoinositide binding' are also highlighted. transcription factor binding sites present in de genes which are significantly over-represented were also predicted. figure 4c shows that genes up-regulated during the host response have a high proportion of irf7 and isre binding sites. irf7 is a transcriptional activator, which binds to the interferon-stimulated response element (isre) in ifn promoters and functions as a molecular switch for antiviral activity. analysis of the gene expression differences between infected and control birds across the two lines has provided us with information on how these lines differ in their response to infection. examination of the gene expression profiles in the control birds of the two different lines also allowed us to identify genes, which are inherently different between the susceptible and resistant birds. it can be seen that there are numerous genes, which show large expression differences between the two lines, even before infection. dramatic differences in gene expression of certain genes, including ddt, sri, blb1, hscb, bf1, bf2, suclg2, mx1 and sri, which are more highly expressed in the resistant n line table s2 shows all 1930 de probes) so, it can be seen that these are genes which have inherently different expression levels between susceptible and resistant birds, even before infection occurs. we therefore postulate that some of these genes may play an important role in disease resistance. the potential interactome of ibv has recently been investigated by stable isotope labelling with amino acids in cell culture (silac) coupled to a green fluorescent proteinnanotrap pull-down methodology [44] . host proteins, which bind to the ibv n protein were identified, some of the genes for which, we see as being inherently expressed at higher levels in susceptible birds in this study. these genes include myh9, caprin1, dhx57, hnrnph3, rpl27a, fmr1, c22orf28, hnrpdl, sfrs3, rpl31, npm1 and rpsa. this may therefore be one of the reasons why line 15i is more susceptible to ibv infectionthere are more host proteins to which the virus binds, compared with the resistant line n. upon infection, differences in response are also seen between the two lines. interestingly, apart from cd38 and cd4 at 3 dpi and fkbp5 at 4 dpi, all other differential gene expression between the lines is seen at 2 dpi in this study (additional file 3: table s3 ). cd38 is a glycoprotein found on the surface of many immune cells including cd4+, cd8+, b lymphocytes and natural killer cells and is a marker of cell activation. it functions in cell adhesion, signal transduction and calcium signalling. cd4 is found on the surface of immune cells such as t helper cells, monocytes, macrophages and dendritic cells. it is a membrane glycoprotein which interacts with mhcii antigens. the protein functions to initiate or augment the early phase of t-cell activation. the protein encoded by fkbp5 is a member of the immunophilin protein family, which play a role in immuno-regulation and basic cellular processes involving protein folding and trafficking. early defence by the host is a key mechanism for combatting viral infection, and induction of ifnb and other innate genes in response to ibv infection has been shown to peak around 18-36 hr post infection [42] . in this study, genes more highly expressed (or less down-regulated) in the resistant n line at 2 dpi include a number of collagen genes (col3a1, col1a2, col9a1, col9a2, col6a1 and col4a1) and other genes such as acan, fstl1, comp, eif3a, stat3 and igfbp5. genes seen to be more highly expressed (or less down-regulated) in the susceptible 15i line include rbm39, mafb, nnk2, ccn1, mgat5 and thrap3. one consequence of ibv infection is the production of poor quality, misshapen eggs by infected birds [45] . some of the genes previously identified as being important for the creation of a healthy eggshell are seen to be more highly expressed by the resistant n line birds after infection in this study. these genes include col1a2, creld2, hsp90b1, p4hb and erp29 [46] . for a full list of genes differentially expressed between the two lines in trachea (409 de probes) see additional file 3: table s3 . ipa analysis of genes showing different inherent expression between lines 15i and n shows that the molecular functions of these genes is primarily concerned with their involvement in cell death and cell adhesion (fig. 5) , two processes previously shown to be significant in infected kidneys [47] . when the differential host responses to infection are examined, it is seen that genes involved in proliferation of t-lymphocytes and genes concerned with cell attachment and cytoplasmic organization are more highly expressed in the resistant line n. other processes significantly involved are apoptosis and necrosis (fig. 6a) , which have been previously documented in ibv-infected vero cells by liu et al. [48] . one of the most perturbed biological networks noted in this analysis is that involving genes related to connective tissue disorders and involve many collagen genes. these genes are more highly expressed in susceptible line 15i birds compared to resistant line n birds (fig. 6b) suggesting that ibv infection might cause more disorder of eggshell formation in this line [49] . the production of poor quality eggs by ibv infected birds may, in part be a reflection of the expression of these kinds of gene networks compared to that seen in resistant birds. twenty-one genes were selected for qrt-pcr validation ( table 3) . these genes were chosen based on their involvement in the host response and whether they were differentially expressed between the susceptible and resistant lines (either inherently or during the course of infection). of the 21 genes tested, 19 showed comparable higher expression in response to infection in the resistant than in the susceptible line c inherently higher expression in the resistant line differential expression to that determined by the arrays. however, the results for ifnar2 and igfbp5 were not confirmed (additional file 4: figure s1 ). besides knowing that the mhc b locus has a bearing on disease resistance, the lack of any genetic information or identified qtl meant that we had to rely upon the gene expression differences we saw between susceptible and resistant lines to give us clues as to genes potentially involved in resistance to ibv infection. identifying genes which were expressed at different levels in the two lines of birds highlighted b-locus genes (blb1, bf1, bf2 , b-g) as well as bringing to our attention various other non-mhc genes which, due to their known biology, could be candidates for being involved in resistance to ibv infection (table 4) . mx1, c1s, irf7, tlr3, c1r, ccli7, isg12-2 and ifitm3 are all strongly induced during the host response to ibv infection. they are all innate immune genes which could potentially have a role in determining susceptibility to the virus. mx1 and ifitm3 are already established as anti-viral molecules [50] [51] [52] . cd38, cd4, fkbp5 and stat3 all show a higher level of expression during the host response in the resistant birds compared to that of the susceptible birds, indicating their involvement in the host defence mechanism. cd38 and cd4, with their role as receptors on immune cells, as described above, are obvious candidates, along with fkbp5 as an immune-regulator. stat3 is activated by various cytokines and growth factors and functions in cellular processes such as cell growth and apoptosis. even before infection, many genes are seen to be highly differentially expressed between lines 15i and n. oasl is an interferon-induced molecule known to have anti-viral activity against certain viruses such as hepatitis c virus. ddt is highly homologous to the macrophage migration inhibition factor, mif. we have also shown it to be highly differentially expressed in other chicken lines, which are susceptible or resistant to marek's disease virus [53] . ifnar2 is an obvious candidate prediction, as the interferon response is central to the host's defence against ibv infection. tpd52l1, bcl2l1, faim2 and ciapin1 are all known to be involved in regulation of apoptosis, a process seen to be important during ibv infection. hscb, sri, and suclg2, although not having an obvious potential biological role in disease resistance, are highly differentially expressed between susceptible and resistant lines and should thus be considered as potential candidates. resistance to ibv infection is brought about by the immune response after the virus has entered the host and is not due to prevention of initial viral infection. there is a small initial innate response at 2 dpi, with much more gene expression seen at 3 and 4 dpi. analysis of genes being activated or inhibited upon infection shows that the biological pathways primarily affected during ibv infection include mapk signalling, those involved in the interferon response and those involving pattern recognition receptors. susceptible and resistant lines show a differential host response mostly at 2 dpi. there are also genes which are inherently different between the two lines studied, including many genes, which control the apoptotic potential of the host. these differences seen in gene expression levels, allow us to postulate on many candidate genes for disease resistance. some potential candidates for involvement in disease resistance include genes already known to confer resistance to other viral infections (mhc-b locus genes, mx1, oasl and ifitm3), genes involved in apoptotic processes (tpd52l1, bcl2l1, faim2 and ciapin1) and others which could be potential candidates due to their known biology (e.g. ddt and cd4). array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm-1128. additional file 1: table s1 . gene expression seen during the host response to ibv infection in the trachea of susceptible birds. (xlsx 24 kb) additional file 2: table s2 . gene expression differences found to be inherent between susceptible and resistant lines in the trachea. (xls 386 kb) additional file 3: table s3 . genes found to be differentially expressed between susceptible and resistant lines in response to ibv infection in the trachea. 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research council (bbsrc), as part of grant numbers bb/d013704/1, bb/ d013704/2 and bb/d010705/1. the authors would like to thank alison downing (edinburgh genomics, edinburgh, uk) for excellent technical assistance with the affymetrix microarray experiments. authors' contributions js performed the arrays, analysed the results and wrote the manuscript; dc carried out challenge experiments, j-rs prepared rna, measured viral load and performed qrt-pcr; db and pk conceived and supervised the project and revised the manuscript. all authors read and approved the final manuscript. key: cord-331740-yjt3q9ph authors: jones, r. m.; ellis, r. j.; cox, w. j.; errington, j.; fuller, c.; irvine, r. m.; wakeley, p. r. title: development and validation of rt‐pcr tests for the detection and s1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples date: 2011-04-07 journal: transbound emerg dis doi: 10.1111/j.1865-1682.2011.01222.x sha: doc_id: 331740 cord_uid: yjt3q9ph two tests were developed that allow the detection and genotyping of infectious bronchitis virus (ibv) and other closely related gammacoronaviruses. the first test employs a one‐step, reverse transcription‐polymerase chain reaction (rt‐pcr) assay in which the amplification is monitored in real time using a taqman(®) probe. this real‐time rt‐pcr test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls’ eggs. a total of 323 field samples were tested; 176 samples were positive using the real‐time rt‐pcr method, but only three were positive by virus isolation. sequencing was used to confirm the positive real‐time rt‐pcr results for a subset of samples. the test is suitable for swabs and post‐mortem samples and has been shown to be highly sensitive and specific. the second test, a genotyping method, was developed for identification of the strain of ibv present in field samples based on nucleotide variations within the gene encoding the s1 subunit of the surface spike (s) glycoprotein. this method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of ibv within the uk. the performance of the test was evaluated using laboratory isolates of ibv and field samples. both tests are suitable for use in a high‐throughput diagnostic laboratory. gammacoronaviruses are known to infect many species of birds, including chickens, turkeys, pheasants, greylag geese, mallard ducks and pigeons, often with different tissue tropisms (cavanagh et al., 2001 (cavanagh et al., , 2002 cavanagh, 2005; jonassen et al., 2005) . more specifically, turkey coronavirus (tcov) causes a highly contagious enteric infection, which can lead to mortality and growth retardation (guy, 2000; cavanagh, 2005) , while, in contrast, pheasant coronavirus infection (phcov) typically affects renal and respiratory tissues with consequent disease signs related to those body systems (cavanagh, 2005) . infectious bronchitis virus (ibv), recognized as the prototypic gammacoronavirus, primarily causes disease in chickens that is characterized by upper respiratory tract signs, including nasal discharge, 'snicking', 'râles', watery eyes and lethargy (ignjatovic and sapats, 2000; cavanagh, 2007) . infectious bronchitis virus is of huge economic importance to the poultry industry worldwide, and the economic impact of disease is exacerbated in part by the existence of multiple serotypes of the virus, a property which complicates the detection and prevention of the disease (ignjatovic and sapats, 2000) . multiple serotypes are known to circulate within a location , while new variants frequently spread to different geographical regions. for example, the qx strain, which was initially identified in china, was first detected in western europe in late 2003, but was not identified in the uk until 2007 (landman et al., 2005; summary two tests were developed that allow the detection and genotyping of infectious bronchitis virus (ibv) and other closely related gammacoronaviruses. the first test employs a one-step, reverse transcription-polymerase chain reaction (rt-pcr) assay in which the amplification is monitored in real time using a taqman ò probe. this real-time rt-pcr test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. a total of 323 field samples were tested; 176 samples were positive using the real-time rt-pcr method, but only three were positive by virus isolation. sequencing was used to confirm the positive real-time rt-pcr results for a subset of samples. the test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. the second test, a genotyping method, was developed for identification of the strain of ibv present in field samples based on nucleotide variations within the gene encoding the s1 subunit of the surface spike (s) glycoprotein. this method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of ibv within the uk. the performance of the test was evaluated using laboratory isolates of ibv and field samples. both tests are suitable for use in a high-throughput diagnostic laboratory. gough et al., 2008; worthington et al., 2008) . different serotypes of the virus derive from variation in the s1 subunit of the spike (s) glycoprotein. the s1 subunit induces neutralizing antibodies in the host, and differences of only a few amino acids may exist between the s1 subunits of different ibv serotypes (cavanagh, 2003 (cavanagh, , 2005 . the poultry industry has adopted vaccination as a disease control strategy to limit the economic impacts of ibv infections, although the situation is complicated as different serotypes may not offer cross-protection. consequently, birds may have to be vaccinated with multiple serotypes of the virus, including those contemporaneously circulating in the field (cavanagh, 2003) . this makes constant surveillance and testing necessary to identify cases of ibv and to determine the strains of ibv currently circulating within a location to ensure that the most suitable and cost-effective strategy is implemented. detection of ibv infection has traditionally been carried out using virus isolation, the current gold standard test (world organisation for animal health (oie) (2008). however, this method is expensive and timeconsuming and is unable to deliver a rapid diagnosis in a cost-effective manner (de wit, 2000) . more recently, nucleic acid detection methods such as rt-pcr have been shown to be rapid, cost-effective and suitable for use in a high-throughput diagnostic environment. real-time rt-pcr has been used as a diagnostic method for ibv (jackwood et al., 2003; callison et al., 2006; escutenaire et al., 2007) , and the use of a taqman ò probe to follow the pcr provides an additional level of specificity. nucleic acid amplification methods can also be used for ibv strain identification, as the amplicons generated by rt-pcr can be sequenced to provide phylogenetic data (de wit, 2000; ignjatovic and sapats, 2000) . we describe the development and validation of a one-step real-time rt-pcr for the detection of ibv-like gammacoronaviruses from a range of sample types. the diagnostic test amplifies a 175-bp region within the 3¢ untranslated region (utr) of the viral genome. this diagnostic test can be used in conjunction with amplification and sequencing of the s1 viral gene to identify the strain of ibv present within a sample. these assays have been designed specifically to be used routinely, in a highthroughput diagnostic laboratory, for the rapid detection and strain identification of ibv from clinical samples. transport and initial processing of field samples tissue samples (kidney, brain, liver, oviduct, bursa and respiratory and intestinal tracts) were collected from a variety of bird species (chicken, turkey, pheasant and pigeon) at post-mortem from cases with respiratory disease or suspected coronavirus infection based on clinical observations. the samples were transported in eagle's minimum essential virus transport medium (vtm). tissue samples were homogenized, ground or cut into small pieces before making a 10-20% (w/v) suspension in phosphate-buffered saline (pbs) containing antibiotics (0.1 m pbs solution, ph 7.2 containing 50 mg/l gentamycin, 1 · 10 6 units/l penicillin g, 10 g/l streptomycin sulphate and 5 · 10 6 units/l of nystatin). the suspension was left for a minimum of 60 min at room temperature before centrifugation to deposit debris and the supernatant decanted for testing by virus isolation and real-time rt-pcr. wire-stemmed ent swabs (mw&e, corsham, wiltshire, uk) were used for cloacal or tracheal sampling and were transported either 'dry' (no transport medium) or 'wet' in eagle's minimum essential medium (vtm). for wet swabs, the eagle's minimum essential vtm was diluted 1 : 2 with pbs with antibiotics (as previously described), and the resulting liquid was tested by virus isolation and real-time rt-pcr. dry swabs were processed by being introduced into 1 ml of brain heart infusion broth (bhib) with antibiotics (1000 iu/ml penicillin g, 10 lg/ml amphotericin b and 1 mg/ml gentamycin) and agitated briefly. this liquid was used for testing by virus isolation and real-time rt-pcr. for virus isolation, 0.2 ml of processed sample was inoculated into either the allantoic or amniotic cavity of 9-to 11-day-old embryonated specified pathogen-free (spf) fowls' eggs. eggs were examined daily by 'candling', using a bright light shone on the egg to examine the development of the embryo, which is compromised or altered if virus replication has occurred. samples underwent a minimum of two passages, while three passages were used when testing samples from cases where the clinical signs strongly suggested the involvement of coronaviruses. samples were initially processed as described above, although for 'wet' swabs, the undiluted eagle's minimum essential vtm was tested. for tissue samples and wet swabs, an automated nucleic acid extraction was carried out using the magna pure lc extraction robot, with dedicated buffers contained in the magna pure lc total nucleic acid isolation kit (roche, burgess hill, west sussex, uk). nucleic acid extractions were prepared from 200 ll of sample following the manufacturers' instructions. for material eluted from dry swab samples, an automated extraction was made from 140 ll of the bhib (see transport and initial processing of field samples) using the biorobot universal extraction robot (qiagen, crawley, west sussex, uk) according to the manufacturers' instructions using the 'single-plate swab extraction' protocol. briefly, 420 ll lysis buffer avl (qiagen) was added to the bhib. this solution was then applied to a silica filter, the filter was washed to remove impurities, and the rna was eluted from the filter in elution buffer ave (qiagen). this real-time rt-pcr assay used the following primer and probe sequences: ibvrt1 forward primer cta tcg cca ggg aaa tgt c, ibvrt2 reverse primer gcg tcc tag tgc tgt acc c, ibvrt3 taqman ò probe fam -cct gga aac gaa cgg tag acc ct -tamra. the primer sequences are truncated versions of those described by cavanagh et al. (2002) and were previously shown to detect phcov (cavanagh et al., 2002) . a novel taqman ò real-time pcr probe that recognizes ibv, turkey coronavirus and pheasant coronavirus sequences was designed and incorporated into the test allowing amplification to be followed in real time. one-step rt-pcr reactions were performed using the qiagen onestep rt-pcr kit. each 25 ll reaction contained the following components: 5 ll one-step reaction buffer, 1 ll 25 mm mgcl 2 , 1 ll 10 mm dntp mix, 0.8 lm ibvrt1 primer, 0.8 lm ibvrt2 primer, 0.2 lm ibvrt3 taqman probe, 0.25 ll rnasin ribonuclease inhibitor (between 5 and 10 units of enzyme), 1 ll one-step enzyme mix, 11.75 ll nuclease-free water and 2 ll extracted nucleic acid. the reactions were run on the following programme using a stratagene mx3000p real-time pcr instrument: 50°c for 30 min, 95°c for 15 min, followed by 50 cycles at 95°c for 20 s and 50°c for 30 s. fluorescence data were collected during the 50°c step. this reaction was performed in triplicate on each nucleic acid extract, and samples were deemed to be positive if amplification was recorded in two or more of the triplicate reactions. the amplicons formed by the diagnostic ibv real-time rt-pcr assay were purified using a multiscreen hts 96 well plate (millipore, watford, uk) according to the manufacturers' instructions. the purified pcr products were sequenced by dye-terminator cycle sequencing using bigdye v3.1 kits (applied biosystems, warrington, cheshire, uk) with either the ibvrt1 or the ibvrt2 primer. the products of these reactions were analysed using an abi prism 3130xl dna analyzer (applied biosystems). the sequence data generated were then used to search the ncbi public sequence database using the blastn search tool (altschul et al., 1990) . determination of the sensitivity of the diagnostic ibv real-time rt-pcr assay using in vitro transcribed rna to determine the analytical sensitivity of the ibv realtime rt-pcr, the amplicon produced by the assay was cloned into the pgem t-easy plasmid vector (invitrogen, paisley, renfrewshire, uk), and the identity of the plasmid was confirmed by sequencing. this plasmid was then used as the template to produce rna transcripts by in vitro transcription using the megascript ò kit (applied biosystems). the dna template was digested using turbo dnase (applied biosystems), and the rna was then purified using trizol ò (invitrogen). the concentration of the rna was then determined using a nanodrop instrument (fisher scientific, loughborough, leicestershire, uk), and the value obtained was used to calculate the number of rna template molecules present in the rna preparation, based on the molecular weight of the transcript and avogadro's number. this was used to set up a 1 in 10 serial dilution of rna template containing a known number of template molecules. this dilution series was then used as the template for duplicate diagnostic ibv real-time rt-pcr reactions, and also for duplicate reactions containing the primers and probes in the absence of reverse transcriptase. amplification and sequencing of the s1 gene of ibv the genotyping rt-pcr uses a panel of ten forward and eighteen reverse primers (table 1) . each primer contains a specific sequence to amplify the s1 gene of certain known ibv strains, together with a generic m13 primer sequence (either an m13 forward or reverse sequence, as appropriate). the primers were designed to amplify the strains of ibv currently in circulation in europe , along with other strains such as vics and v18-91 that are not commonly found in europe. to produce amplicons for sequencing, one-step rt-pcr reactions were performed using the qiagen one-step rt-pcr kit. each 25 ll reaction contained the following components: 5 ll one-step reaction buffer, 1 ll 25 mm mgcl 2 , 1 ll 10 mm dntp mix, 0.4 lm of primer s1for2, 0.4 lm of primer s1rev3, 0.04 lm of each of the remaining primers, 0.25 ll rnasin ribonuclease inhibitor (between 5 and 10 units of enzyme) (promega, southampton, hampshire, uk), 1 ll one-step enzyme mix, 12.75 ll nuclease-free water and 2 ll of extracted nucleic acid. the reactions were run on the following programme using an mx3000p real-time pcr instrument: 50°c for 30 min, 95°c for 15 min, followed by 50 cycles at 94°c for 30 s, 54°c for 30 s and 72°c for 30 s. the amplicons generated were sequenced using the m13f (gtaaaacgacggccagtg) and m13r (cagg-aaacagctatgaccatg) generic primers. the forward and reverse sequences were aligned, and a 140-bp region of the sequence data generated was compared to a library of 36 sequences of well-characterized ibv strains using the abi prism seqscape v2.6 software (applied biosystems). the ibv s1 sequence files were taken from the public access genbank database and included representatives of the major groups of ibv strains that are currently circulating in europe alongside other ibv strains (see fig. 1 ). a neighbour-joining phylogenetic tree was constructed using the molecular evolutionary genetics analysis package (mega v4) with the kimura 2-parameter algorithm (tamura et al., 2007) . design and calibration of ibv real-time rt-pcr to confirm that the modified test was suitable for detecting contemporary uk field strains of ibv, a panel of laboratory isolates of ibv representing the major genotypes currently circulating in the uk was tested . the panel included m41, italy-02, 4/91, d1466, d274 and three uk qx-like strains isolated from field samples originating from poultry flocks in great britain. the modified real-time rt-pcr successfully detected all of the ibv strains tested. determination of the analytical specificity of the realtime rt-pcr assay to detect ibv-like coronaviruses in chicken, turkeys and pheasants a wide range of microorganisms, both bacterial and viral, can be responsible for respiratory disease in avian species and might be present in clinical submissions. to investigate the possibility of cross-reactivity of the assay, nucleic acid extracts from the organisms listed in table 2 were prepared and used as the template for the gammacoronavirus diagnostic test. no cross-reaction with any of the organisms was detected, indicating that the test is specific for ibv-like gammacoronaviruses and false-positive results will not be caused by the presence of these additional organisms in field samples. the importance of this was reinforced following testing of field samples, as table 1 . primers used in the s1 genotyping rt-pcr primer name primer sequence s1 for 1 gtaaaacgacggccagtggtttactactaccagagtgc s1 for 2 gtaaaacgacggccagtggtgtactactaccaaagtgc s1 for 3 gtaaaacgacggccagtgacatactattaccagagtcag s1 for 4 gtaaaacgacggccagtggtttactactaccaaagtgc s1 for 5 gtaaaacgacggccagtgaattattactaccaaagtgc s1 for 6 gtaaaacgacggccagtggtgtactactaccagagtgg s1 for 7 gtaaaacgacggccagtggtgtattactaccagagtgc s1 for 8 gtaaaacgacggccagtggtctactactaccaaagcgc s1 for 9 gtaaaacgacggccagtggtgtactactaccaaagcgc s1 for 10 gtaaaacgacggccagtggtctactactaccaaagtgc s1 rev 1 caggaaacagctatgaccatgacatcttgtgcagtaccattaac s1 rev 2 caggaaacagctatgaccatgacatcttgtgctgtaccattaac s1 rev 3 caggaaacagctatgaccatgacatcttgtgcggtgccattaac s1 rev 4 caggaaacagctatgaccatgacttcaacagcagtgccatttac s1 rev 5 caggaaacagctatgaccatgcttgtgcggtaccattaataaag s1 rev 6 caggaaacagctatgaccatgatatcttgcgcagtaccattttc s1 rev 7 caggaaacagctatgaccatgacatcctgtgcagtaccattaac s1 rev 8 caggaaacagctatgaccatgacatcatgtgcagtaccattgac s1 rev 10 caggaaacagctatgaccatgacgtcttgtgcagtaccattaac s1 rev 11 caggaaacagctatgaccatgacatcttgtgcggtaccattaac s1 rev 12 caggaaacagctatgaccatgacgtcttgtgcggtaccattaac s1 rev 13 caggaaacagctatgaccatgacgtcttgtgcagtaccattacc s1 rev 14 caggaaacagctatgaccatgagaataacatcttgcgcagtacc s1 rev 15 caggaaacagctatgaccatgaaaataacatcttgtgcagtacc s1 rev 16 caggaaacagctatgaccatgacatcatgtgcggtgccattaac s1 rev 17 caggaaacagctatgaccatgcttgtgcggtgccattaataaag s1 rev 18 caggaaacagctatgaccatgaaaataatatcctgtgcagtacc several samples that were negative in the molecular diagnostic test were shown to contain other viruses, i.e. infectious laryngotracheitis virus and adenoviruses, using alternative testing methods (data not shown). two approaches were used to estimate the analytical sensitivity of the diagnostic real-time rt-pcr test. to investigate the analytical sensitivity of the test in toto including the extraction process, its performance was correlated with the current standard test, virus isolation in embryonated spf fowls' eggs (world organisation for animal health (oie) (2008). a tenfold dilution series of the ibv laboratory isolate 793/b was made, and 0.2 ml of each dilution was tested in parallel by both tests. virus isolation had a limit of detection between the 10 )5 and 10 )6 dilutions, and it was calculated that the original 793/b virus pool had a titre of 10 5.5 median egg infectious doses (eid 50 ) in 0.2 ml. the limit of detection of the real-time rt-pcr test was the 10 )6 dilution. to investigate the analytical sensitivity of the real-time rt-pcr alone, in vitro transcription of a cloned target sequence was used to prepare an rna target template. the rna produced was used to introduce a known number of rna molecules into the real-time rt-pcr assay. parallel reactions lacking reverse transcriptase (rt) enzyme were also run. this experiment demonstrated that the limit of detection of the real-time rt-pcr reaction was between 10 and 100 copies of template. the absence of any amplification in the 'no-rt' control wells demonstrated the absence of contaminating plasmid dna in the preparation of the rna transcribed in vitro. to further determine the performance of the diagnostic test, it was necessary to test its ability to identify positive and negative field samples correctly. samples received for routine testing using virus isolation were tested in parallel using the diagnostic ibv real-time rt-pcr. the panel contained field samples from a variety of species of birds (chickens, turkeys, pheasants and pigeons) and also a range of sample types including both swabs and postmortem tissue samples. a total of 323 samples were analysed during the course of this study, and while only three samples were found to be positive using virus isolation, 176 samples were positive by real-time rt-pcr, including in this number the three samples that were positive by virus isolation (table 3) . as the results obtained using virus isolation were not comparable to those obtained using the realtime rt-pcr, some of the amplicons formed using the diagnostic test were sequenced. for 118 of the 176 realtime rt-pcr-positive samples, amplicons were generated using the ibvrt1 and ibvrt2 primers and sequenced using these primers. because of sample degradation, amplicons were no longer generated when retesting the remainder of the samples. good-quality sequence data were generated from 41 of the 118 amplicons, but the sequence data obtained for the other samples were of insufficient quality or length to interpret. for 40 samples, the sequence data generated were shown to be most similar to ibv, while for one sample, the most similar sequence within the genbank database was shown to be turkey coronavirus. the original samples for which the real-time rt-pcr results could be confirmed by sequencing included cloacal swabs (16 samples), oropharyngeal and respiratory tract swabs (four samples), intestines and intestinal contents (eight samples), caecal tonsils and caecal contents (five swabs), and trachea and tracheal contents (three samples). real-time rt-pcr-positive results were generated for several poultry species (turkeys, pheasants and chickens), but the ten pigeon samples tested were all negative (table 3) . it was also demonstrated that gammacoronaviruses could be detected from a range of sample types using the molecular ibv real-time rt-pcr (table 4) . positive results were obtained from chickens using oropharyngeal and cloacal swabs (both dry and in vtm). positive results were also obtained from a variety of postmortem sample types, including kidney, small intestine, trachea, caecal tonsils, mixed kidney and oviduct, and caecal contents from chickens. positive post-mortem sample types from turkeys included intestine and intestinal contents, while coronavirus was detected in mixed lung and trachea samples from pheasants. the diagnostic ibv real-time rt-pcr described here has been demonstrated to be a highly sensitive and specific test, which is eminently suitable for demonstrating the presence of gammacoronavirus nucleic acid in field samples. however, because of its design, it will detect both field and vaccine strains of ibv. to allow determination of the strain of ibv present within a field sample, an s1 genotyping method was designed for routine use within a diagnostic laboratory setting. samples of live attenuated ibv vaccines currently in use in the uk were sequenced using this method, and the sequence data generated used to populate the sequence library used during interpretation of the sequence data generated by the s1 genotyping test. analysis of the sequences present in the sequence library demonstrated the sequences clustered in several main ibv serotypes including the so-called massachusetts, italy 02, 4/91 (also known as 793/b and cr88), arkansas and qx-like serotypes (fig. 1) . the variation within sequences in these different groups has been shown to be <5%, equivalent to seven base changes in the 140 bp region used for sequence analysis. in contrast, greater variation is seen between the different serotypes, with the amount of variation between serotypes exceeding 15% of the nucleotides within the region sequenced. initially, to demonstrate that this method was performing well, it was used to test a panel of laboratory isolates representing ibv strains currently circulating in the uk . the panel included m41, italy 02, d1466, d274 and 4/91 along with three qx-like isolates derived from poultry in great britain. gel electrophoresis was used to demonstrate that the s1 rt-pcr was able to amplify the s1 gene of these ibv strains. the amplicons generated were also sequenced, and when the data generated were compared to the ibv sequence library, the ibv strain present within each sample was correctly identified. to determine whether the ibv genotyping method was capable of identifying the strain of ibv present in a field sample, the method was used to test a panel of ten field samples, which had previously tested positive using the diagnostic ibv real-time rt-pcr. the panel included both swabs and post-mortem tissue samples from chickens. the results from this testing are shown in table 5 . five of the field strains of ibv were most closely matched at the sequence level to strains of the 4/91 serotype, three of the field samples were identified as being qx-like strains, and the remaining two field samples were identified as having closest sequence matches within the d274 and italy 02 serotypes, respectively. this paper describes the development and validation of a taqman ò real-time rt-pcr for the detection of ibv, turkey coronavirus and pheasant coronavirus, and an ibv s1 genotyping method to enable the subsequent identification of the strain of ibv present. unfortunately, it was not possible within the scope of this study to determine whether the diagnostic ibv real-time rt-pcr assay can detect pigeon coronaviruses. although clinical samples from pigeons with suspected disease were tested, these were not positive using the diagnostic real-time rt-pcr. comparison of the sequence of the pigeon coronavirus 3¢ utr to the sequence of the primers and probe used within this study shows several differences at the nucleotide level, suggesting that although amplification may occur, this could be inefficient and it may be preferable to use an alternative rt-pcr to detect pigeon coronavirus. the real-time rt-pcr assay targets the 3¢ utr of the ibv genome, adjacent to the poly a tail, which, unlike some other regions of the genome, is highly conserved in all gammacoronaviruses (williams et al., 1993; sapats et al., 1996; dalton et al., 2001) . because of the transcriptional strategy employed by coronaviruses in which sets of 3¢ coterminal nested rna molecules are produced, the 3¢ utr of the virus is present not only in the genomic rna but also in the mrna molecules produced by the virus. this means that there may be more copies of the viral 3¢ utr present within a field sample than other regions of the viral genome, making this region an ideal target for a diagnostic rt-pcr where a high level of sensitivity is required. during this work, for each of the three samples that tested positive by virus isolation, a positive result was also seen using the diagnostic ibv real-time rt-pcr assay, and the presence of ibv within these samples was additionally confirmed by sequencing the amplicon generated by the diagnostic assay. furthermore, a substantial number of field samples were positive using the molecular diagnostic assay, but no virus was isolated following attempted virus isolation in spf embryonated fowls' eggs. unlike virus isolation, real-time rt-pcr does not require the presence of viable virus particles. therefore, the molecular method will be less affected than virus isolation by adverse storage and transport conditions that field submissions may be subjected to prior to receipt for testing which may result in degradation of the viral particles. this could account for the discrepancy between the results obtained with virus isolation and the realtime rt-pcr. the validity of the result obtained for 38 of the 173 real-time rt-pcr positive, virus isolation negative samples could be confirmed by sequencing of the amplicon generated by the diagnostic rt-pcr. how-ever, it was not possible to generate good-quality sequence data from all of the 116 amplicons tested, and we hypothesized that this may be attributed to degradation of the viral rna after the long periods of storage and repeated cycles of freeze-thawing that the rna extracts were subjected to. alternatively, this may be caused by the short length of the amplicon used, as short stretches of sequence information could be difficult to identify using the blastn-based method utilized within this study. infectious bronchitis virus rna has been detected in tracheal swab samples by other real-time rt-pcrs for at least 21 days post-vaccination (callison et al., 2006) and has been isolated from faecal samples in some infected birds as long as 227 days post-infection gough, 1977, 1978) , making it essential to be able to differentiate between vaccine and field strains for diagnostic purposes. s1 genotyping to identify an ibv strain is used widely (kingham et al., 2000; farsang et al., 2002; bochkov et al., 2006; worthington et al., 2008) and has been found to correlate with serotyping of the viral strain (wang and tsai, 1996; keeler et al., 1998) , although it has been demonstrated (bochkov et al., 2007 ) that genotyping ibv strains using either the s or n genes can offer a different perspective on the relationship of ibv strains. genotyping of the hypervariable region (hvr) of the s1 gene has also been reported to yield the same result as genotyping the entire s1 gene sequence (wang and huang, 2000) . the s1 genotyping method described in this paper amplifies a region, including the hvr 2, which is known to show variation at the sequence level between different ibv strains (cavanagh et al., 1988) . the use of a panel of both forward and reverse primers should allow the identification of the majority of ibv strains currently circulating in europe, as well as offering the opportunity to detect novel ibv genotypes as they arise, which is crucial for informing vaccination strategies. it is also recognized that performing such a genotyping assay in a routine testing environment offers an extra challenge in the form of data interpretation, particularly when distinguishing field from vaccine strains. it seems likely, as discussed , that identification of strains with sequences identical to those of vaccine strains is likely to indicate the presence of the vaccine strain within the sampled birds. however, it cannot be discounted that theoretically, these could be separate field strains. in the present study, comparison of the sequence of a 140-bp region of the s1 gene of ibv allowed field strains to be clustered within the main recognized ibv serotypes. the sequence of strains within a serotype was shown to differ with <5% nucleotide identity, which is comparable with previous studies looking at different strains from table 5 . results from testing a panel of field samples using s1 genotyping method. the results show the closest sequence in the s1 sequence database, along with the number of mismatches between the sample sequence and the closest match within the database for the 140-bp region compared caecal tonsils 4/91 pathogenic 2 s1fs10 trachea qx (av2150/107) 2 s1fs84 oropharyngeal swab fr/l-1450t/05 2 s1fs85 oropharyngeal swab nl/l-1449t/04 2 s1fs31 oropharyngeal swab 4/91 pathogenic 0 diagnostic rt-pcr and genotyping methods for gammacoronavirus diagnosis r. m. jones et al. within a particular serotype and comparing the sequence of the entire s1 gene (cavanagh et al., 1988 (cavanagh et al., , 1992 . analysis based on the sequence of a 140-bp region suggested that different groups of serotypes differed at the level of nucleotide identity at >15% in this region, consistent with published data that suggested that different serotypes varied at approximately 70% when comparing the first 560 nucleotides of the s1 gene . one of the drawbacks to this method of ibv strain identification is the possibility of multiple ibv strains being present within a single field sample, as may occur after routine vaccination of birds with a live attenuated ibv vaccine followed by a field challenge with a genetically distinct strain of ibv. while this remains a limitation of the method, evidence exists that more virulent strains of ibv may replicate to higher titres than more attenuated strains in infected birds (cavanagh and gelb, 2008) and should therefore be detected preferentially by this method. the coupling of a generic diagnostic real-time rt-pcr to detect ibv with a strain genotyping method based on the s1 hvr provides a rapid and cost-effective method suitable for the routine detection and identification of ibv and closely related gammacoronaviruses in field samples, and use of such tests within a diagnostic setting enhances existing scanning surveillance capabilities to detect both extant, contemporary ib viruses and new and emerging ibv variants in poultry. isolation of avian infectious bronchitis virus from experimentally infected chickens a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus lipman, 1990: basic local alignment search tool molecular epizootiology of avian infectious bronchitis in russia phylogenetic analysis of partial s1 and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity and recombination development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus infectious bronchitis amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs 2001: cis-acting sequences required for coronavirus infectious bronchitis virus defective-rna replication and packaging detection of infectious bronchitis virus sybr green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain chinese qx strain of infectious bronchitis virus isolated in the uk. vet. rec turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review avian infectious bronchitis virus detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the beaudette strain molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s-1) gene identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s-1 gene high incidence of false layers in (re)production hens supposedly attributed to a juvenile infectious bronchitis virus infection novel variation in the n protein of avian infectious bronchitis virus mega4: molecular evolutionary genetics analysis (mega) software version 4.0 relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan analysis of a hypervariable region in the 3¢ non-coding end of the infectious bronchitis virus genome avian infectious bronchitis. chapter 2.3.2. manual of diagnostic tests and vaccines for terrestrial animals a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from this work was funded by the veterinary laboratories agency (vla) test development projects, td0022 and td0069. the authors thank staff from the vla regional laboratories and from private veterinary practices who submitted samples for this study, and the vaccine companies who provided vaccines for use in this work. the authors also thank robin sayers for his help with the statistical analysis of the data, jo mayers, vaneesa ceeraz and shital patel for technical assistance, and richard gough, mike collins, jason sawyer and kath webster for their advice and support. key: cord-322516-wekvet6f authors: maceyka, michael; machamer, carolyn e. title: ceramide accumulation uncovers a cycling pathway for the cis-golgi network marker, infectious bronchitis virus m protein date: 1997-12-15 journal: j cell biol doi: nan sha: doc_id: 322516 cord_uid: wekvet6f the m glycoprotein from the avian coronavirus, infectious bronchitis virus (ibv), contains information for localization to the cis-golgi network in its first transmembrane domain. we hypothesize that localization to the golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. because the site of sphingolipid synthesis overlaps the localization of ibv m, we asked whether perturbation of sphingolipids affected localization of ibv m. short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of ibv m or other golgi markers. thus, ongoing synthesis of these lipids was not required for proper localization. surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino1-propanol (pdmp), shifted the steady-state distribution of ibv m from the golgi complex to the er. this effect was rapid and reversible and was also observed for ergic-53 but not for golgi stack proteins. at the concentration of pdmp used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. pretreatment with upstream inhibitors partially reversed the effects of pdmp, suggesting that ceramide accumulation mediates the pdmp-induced alterations. indeed, an increase in cellular ceramide was measured in pdmp-treated cells. we propose that ibv m is at least in part localized by retrieval mechanisms. further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics. 1-propanol (pdmp), shifted the steady-state distribution of ibv m from the golgi complex to the er. this effect was rapid and reversible and was also observed for ergic-53 but not for golgi stack proteins. at the concentration of pdmp used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. pretreatment with upstream inhibitors partially reversed the effects of pdmp, suggesting that ceramide accumulation mediates the pdmp-induced alterations. indeed, an increase in cellular ceramide was measured in pdmp-treated cells. we propose that ibv m is at least in part localized by retrieval mechanisms. further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics. t he organelles of the classical secretory pathway must maintain their identity despite a large flux of lipids and proteins. two models have emerged to explain how proteins can be maintained in specific compartments (machamer, 1993; nilsson and warren, 1994 ). the retention model proposes that proteins are efficiently anchored in the appropriate compartment. the retrieval model proposes that proteins are continually recycled from later compartments. the two models are not mutually exclusive; indeed most proteins within the secretory pathway probably use both mechanisms for localization, albeit to differing extents. an example of this is the localization of the er resident protein bip, which contains a kdel retrieval signal but is only slowly secreted when this signal is removed (munro and pelham, 1987) . this suggests that other parts of the molecule may contain retention information. the m glycoprotein from the avian coronavirus, infectious bronchitis virus (ibv), 1 is a model protein for studying localization to the early secretory pathway. immunoelectron microscopy showed that ibv m expressed in the absence of other ibv proteins was found in the tubulovesicular structures at the entry or cis face of the golgi stack, as well as the first or second cisterna of the golgi stack (machamer et al., 1990; sodeik et al., 1993) . we will refer to this region as the cis -golgi network (cgn; mellman and simons, 1992) . defined in this way, we would consider the cgn to at least partially overlap with the intermediate compartment (ic), defined by such markers as ergic-53 or p58 (schweizer et al., 1988; lahtinen et al., 1996) . the first transmembrane domain of ibv m is sufficient to target chimeric proteins to the cgn (swift and machamer, 1991; machamer et al., 1993) . although the transmembrane domains of other golgi proteins also contain targeting information, no consensus motif for localization has been identified (for review see colley, 1997) . we are intrigued by the possibility that the targeting of golgi membrane proteins may in part depend on interactions between their transmembrane domains and specific membrane lipids. independent studies demonstrate that the lipid compositions of membranes differ at each stage of the secretory pathway (keenan and morre, 1970; cluett et al., 1997; for review see van meer, 1993) . sphingolipids, including sphingomyelin (sm) and glucosylceramide (glccer), the precursor to all gangliosides, are one class of lipids thought to increase in relative concentration through the secretory pathway. ceramide, the precursor of sphingolipids, is synthesized in the er. in rat liver, ceramide is converted into the different classes of sphingolipids by enzymes localized to the cgn and the cis -and medial -golgi cisternae (futerman et al., 1990; futerman and pagano, 1991) . when expressed from a recombinant vaccinia virus, ibv m is localized to the cgn in several cell types, and its localization presumably overlaps with that of sm and glccer synthase activities. this led us to speculate that there may be a link between sphingolipid synthesis and the localization of ibv m. to address this question we tested the effects of three sphingolipid synthesis inhibitors on the steady-state localization of ibv m. we observed a dramatic redistribution of ibv m induced by one of these inhibitors, the glucosylceramide analogue d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (pdmp). use of upstream inhibitors coupled with lipid analysis suggested that the pdmp effects are mediated by the accumulation of the precursor ceramide. because ibv m can be induced to move to the er, we propose that ibv m is at least in part localized by retrieval mechanisms. fcs was from atlanta biologicals (norcross, ga); fumonisin b1 was from calbiochem (la jolla, ca); chromatography plates were from e. merck (darmstadt, germany); endoglycosidase h (endo h) from new england biolabs (beverly, ma); n -hexanoylsphingosine (c 6 cer), lipid standards, and pdmp were from matreya (pleasant gap, pa); [ 3 h]palmitate (50 ci/mmol) was from dupont-nen (wilmington, de); pro-mix ( ͼ 1,000 mci/mmol [ 35 s]methionine) was from amersham intl. (arlington heights, il); dme, tissue culture-grade trypsin, and penicillin-streptomycin were from gibco brl (gaithersburg, md); all other reagents were from sigma chemical co. (st. louis, mo). antibodies were obtained as follows: monoclonal anti-bip, stressgen (victoria, bc, canada); monoclonal anti-giantin and monoclonal anti-ergic-53, hans-peter hauri (basel, switzerland); polyclonal anti-␤ -cop, jennifer lippincott-schwartz (national institutes of health, bethesda, md); polyclonal ␣ -mannosidase ii, marilyn farquhar (university of california at san diego, la jolla, ca) and kelley moreman (university of georgia, athens, ga); polyclonal antibodies to ibv m and vesicular stomatitis virus (vsv) were prepared as described (machamer and rose, 1987; weisz et al., 1993, respectively) ; texas red-and fitc-conjugated secondary antibodies, jackson immunoresearch (west grove, pa). cell culture. tissue culture cells were grown in dme supplemented with 5% (bhk-21) or 10% (vero) fcs. cells were grown at 37 њ c in an atmosphere of 5% co 2 . pulse-chase labeling. pulse-chase experiments were performed as previously described (rosenwald et al., 1992; weisz et al., 1993) . briefly, cells were plated in 35-mm dishes the night before the experiment to be 90% confluent the next day. vsv (san juan strain, indiana serotype) was adsorbed for 30 min in 0.5 ml of serum-free dme. at 4 h after infection, the cells were starved for methionine for 15 min. cells were then pulsed for 5 min with 50 ci 35 s-pro-mix and chased for the indicated amount of time in the presence or absence of the indicated drugs. the isopropanol carrier alone had no effect on the rate of transport of vsv g protein. after the chase, cells were washed once with cold pbs and lysed in detergent. vsv g protein was then immunoprecipitated, treated with endo h as described (rosenwald et al., 1992) , separated by sds-page, and visu-alized by fluorography. endo h-sensitive and -resistant forms of the vsv g protein were quantitated by densitometry. indirect immunofluorescence microscopy. these experiments were performed as previously described (swift and machamer, 1991) . briefly, cells were infected for 30 min with a recombinant vaccinia virus encoding ibv m, and the indicated treatments were begun 4 h after infection. for experiments using exogenous ceramides, the soluble short-chain analogues of ceramide were added to cells as a complex with 0.34 mg/ml defatted bsa as described (pagano and martin, 1988) . after treatment, cells were fixed, permeabilized, and stained with the appropriate antibody. images were acquired using a microscope (axioskop; zeiss, inc., thornwood, ny) equipped with epifluorescence and a ccd camera (photometrics sensys, tucson, az) using ip lab software (signal analytics corp., vienna, va). all images shown are the raw data collected at 1 ϫ 1 binning with a gain of 1. lipid synthesis assays. cells were seeded onto 6-cm plastic dishes 2 d before the experiment so that they were 90% confluent the day of the experiment. cells were incubated in dme supplemented with 50 g/ml cycloheximide, and half of the dishes were also incubated with 5 mm ␤ ca. 1 h later, fresh medium containing cycloheximide and [ 3 h]palmitate (100 ci/ dish) was added, along with either 1% isopropanol (control and ␤ ca) or 100 m pdmp and 5 mm ␤ ca if indicated. 1 h later, cells were trypsinized and washed off the plate in 0.8 ml cold pbs. to normalize lipid levels, 50 l were removed for a protein assay by the method of bradford (1976) . lipids were then extracted by the method of bligh and dyer (1959) . labeled samples were doped with 10 g cold ceramide to allow for visualization. samples were run on 10 ϫ 10 cm high performance thinlayer chromatography plates with a mobile phase of chloroform/glacial acetic acid (9:1; abe et al., 1992) . plates were sprayed with water to visualize the ceramide bands that were scraped and counted after the addition of scintillation fluid. alternatively, plates were dipped in 10% 4,5-diphenyloxazole in chloroform for visualization by fluorography (henderson and tocher, 1992) . to test whether ongoing sphingolipid synthesis was necessary for the correct localization of the ibv m protein, we used three inhibitors of sphingolipid synthesis: ␤ -chloroalanine ( ␤ ca; medlock and merrill, 1988) , fumonisin b1 (fb1; wang et al., 1991) , and pdmp (vunnam and radin, 1980; inokuchi and radin, 1987) . the biosynthetic pathway for sphingolipids with the sites of inhibition of these drugs is shown in fig. 1 . both ␤ ca (5 mm) and fb1 (100 m) inhibited the incorporation of radiolabeled precursors into sphingolipids in bhk-21 cells by ͼ 90% (data not shown). consistent with previous results (rosenwald et al., 1992) , we found that pdmp (100 m) inhibited glccer synthesis by ‫ف‬ 90% and sm synthesis by ‫ف‬ 50% in bhk-21 cells (data not shown). to express the ibv m protein, bhk-21 cells were infected with a recombinant vaccinia virus encoding ibv m (machamer and rose, 1987) . 4 h after infection the cells were treated with cycloheximide for 1 h to chase newly synthesized m protein out of the er. sphingolipid synthesis inhibitors were then added and the cells incubated for another 1 h before processing for immunofluorescence ( fig. 2) . in control cells, ibv m exhibited a tight, juxtanuclear staining pattern that colocalized with golgi markers. the golgi localization pattern of ibv m was unchanged by treatment with either ␤ ca or fb1, suggesting that ongoing sphingolipid synthesis is not required for proper localization of ibv m. in contrast, pdmp-treated cells showed a marked change in the staining pattern of the ibv m pro-tein after 1 h. the localization of ibv m in the presence of pdmp changed from golgi to er based on the absence of strong juxtanuclear staining and the presence of nuclear rim staining and a tubulo-reticular staining pattern that colocalized with er markers. treatment of infected cells with lower concentrations of pdmp had no effect on ibv m localization and very little effect on sm synthesis (data not shown). we next tested the kinetics of pdmp-induced mislocalization of ibv m. cells were infected as before and treated for 1 h with cycloheximide. then cells were treated with pdmp for varying lengths of time up to 1 h (fig. 3) . redistribution was first observed at 15 min and became maximal at 60 min. this redistribution was rapidly reversible, such that 30 min after washing out pdmp, ibv m had moved back to the golgi region. we next asked if pdmp mislocalized other golgi markers. cells were infected and treated as described above and then stained with antibodies to two integral membrane proteins of the golgi stack, giantin (linstedt and hauri, 1993) and mannosidase ii (man ii; moremen and robbins, 1991; velasco et al., 1993) . treatment of cells with 100 m pdmp for 1 h had no effect on the localization of either protein (fig. 4) , suggesting that the morphology of the golgi was not greatly altered. we also examined the localization of ␤ -cop, a peripheral membrane protein of the stack and cgn involved in vesicular traffic (oprins et al., 1993) . as seen in fig. 4 , the distribution of ␤ -cop appeared unaffected in treated cells. we then looked at a protein which cycles through the cis -golgi. as we did not have access to antibodies that crossreact with known markers in bhk-21 cells, we examined ergic-53 in vero cells. ibv m localizes to the cgn in these cells and is also redistributed to the er by pdmp (data not shown). ergic-53 is an ic protein that cycles between the er, ic, and golgi stack (lippincott-schwartz et al., 1990; schindler et al., 1993) . vero cells were treated with 50 g/ml cycloheximide for 1 h. pdmp (100 m) was added, and dishes were fixed at the indicated times and prepared for indirect immunofluorescence. after 1 h in pdmp, the remaining dishes were washed three times with 1 ml dme with serum and incubated with cycloheximide-containing medium for the indicated time before being prepared for indirect immunofluorescence. for each experimental series, the nomarski image is shown on the left and the fluorescence image on the right. bar, 10 m. with cycloheximide for 1 h and then with or without 100 m pdmp for an additional hour before being fixed and prepared for immunofluorescence (fig. 5) . similar to the effects on ibv m, pdmp treatment shifted the steady-state distribution of ergic-53 to the er. in addition, neither ␤ ca nor fb1 had any effect on the localization of er-gic-53 (data not shown). the above immunofluorescence results suggested that pdmp might generally alter the distribution of proteins that have a dynamic localization mechanism. interestingly, it has been shown that pdmp slows the rate of both anterograde vesicular traffic (rosenwald et al., 1992) and endocytosis (chen et al., 1995) in cho cells. we tested whether pdmp also slowed anterograde traffic in bhk-21 cells. we used a plasma membrane protein, the well-characterized g protein from vsv. a pulse-chase experiment was performed either in the presence or absence of pdmp, ␤ ca, or fb1. the rate of accumulation of endo h-resistant vsv g was used as an assay for the rate of arrival at the medial -golgi. neither ␤ ca nor fb1 had any effect on the rate of anterograde traffic. however, pdmp increased the halftime for transport by ‫ف‬ 2.5-fold from 18 to 44 min (fig. 6 ). fig. 6 shows that neither ␤ ca nor fb1 had any effect on the transit rate of vsv g, suggesting that the decreased rate with pdmp was not due to a block in the ongoing synthesis of sphingolipids. pdmp inhibits the conversion of ceramide into glycosphingolipids and sphingomyelin. one possible explanation for the effects observed with pdmp was an accumulation of the precursor ceramide. ␤ ca and fb1 act upstream of pdmp (fig. 1) , and the intermediates in sphingolipid synthesis between these steps and ceramide production are thought to be short lived (merrill and wang, 1986; medlock and merril, 1988) . to ask if ceramide accumulation might mediate the pdmp-induced slowing of anterograde traffic, cells were treated with ␤ ca or fb1 before pdmp. we performed a pulse-chase labeling experiment with cells pretreated with either ␤ ca or fb1 for 1 h before being chased in the presence or absence of ␤ ca or fb1 and pdmp (fig. 6) . pretreatment with the earlier inhibitors reduced the pdmp-induced slowing of anterograde traffic by about half, suggesting that accumulation of newly synthesized ceramide at least partially mediates this effect. to test whether ceramide was likely to mediate the pdmpinduced mislocalization of ibv m, indirect immunofluorescence was performed as above with cells pretreated with either ␤ ca or fb1 for 1 h before the addition of pdmp. to simplify the quantification, the localization of ibv m was classified into one of three staining patterns. class 1 was the most commonly seen pattern in untreated cells, i.e., tight juxta-nuclear staining that colocalized with golgi markers. class 3 was the most commonly seen pattern in pdmp-treated cells, i.e., diffuse staining with prominent nuclear envelope staining that colocalized with er markers. class 2 was intermediate between the class 1 and class 3. whether this pattern represents an overlap of er and golgi staining patterns or an increase in ic staining is not clear. for these experiments, coded samples were quantified by counting at least 100 cells for each sample. the experiment was performed five times, with one representative experiment shown (fig. 7) . interestingly, when cells were quantified in this way, we observed that both ␤ ca and fb1 caused a slight shift from class 1 to class 2. the significance of this observation is not clear. pdmp dramatically shifted the distribution of ibv m from mainly class 1 in the control cells to mainly class 3. pretreatment with either ␤ ca or fb1 significantly reduced this shift in the staining pattern of ibv m, suggesting that accumulafigure 5 . pdmp induced the redistribution of the endogenous ic protein, ergic-53, to the er. vero cells were incubated with cycloheximide for 1 h and then incubated for 1 h in the presence either 1% isopropanol (control) or 100 m pdmp. cells were then fixed and prepared for indirect immunofluorescence with antibodies to ergic-53. for each experimental group, the nomarski image is shown on the left and the fluorescence image on the right. bar, 10 m. the pretreatment experiments suggested that increased levels of ceramide mediate the pdmp-induced effects on ibv m localization. ceramide can act as a second messenger (for review see hannun, 1994) and has been suggested to be the mediator of some of the effects of pdmp in cho cells (rosenwald and pagano, 1993) . furthermore, in several systems, cellular levels of ceramide increased upon treatment with pdmp or one of its more soluble analogues (for example see rani et al., 1995; posse de chaves et al., 1997) . we measured the levels of ceramide after various treatments by pulse-labeling with [ 3 h]palmitate. after labeling and treatment, cellular lipids were extracted and run on chromatography plates. the appropriate spots corresponding to ceramide were scraped and counted. consistent with our hypothesis, fig. 8 a shows that pdmp treatment increased the levels of ceramide 2.5-fold over control levels. pretreatment with ␤ ca reduced pdmpinduced ceramide accumulation to roughly half of control levels, but this level was still threefold higher than cells treated with ␤ ca alone. fig. 8 b is a representative fluorograph showing the ceramide region that was quantified in fig. 8 a. interestingly, we noticed the appearance of a lower migrating ceramide band in pdmp-treated cells. the pretreatment experiments as well as lipid analysis implicated ceramide as the mediator of pdmp effects. if this were true, addition of a soluble, short-chain analogue of ceramide might mimic the effects of pdmp. indeed, one such cell-permeable analogue of ceramide, n -hexanoylsphingosine (c 6 cer), reproduces the effects of pdmp on anterograde traffic of vsv g in cho cells (rosenwald and pagano, 1993) . we first asked whether c 6 cer slowed anterograde traffic in bhk-21 cells. a pulse-chase labeling experiment with vsv-infected cells was performed, with cells chased in the absence or presence of 25 m c 6 cer. c 6 cer slowed the rate of anterograde traffic in bhk-21 cells ‫-5.2ف‬fold (data not shown). we went on to use c 6 cer in our indirect immunofluorescence assay. as expected, c 6 cer had no effect on the localization of either man ii or ␤-cop (fig. 9) . however, contrary to our expectation, c 6 cer did not alter the localization of either ibv m or er-gic-53. this suggests that exogenously added c 6 cer and ceramide generated by treatment with pdmp do not have the same effects. two other soluble analogues of ceramide, c 2 cer and c 8 cer, were also tested and found to have no effect on the localization of ibv m (data not shown). there are two possible mechanisms to maintain the steady-state localization of proteins to specific sites along the secretory pathway: (a) retention in the particular compartment, and (b) retrieval from other compartments (machamer, 1993; nilsson and warren, 1994) . the redistribution of ibv m induced by pdmp was surprising, as we expected that if we disrupted its localization, it would move with "bulk flow" to the plasma membrane. that ibv m was redistributed to the er suggests that it has specific targeting information for retrieval to the er. coupled with earlier work in our laboratory on the role of oligomerization of ibv m chimeras in cgn targeting (weisz et al., 1993) , our findings suggest that ibv m maintains its steady-state distribution by both retention and retrieval ( fig. 10 a) . we propose two models to explain the redistribution of ibv m protein induced by pdmp. the simplest model (fig. 10 b, left arrow) is that the m protein normally cycles between the er and the golgi at a significant rate, as has been shown for ergic-53 (lippincott-schwartz et al., 1990; schindler et al., 1993) . the slowing of anterograde traffic would cause the protein to shift its steady-state distribution to the er, assuming that retrograde traffic is unaffected or perhaps increased by pdmp. a second possibility is that pdmp disrupts retention of the m protein, which then cycles back to the er by normal retrieval mechanisms (fig. 10 b, right arrow) . these two models are not mutually exclusive, and both effects of pdmp may be necessary to redistribute ibv m to the extent observed. implicit in both models is the idea that ibv m contains retrieval information. pdmp-induced er redistribution was also observed for ergic-53, a dilysine-containing ic marker known to cycle through the er (lippincott-schwartz et al., 1990; schindler et al., 1993) . double-label immunoelectron microscopy in hela cells showed that the distri-bution of ergic-53 and ibv m overlapped significantly, though not completely (sodeik et al., 1993) . this suggests that ibv m may in part use the same, as yet unknown, cellular machinery used by ic proteins to maintain its steadystate localization. using deletion mutants and chimeric molecules, dissection of ibv m retention and retrieval information should be possible using pdmp as a tool. the pathway followed by ibv m during retrieval will also be important to decipher. experiments in nontreated cells suggest that the protein may move through later golgi compartments even though its steady-state distribution is the cgn, because its oligosaccharides are slowly processed (machamer et al., 1990) . interestingly, we did not observe redistribution of two golgi stack markers (man ii and giantin) to the er with pdmp treatment. golgi membrane proteins were recently suggested to be highly mobile (cole et al., 1996b) and to cycle through the er (cole et al., 1996a) . our results with pdmp suggest that cycling of these proteins does not occur during the time scale of our experiments, and/or that its predominant effect on ibv m is a loss of retention. interestingly, it has been shown that myriocin, an inhibitor of sphingolipid synthesis thought to inhibit the same step as ␤ca, reduces the rate of transport of a glycosylphosphatidylinositol-linked protein but not other proteins out of the er in yeast (horvath et al., 1994) . it would be interesting to test whether inhibition of ceramide synthesis in mammalian cells also slows the rate of transport of glycosylphosphatidylinositol-linked proteins in mammalian cells. however, neither ␤ca nor fb1 had an effect on the rate of anterograde traffic of vsv g or the localization of ic, cgn, or golgi stack proteins. when we quantified the effects of these two inhibitors on the immunofluorescence figure 9 . exogenous c 6 cer did not redistribute ibv m or er-gic-53. bhk cells (ibv m, man ii, and ␤-cop) or vero cells (ergic-53) were infected with a recombinant vaccinia virus expressing ibv m. 4 h after infection the cells were treated with 50 g/ml cycloheximide. at 5 h after infection cells were incubated for 1 h in serum-free dme with 0.34 mg/ml defatted bsa with or without 25 m c 6 cer. cells were then prepared for indirect immunofluorescence and stained with the indicated antibodies. for each experimental series, the nomarski image is shown on the left and the fluorescence image on the right. bar, 10 m. figure 10 . current models for how ibv m maintains its steady-state distribution and how pdmp might alter this distribution. (a) evidence presented here shows that ibv m can be induced to redistribute to the er, indicating that ibv m has information necessary for traffic to the er. we propose that ibv m normally maintains its steady-state distribution by cycling through the er at some basal rate. (b) if ibv m is cycling through the er normally, then its localization would depend on the balance of anterograde and retrograde traffic rates. as pdmp slows anterograde traffic, ibv m could accumulate in the er when the balance of membrane traffic is disrupted (left arrow). alternatively, pdmp may act by disrupting the retention of ibv m, causing it to move out of the golgi (right arrow). it is also possible that redistribution of ibv m requires both effects of pdmp. staining pattern of ibv m, we did detect a slight shift toward the intermediate staining pattern (fig. 7) . whether this represents an altered distribution pattern or subtle effects on golgi structure caused by these inhibitors is unclear. from these results we conclude that ongoing sphingolipid synthesis is not required for either normal rates of anterograde traffic or proper localization of golgi proteins. however, the glucosylceramide analogue pdmp, at a concentration that inhibits both glccer and sm synthases, causes a redistribution of ibv m to the er and a slowing of anterograde traffic in bhk-21 cells. why does pdmp have these effects while ␤ca and fb1 do not? it is likely that pdmp exerts its effects by causing the accumulation of ceramide. other groups have found that the various effects of pdmp and its active analogues were concomitant with (uemura et al., 1990; shayman et al.; rani et al., 1995) or actually caused by increases in ceramide concentrations (abe et al., 1996; posse de chaves et al., 1997) . in bhk-21 cells treated with 100 m pdmp for 1 h we measured an increase in newly synthesized ceramide (fig. 8 a) , showing at least a correlation between levels of newly synthesized ceramide and the effects of pdmp on protein traffic and localization. the fact that pretreatment with either ␤ca or fb1, both of which block the synthesis of ceramide, can ameliorate the effects of pdmp demonstrates that ceramide is at least in part the mediator of these effects. however, the effects of pdmp are not completely abolished by pretreatment, even though ␤ca pretreatment reduced the level of newly synthesized ceramide by 50% compared to control. one explanation is that ceramide accumulation is only one of the ways that pdmp exerts its effects. another possibility is that ceramide is being generated by the degradation of preexisting sphingolipids, e.g., at the cell surface or in lysosomes. this ceramide cannot be made back into sphingolipids because the enzymes sm and glccer synthases are inhibited in the presence of pdmp. interestingly, the fluorograph of the ceramide bands generated after the various treatments (fig. 8 b) shows different relative amounts of ceramide species. we suspect that these ceramides differ in acyl chain lengths (abe et al., 1992) and that this may be an indication of different sources of ceramide, i.e., de novo synthesis or sphingolipid degradation. we are currently investigating this possibility. based on the pretreatment results, we expected that soluble analogues of ceramide would mimic the effects of pdmp. rosenwald and pagano (1993) showed that like 100 m pdmp, 25 m c 6 cer decreased the rate of transit of an itinerant protein to the medial-golgi in cho cells. consistent with these results, 25 m c 6 cer also slowed anterograde traffic in bhk-21 cells. however, 25 m c 6 cer had no effect on the localization of either the m protein or ergic-53. other short-chain ceramide analogues (c 2and c 8 -ceramide) also had no effect on ibv m localization. how does this fit with our model that ceramide mediates pdmp effects? one explanation is that the effect on protein localization is independent of the effect on antero-grade traffic rates. in this case, it may be that c 6 cer and endogenous ceramide have similar ability to bind and affect proteins that regulate anterograde traffic, but c 6 cer is unable to bind or affect proteins that regulate protein localization. it has been shown that some short chain analogues of ceramide do not have the same effects as endogenous ceramide (e.g., wolff et al., 1994) . a second possibility is that effects of ceramide are limited to the bilayers in which its concentration increases, and the difference seen between endogenous ceramide and exogenous c 6 cer is due to the different intracellular distribution of these molecules. c 6 cer would be unlikely to accumulate in compartments where ibv m and ergic 53 reside, because it is rapidly converted to sphingolipids there (rosenwald et al., 1992) . effects on membrane traffic might be more dramatic in compartments where c 6 cer accumulates, possibly the trans-golgi and tgn (pagano et al., 1989) . consistent with this idea, the rate of movement of vsv g through the late golgi (measured by sialylation) was slowed to a much greater extent by c 6 cer than by pdmp (rosenwald and pagano, 1993) . it is not yet clear how ceramide induces the changes in anterograde traffic and protein localization. recent studies have shown that ceramide (hannun, 1994) and its metabolites (hakomori, 1990 ) play a major role as second messengers. preliminary experiments with both the ceramidase inhibitor n-oleoylethanolamine and exogenously added sphingosine suggest that the ceramide breakdown product sphingosine does not play a role in the localization of ibv m (maceyka, m., and c. machamer, unpublished observations). ceramide activates a cytosolic protein phosphatase (dobrowsky and hannun, 1992; wolff et al., 1994) that can be inhibited by okadaic acid. preliminary experiments suggest that okadaic acid does not block the pdmpinduced changes (maceyka, m., and c. machamer, unpublished observations). ceramide could be activating a protein kinase (mathias et al., 1991) , but this kinase has not been fully characterized. another possibility is that increased ceramide within the lipid bilayer directly affects the localization of ibv m. earlier work from our laboratory showed that the first transmembrane domain of ibv m can target chimeras to the cgn (swift and machamer, 1991; machamer et al., 1993) . it is possible that cgn bilayers have distinct lipid domains, and that these domains contain different sets of proteins, analogous to glycosphingolipid rafts. segregation of membrane proteins involved in vesicular traffic, such as snares, would lead to mobile and immobile domains. we hypothesize that under normal conditions, ibv m would be targeted to an immobile domain and escaped molecules would be cycled back through the er. elevated levels of ceramide could disrupt these immobile lipid domains, inducing ibv m to cycle. alternatively, perhaps ceramide binds to ibv m transmembrane domains, preventing an interaction with immobile lipid domains. metabolic effects of shortchain ceramide and glucosylceramide on sphingolipids and protein kinase c inhibition of glucosylceramide synthase by synthase inhibitors and ceramide a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding a rapid method of total lipid extraction and purification ceramide as a modulator of endocytosis heterogeneous distribution of an unusual phospholipid in the golgi complex golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites diffusional mobility of golgi proteins in membranes of living cells golgi localization of glycosyltransferases: more questions than answers ceramide stimulates a cytosolic protein phosphatase determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the golgi apparatus bifunctional role of glycosphingolipids. modulators for transmembrane signaling and mediators for cellular interactions the sphingomyelin cycle and the second messenger function of ceramide thin-layer chromotography ceramide synthesis enhances transport of gpi-anchored proteins to the golgi apparatus in yeast preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase phospholipid class and fatty acid composition of golgi apparatus isolated from rat liver and comparison with other cell fractions molecular cloning and expression of a 58-kda cis-golgi and intermediate compartment protein giantin, a novel conserved golgi membrane protein containing a cytoplasmic domain of at least 350kda microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway golgi retention signals: do membranes hold the key? a specific membrane-spanning domain of a coronavirus e1 glycoprotein is required for its retention in the golgi region the avian coronavirus e1 protein is targeted to the cis golgi retention of a cis golgi protein requires polar residues on one face of a predicted ␣-helix in the transmembrane domain characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor ␣ inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact chinese hamster ovary cells by ␤-chloroalanine the golgi complex: in vitro veritas? biosynthesis of long-chain (sphingoid) bases from serine by lm cells. evidence for introduction of the 4-trans-double bond after de novo biosynthesis of n-acylsphinganines isolation, characterization and expression of cdnas encoding murine ␣-mannosidase ii, a golgi enzyme that controls conversion of high mannose to complex n-glycans a c-terminal signal prevents secretion of luminal er proteins retention and retrieval in the endoplasmic reticulum and golgi apparatus ␤-cop localizes mainly to the cis-golgi side in exocrine pancrease use of fluorescent analogs of ceramide to study the golgi apparatus of animal cells molecular trapping of a fluorescent ceramide analogue at the golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-golgi marker for both light and electron microscopy elevation of ceramide within distal neurites inhibits neurite growth in cultured rat sympathetic neurons cell cycle arrest induced by an inhibitor of glucosylceramide synthase. correlation with cyclin-independent kinases inhibition of glycoprotein traffic through the secretory pathway by ceramide effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway ergic-53, a membrane protein of the er-golgi intermediate compartment, carries an er retention motif identification, by a monoclonal antibody, of a 53-kd protein associated with a tubulovesicular compartment at the cis-side of the golgi apparatus modulation of renal epithelial cell growth by glucosylceramide. association with protein kinase c, sphingosine, and diacylglycerol assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks a golgi retention signal in a membrane-spanning domain of coronavirus e1 protein effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts transport and sorting of membrane lipids cell type-dependent variations in the subcellular distribution of ␣-mannosidase i and ii analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain inhibition of sphingolipid biosynthesis by fumonisins: implications for diseases associated with fusarium moniliforme oligomerization of a membrane protein correlates with its retention in the golgi complex role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction we thank drs. e. cluett, d. raben, and a. hubbard for useful discussions and the members of the machamer lab for critical reading of the manuscript. we also thank h.-p. hauri, m. farquhar, k. moremen, and j. lippincott-schwartz for antibodies. this work was supported by grant gm42522 from the national institutes of health.received for publication 29 august 1997 and in revised form 6 october 1997.the journal of cell biology, volume 139, 1997 key: cord-328046-5us4se5o authors: xu, h. y.; lim, k. p.; shen, s.; liu, d. x. title: further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein date: 2001-09-30 journal: virology doi: 10.1006/viro.2001.1098 sha: doc_id: 328046 cord_uid: 5us4se5o abstract the coronavirus 3c-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. in cells infected with avian coronavirus infectious bronchitis virus (ibv), three proteins of 100, 39, and 35 kda, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3c-like proteinase. in this report, we show the identification of two more cleavage products of 68 and 58 kda released from the same region of the polyprotein. in addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kda, respectively, were identified in ibv-infected cells. the 160-kda protein was shown to be an intermediate cleavage product covering the 100and 68-kda proteins, and the 132-kda protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kda proteins. immunofluorescent staining of ibv-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum, and the 39and 35-kda proteins displayed diffuse distribution patterns. coronavirus gene expression involves the expression of six to seven mrna species. in cells infected with the prototype species of the coronaviridae, avian coronavirus infectious bronchitis virus (ibv), six mrna species are detected. these include the genome-length mrna (mrna1) of 27.6 kilobases (kb) and five subgenomic mrna species (mrnas 2-6) with sizes ranging from 2 to 7 kb. the 5ј-terminal unique region of mrna 1 contains two large orfs, 1a and 1b, encoding the 441-kda 1a and 741-kda 1a/1b polyproteins (boursnell et al., 1987) (fig. 1) . the two polyproteins are cleaved by two viral proteinases to produce functional products associated with viral replication (ziebuhr et al., 2000) (fig. 1) . the first proteinase was identified to be included in a 195-kda cleavage product, which contains a papain-like proteinase domain encoded by orf 1a from nucleotides 4242 to 5553 (lim et al., 2000) . this proteinase was shown to be involved in cleavage of the n-terminal region of the 1a and 1a/1b polyproteins at two g-g dipeptide bonds (g 673 -g 674 and g 2265 -g 2266 ) to release two mature products of 87 and 195 kda lim and liu, 1998; lim et al., 2000) . the second proteinase was identified as a 33-kda protein in ibv-infected cells lim et al., 2000; ng and liu, 2000) . this serine proteinase belongs to the picornavirus 3c proteinase group (3c-like proteinase) and was shown to mediate cleavage of the 1a and 1a/1b polyproteins at more than 10 q-s(g, n) dipeptide bonds to release mature cleavage products (liu et al., 1994 (liu et al., , 1997 liu, 1998, 2000) . in addition to the viral proteinases, understanding of the functions of other cleavage products from the 1a and 1a/1b polyproteins is emerging. for example, the 71-kda protein released from the human coronavirus 1a/1b polyprotein was shown to possess atpase and rna duplex-unwinding activities, confirming the previous predication that the protein may be the viral helicase (heusipp et al., 1997a; seybert et al., 2000a,b) . immunofluorescence and biochemical studies demonstrated that several cleavage products are membrane-associated and colocalize with the viral rna replication-transcription machinery (bost et al., 2000; denison et al., 1999; ng and liu, 2000; schiller et al., 1998; shi et al., 1999; sims et al., 2000; ziebuhr and siddell, 1999; , suggesting the involvement of these products in viral rna replication. more recent studies showed that the first cleavage product of mouse hepatitis virus-a59 (mhv-a59) 1a and 1a/1b polyproteins, p28, might play a direct role in viral rna synthesis together with polymerase and helicase (bost et al., 2000; sims et al., 2000) . other cleavage products, such as the mhv 22-kda protein, may segregate into different but tightly associated membrane populations which may serve independent functions during viral replication (bost et al., 2000; sims et al., 2000) . in previous reports, we demonstrated that four previously predicted q-s(g) dipeptide bonds located in the 1b region of the 1a/1b polyprotein are genuine cleavage sites of the 3c-like proteinase (liu et al., 1994 . taken together with the n-terminal cleavage site identified for releasing the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda, respectively (fig. 1 ). among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells. in this communication, we report the identification of the two previously unidentified products, the 68-and 58-kda proteins, in ibvinfected cells with newly raised region-specific antisera. meanwhile, two relatively stable intermediate cleavage products with molecular masses of approximately 160 and 132 kda were identified. analysis of the expression and processing kinetics showed that both the 160-and 132-kda intermediate cleavage products coexist with their final cleavage products during the viral replication cycle. furthermore, immunofluorescence analysis showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum (er), while the 39-and 35-kda proteins displayed diffuse distribution patterns. in our previous reports, four q-s(g) dipeptide bonds, q 891(1b) -s 892(1b) , q 1492(1b) -g 1493(1b) , q 2012(1b) -s 2013(1b) , and q 2350(1b) -s 2351(1b) , located in the orf 1b region and encoded by nucleotides 15129-15134, 16929-16934, 18492-18497, and 19506-19511, respectively , were demonstrated to be the cleavage sites of the 3c-like proteinase (liu et al., 1994 (fig. 1 ). taken together with the q 3928 -s 2929 dipeptide bond (encoded by nucleotides 12310-12315) identified as the n-terminal cleavage site of the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda. among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells (liu et al., 1994 . to further identify and characterize the cleavage products, four new region-specific antisera, anti-100, anti-68, anti-58, and anti-35, were raised in rabbits against bacterially expressed viral proteins. the ibv proteins used to raise these antisera were encoded by nucleotides 12447-15131, 15536-16787, 16932-18494, and 19509-20414, respectively (fig. 1) . anti-100 was raised to replace v-58, which was raised against the ibv sequence encoded by nucleotides 14492-15520, and was used to identify the 100-kda protein in ibvinfected cells (liu et al., 1994) . the specificities of these antisera were tested by immunoprecipitation assay, showing that they could specifically precipitate their target proteins synthesized in both the in vitro system and intact cells (data not shown). to identify the cleavage products in ibv-infected cells, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell. to reduce the background, 5 g/ml of actinomycin d was added to the culture medium at 2 h postinfection and cells were labeled with [ 35 s]methionine and cysteine at 6 h postinfection. cell lysates were prepared from cells harvested at 8 h postinfection and subjected to immunoprecipitation with anti-100, anti-68, anti-58, v17, and anti-35. v17 was raised against the ibv polypeptides encoded by nucleotides 19154-20414 and used to detect the 39-and 35-kda proteins in ibv-infected cells in a previous report . immunoprecipitation with anti-100 resulted in the detection of the 100-kda protein and a protein with an apparent molecular mass of 160 kda (fig. 2a, lane 10) . the 160-kda protein was also detected by anti-68 (fig. 2a, lane 9) . no product corresponding to the 68-kda putative helicase domain-containing protein was detected by anti-68 (fig. 2a, lane 9) . the detection of the 160-kda protein with the two n-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product containing the 100-kda and the putative 68-kda proteins. immunoprecipitation of the same lysates with anti-58 resulted in the detection of two products with apparent molecular masses of 58 and 132 kda (fig. 2a, lane 8) . the 132-kda protein was also immunoprecipitated by v17 and anti-35 (fig. 2a, lanes 6 and 7) . in addition, v17 also precipitated specifically the 39-and 35-kda proteins (fig. 2a, lane 7) . once again, very weak immunoprecipitation of the 35-kda protein was observed (fig. 2a, lane 7) . the 35-kda protein was also weakly immunoprecipitated by anti-35 (fig. 2a, lane 6 ). the detection of the 132-kda protein by the three c-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product derived from the c-terminal region of the polyprotein. interestingly, the three c-terminally specific antisera also weakly immunoprecipitated the 160-kda protein (fig. 2a , lanes 6-8). the reason for this result is currently unclear, but it may reflect the interaction among the cleavage products. as immunoprecipitation with anti-68 failed to detect the putative 68-kda protein in ibv-infected vero cells, we then tried to detect the protein by western blot with the same antiserum. for this purpose, cell lysates were prepared from ibv-infected vero cells harvested at 8, 24, and 32 h postinfection, respectively, and subjected to western blotting analysis. as shown in fig. 2b , a polypeptide with an apparent molecular mass of 68 kda was specifically detected in lysates prepared from cells harvested at 24 h postinfection (lane 2), and the expression of the protein was dramatically increased at 32 h postinfection (lane 3). expression and processing kinetics of the orf 1b region of the 1a/1b polyprotein in ibv-infected vero cells as the 160-and 132-kda proteins may represent stable intermediate cleavage products, time-course experiments were carried out to investigate the expression and processing kinetics of the two products in ibv-infected cells. for this purpose, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell and were labeled for 2 h with [ 35 s]methionine at 6 h postinfection. the cells were then washed with complete medium and chased with a 10-fold excess of cold methionine until they were harvested at appropriate times. immunoprecipitation of cell lysates with anti-100 showed the detection of both the 100-and 160-kda proteins throughout the time course (fig. 3a) . the 100-kda protein appeared at the beginning of the time course, peaked after chase for 1.5 h, and remained stable at the end of the time course (fig. 3a, lane 2) . the 160-kda protein peaked at the beginning of the time course and remained detectable after chase for 7.5 h (fig. 3a, lanes 1-6) . slight and gradual reduction of the 160-kda protein over time was observed (fig. 3a) . these results demonstrate that the 160-kda protein is a relatively stable intermediate cleavage product coexisting with the 100-kda mature cleavage product during the ibv infection cycle. similarly, immunoprecipitation of cell lysates with anti58 showed the detection of both the 132-and 58-kda proteins (fig. 3b) . the 132-kda protein appeared at the beginning of the time course, peaked after chased for 3 h, and remained detectable at the end of the time course (fig. 3b, lanes 9-12) . the 58-kda protein was first seen after chased for 3 h and gradually increased over time (fig. 3b, lanes 9-12) . interestingly, a product with an apparent molecular mass of 97 kda, representing an intermediate cleavage product containing the 58-and 39-kda proteins , was weakly detected and briefly observed during the time course (fig. 3b , lanes 9 and 10), indicating that it is not a stable intermediate cleavage product. the coexistence of the 132-and 58-kda proteins over time suggests that the 132-kda protein is a stable intermediate cleavage product. further characterization and definition of the coding sequences of products processed from the cterminal 200-kda region of the 1a/1b polyprotein two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were made and expressed to characterize the expression and processing patterns of the c-terminal 200-kda region of the 1a/1b polyprotein. in these two constructs, the region coding for the 3c-like proteinase was placed between the t7 promoter and the internal ribosome entry site (ires) of encephomyolitis virus (emcv), and the ibv sequences from nucleotides 15132-20506 and 15132-18495, respectively, were cloned downstream of ires (fig. 1) . expression of both constructs in cos-7 cells resulted in the detection of the 33-kda 3c-like proteinase, which comigrates on sds-page with the 33-kda protein detected in ibv-infected cells (fig. 4, lanes 2-4) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with anti-58 led to the detection of three protein species with apparent molecular masses of 200, 132, and 58 kda, respectively (fig. 4, lane 7) . the 200-kda protein represents the full-length product encoded by the ibv 1b sequence present in this plasmid, and the 132-and 58-kda proteins comigrated with the 132-and 58-kda proteins detected in ibv-infected cells (fig. 4, lanes 6 and 7) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv8-transfected cells with anti-58 led to the detection of the 58-kda protein and a product with an apparent molecular mass of 125 kda (fig. 4, lane 8) . the 125-kda protein may represent the full-length product encoded by the 1b sequence present in this construct. immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with antiserum v17 led to the detection of the 200-, 132-, 39-, and 35-kda proteins (fig. 4, lane 11) . the 132-, 39-, and 35-kda proteins comigrated with the three equivalent fig. 4 . comparative analysis of the 132-and 58-kda proteins expressed and processed in cells transfected with p3c-cite-ibv20, p3c-cite-ibv8, and pibv1b4 and in ibv-infected cells. plasmid dnas were expressed in cos-7 cells using the vaccinia virus-t7 expression system (fuerst et al., 1986) . semiconfluent monolayers of cos-7 cells were infected with a recombinant vaccinia virus (vtf7-3), transfected with plasmid dna using dotap according to the instructions of the manufacturer (roche), and labeled with [ 35 s]methionine and cysteine, and lysates were prepared. cell lysates prepared from mock-infected (lanes 1, 5, and 9) and ibv-infected (lanes 2, 6, and 10) vero cells and transfected cells (lanes 3, 4, 7, 8, 11-13) were immunoprecipitated with anti-3c (lanes 1-4) , anti-58 (lanes 5-8, 12, and 13), and v17 (lanes 9-11). the radiolabeled polypeptides were separated on an sds-12.5% polyacrylamide gel and detected by fluorography. numbers indicate molecular mass in kilodaltons. products detected in ibv-infected cells (fig. 4, lanes 9-11) . as mentioned earlier, the apparent molecular mass and processing pattern of the 132-kda protein suggested that it may be derived from the c-terminal region of the 1a/1b polyprotein covering the 58-, 39-, and 35-kda proteins. to further confirm this possibility, plasmid pibv1b4, which covers nucleotides 16932-20490 and therefore encodes the 132-kda product , was expressed in cos-7 cells. as expected, immunoprecipitation of cell lysates prepared from cells transfected with pibv1b4 with anti-58 resulted in the detection of the full-length 132-kda protein, which comigrated on sds-page with the 132-kda intermediate cleavage detected from cells transfected with p3c-cite-ibv20 (fig. 4, lanes 12 and 13) . to gain clues of the functions of the five cleavage products in the viral replication cycle, indirect immunofluorescence analysis of cells expressing individual cleavage products was carried out and representative confocal microscopy images are present in the proteins were labeled with the fitc-conjugated secondary antibodies. panels b, e, h, k, and n refer to cells stained with r6, a dye for the er. the green images represent fitc-derived green fluorescence, and red images represent rhodamine and texas red-derived red fluorescence. colocalization of viral proteins with the organelle markers is represented by the yellow region within each cell in the merged images (c, f, i, l, and o). panels a-o show cos-7 cells transfected with the empty vector pkt0 and stained with antisera or r6 as indicated. the fluorescence was viewed using a confocal scanning zeiss microscope. nofluorescent pattern of anti-pdi (data not shown). these results suggest that the three proteins may be associated with the er membranes. in cells expressing the 39and 35-kda proteins, a diffuse staining pattern was observed for each protein (figs. 5j and 5m), which does not coalign with the staining pattern of r6 (figs. 5j-5o). this diffuse distribution pattern of the 39-and 35-kda proteins was unexpected, as the majority of cleavage products were shown to be membrane-associated. the same antisera were used to stain cells transfected with the empty vector pkt0 (liu et al., 1994) , showing weak background staining (figs. 5a-5o) . the subcellular localization patterns of the five proteins were then analyzed in ibv-infected cells (fig. 6) . immunofluorescent staining of ibv-infected vero cells with anti-100, anti-68, and anti-58, respectively, showed similar er localization profiles (figs. 6a-6i), and a diffuse staining pattern was observed in cells stained with antiserum v17, which reacts with both the 39-and 35-kda proteins (figs. 6j-6l) . similarly, a diffuse distribution pattern was observed in cells stained with anti-35 (figs. 6m-6o). the same antisera were used to stain mockinfected cells, showing weak background staining (figs. 6a-6o). in our previous reports, we showed the identification of three mature cleavage products of 100, 39, and 35 kda, processed from the 1b region of the 1a/1b polyprotein (liu et al., 1994 . however, we were unable to detect the two other cleavage products of 68 and 58 kda. in this study, we report the identification of the 68-and 58-kda proteins in ibv-infected cells. in addition, two stable intermediate cleavage products of 160 and 132 kda, respectively, were identified, which coexist with the mature cleavage products in virus-infected cells. immunofluorescence analysis showed that the polymerase domain-containing 100-kda protein and the helicase domain-containing 68-kda protein as well as the 58-kda protein may be associated with the er and ic membranes, the viral replication and assembly sites. however, the 39-and 35-kda proteins display diffuse distribution patterns in both ibv-infected cells and cells expressing each of the proteins. it is intriguing that immunoprecipitation and western blot of ibv-infected cells harvested at 8 h postinfection failed to detect the 68-kda protein. the protein was detected by western blot in ibv-infected cells harvested at 24 and 32 h postinfection. as the equivalent human coronavirus 71-kda protein was shown to be an rna helicase (heusipp et al., 1997a; seybert et al., 2000a,b) , this protein is likely the ibv helicase. it is expected that such a functional product directly involved in viral rna replication would be expressed at early stages of the viral replication cycle. in fact, the equivalent 71-kda protein of human coronavirus was first seen in virus-infected cells at 5 h postinfection (heusipp et al., 1997a) . the reason for the failure to detect the 68-kda protein in ibv-infected cells at earlier time point is uncertain, but it might reflect the folding property, as discussed later, of the protein. the dramatic increase in the detection of the 68-kda protein at 24 and 32 h postinfection may partially be due to the secondary infection of cells that remain uninfected during the primary infection. as significantly more cells (over 95% of cells) were infected at 24-32 h postinfection, it is understandable that more protein would be detected. a slight, but gradual increase of the accumulation of the human coronavirus 71-kda protein over a time course of 15 h was also observed (heusipp et al., 1997a) . alternatively, the increase in the detection of the 68-kda protein at 24-32 h postinfection may reflect the genuine accumulation pattern of the protein in virusinfected cells at late stages of the viral replication cycle. if this were the case, it may indicate that the protein might also be involved in processes other than viral rna replication. the identification of two stable intermediate cleavage products of 160 and 132 kda coexisting with the mature cleavage products raised two interesting questions. first, the two products may have certain functions during the viral replication cycle. the 160-kda product is particularly interesting in this aspect, considering the fact that it contains both the polymerase and helicase domains and the 68-kda helicase protein is undetectable at early stages of infection. it is plausible that the 160-kda protein might have both polymerase and helicase functions in the replication of viral rna at early stages of the infection. in fact, some smaller positive-stranded rna viruses encode single proteins containing both the polymerase and helicase domains (buck, 1996) . the second interesting question is why only these two intermediate cleavage products were detectable in ibv-infected cells. as shown in fig. 4 as well as in our previous report , other intermediate cleavage products were also observed when this region was expressed in intact cells. one obvious distinct feature of the cleavage site between these two products is that it is a q-g dipeptide bond, while all the other cleavage sites in this region of the polyprotein are q-s dipeptide bonds (fig. 1) . however, no experimental data indicate that cleavage at the q-g site is more efficient than at the q-s sites. the 68-kda protein migrated on sds-page as a multiprotein species, heterogeneous smear, probably due to the formation of protein aggregates, and deletion analysis showed that a stretch of 30 amino acid residues in the c-terminal region was responsible for the aberrant migration property of the protein (data not shown). it suggests that the 68-kda protein may misfold when expressed in intact cells in the absence of other viral components. in recent years, it was found that the proper folding of certain proteins requires the assistance of molecular chaperones (ellis and van der vies, 1991; gething and sambrook, 1992) . as the 68-kda protein may be a component of the viral rna replication complex, the protein is expected to interact with viral rna and other viral proteins. those viral rna/proteins may act as a chaperone for the correct folding of the 68-kda protein in ibv-infected cells. it would be of interest to define if the region of the 30 amino acid residues that were shown to be responsible for the aberrant migration of the 68-kda protein on sds-page contain either rna binding or protein interacting domains. however, no such domains were found in this or the neighboring regions by computer analysis using relevant programs. the 58-kda protein was recently shown to be able to induce programmed cell death when expressed alone in intact cells . this is the first ibv product that was demonstrated to be a proapoptotic protein. in our previous report, we were unable to identify the 58-kda protein in ibv-infected cells due to the cross-reactivity of the antiserum used with a cellular protein . a newly raised antiserum was used in this study, leading to the successful identification of the protein in virus-infected cells. understanding of the expression, processing, and subcellular distribution pattern of the 58-kda protein would help us to further characterize the proapoptotic property of the protein and to study the functions of the protein in the pathogenesis of ibv-induced infection in chicken, the natural host of ibv. currently, no functions have been assigned to the 39and 35-kda proteins. a counterpart of the ibv 39-kda protein, the 41-kda protein of human coronavirus, was shown to exhibit a punctate, perinuclear distribution pat-tern in virus-infected cells (heusipp et al., 1997b) . interestingly, the 39-and 35-kda proteins display a diffuse distribution pattern in both ibv-infected cells and in cells overexpressing the proteins. as the majority of the cleavage products from the 1a and 1a/1b polyproteins were shown to be associated with cellular membranes at or near the viral replication and assembly sites, the diffuse distribution pattern may exclude the direct involvement of the two proteins in the formation of viral replication complexes. the egg-adapted beaudette strain of ibv (atcc vr-22) was obtained from the american type culture collection (atcc) and was adapted to vero cells as described by alonso-caplen et al. (1984) . briefly, the virus was passaged three times in 11-day-old chicken embryos and then adapted to vero cells (atcc ccl-81) by a series of passages at 24-48 h intervals. the cytopathic effects, including syncytium formation and rounding up of cells, were initially observed after three passages in vero cells. virus stocks were prepared after the 36th passage by infecting monolayers of vero cells at a m.o.i. of approximately 0.1 pfu/cell. the virus was harvested at 24 h postinfection and the titer of the virus preparation was determined by plaque assay on vero cells. vero cells were grown at 37°c in 5% co 2 and maintained in glasgow's modified minimal essential medium (gmem) supplemented with 10% newborn calf serum. confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu/cell. prior to being labeled, the cells were incubated in methionine-free medium for 30 min. after 4 h of labeling with 25 ci of [ 35 s]methionine, the cells were scraped off the dishes in phosphate-buffered saline (pbs), recovered by centrifugation, and stored at ϫ80°c. open reading frames placed under control of the t7 promoter were expressed transiently in eukaryotic cells as described previously (liu et al., 1994) . briefly, semiconfluent monolayers of vero cells were infected with 10 pfu/cell of a recombinant vaccinia virus (vtf7-3) which expresses the bacteriophage t7 rna polymerase and then transfected with appropriate plasmid dna using the dotap transfection reagent according to the instructions of the manufacturer (roche). after incubation of the cells at 37°c for 4 h, 25 ci/ml of [ 35 s]methionine was added directly to the medium. the radiolabeled cells were harvested at 18 h posttransfection. appropriate primers and template dnas were used in amplification reactions with pfu dna polymerase (stratagene) under standard buffer conditions with 2 mm mgcl 2 . the pcr conditions were 30 cycles of 95°c for 45 s, x°c for 45 s, and 72°c for x min. the annealing temperature (x°c) and the extension time (x min) were adjusted according to the melting temperature of the primers used and the length of the pcr fragments synthesized. plasmid dna-transfected vero cells were lysed with ripa buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 1% sodium deoxycholate, 0.1% sds, 1% np-40) and precleared by centrifugation at 12,000 rpm for 5 min at 4°c in a microfuge. immunoprecipitation was carried out as described previously (liu et al., 1994) . sds-polyacrylamide gel electrophoresis (sds-page) of virus polypeptides was carried out using 12.5% polyacrylamide gels (laemmli, 1970) . labeled polypeptides were detected by autoradiography or fluorography of dried gels. cells were grown on four-well chamber slides (iwaki) and infected with ibv or transfected with appropriate plasmid dnas. after washing with pbs, the cells were fixed with 4% paraformaldehyde (in pbs) for 15 min at room temperature and permeabilized with 0.2% triton x-100 (in pbs), followed by incubation with specific antiserum at room temperature for 2 h. antibodies were diluted in fluorescence dilution buffer (pbs with 5% normal goat serum). the cells were then washed with pbs and incubated with anti-rabbit igg conjugated to fluorescein isothiocynate (fitc) (sigma) in the fluorescence dilution buffer at 4°c for 1 h before mounting. confocal microscopy was performed on a zeiss axioplan microscope connected to a bio-rad mrc 1024 laser scanner equipped with an argon laser with appropriate filters. fluorescent images were superimposed to allow fine comparison and colocalization of green (fitc) and red (tritc) signals in a single pixel produces yellow, while separated signals are green or red. plasmid pibv1b4, which contains nucleotides 16932-20490, was previously described . plasmid pibv1b3 contains nucleotides 15132-16931 and codes for the 68-kda protein, pibv1b6 contains nucleotides 16930-18495 and codes for the 58-kda protein, pibv1b9 contains nucleotides 18496-19508 and codes for the 39-kda protein, and pibv1b10 contains nucleotides 19509-20506 and codes for the 35-kda protein. these constructs were made by cloning an ncoi-and bamhi-digested pcr fragment into ncoi-and bamhidigested pkt0 (liu et al., 1994) . the sequences of the two primers used to construct pibv1b3 were 5ј-cgact-tccatggcttgtggcgttј-3 and 5ј-ccaaaggatccta-ttgcagacttg-3ј. the sequences of the two primers used to construct pibv1b6 were 5ј-acaagtcccatggg-tacaggtt-3ј and 5ј-tattggatcctacggagagctg-3ј. the sequences of the two primers used to construct pibv1b9 were 5ј-gtttttcagctcccatggctatcgac-aat-3ј and 5ј-aaccacacgtcggatcctattgaagctg-tg-3ј. the sequence of the two primers used to construct pibv1b10 was 5ј-ccacagcttcccatggcatg-gacgtg-3ј. plasmid pibvpol, which contains nucleotides 12451-15131 and codes for the 100-kda protein with a 37-aminoacid truncation at the n-terminus, was constructed by cloning a bamhi-and xhoi-digested pcr fragment into bamhi-and xhoi-digested pet22b(ϩ) (novagen). the sequences of the primers used were 5ј-gtaataag-gatccagctggtatg-3ј and 5ј-aaggcctcgagttgta-aagtcgtaggagc-3ј. the two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were constructed as follows. the ires sequence was obtained by digestion of pcite-1 (novagen) with ecori, end-repair with klenow, and redigestion with ncoi. this 592-bp fragment was then cloned into pvuii-and ncoi-digested pkt0, giving rise to pkt-cite. the ibv sequence that codes for the 3c-like proteinase was obtained by digestion of pibv3c (liu et al., 1997) with bglii and bamhi and was cloned into bglii-digested pkt-cite, giving rise to p3c-cite. digestion of p3c-cite with ncoi produced a 1510-bp fragment containing both the ibv 3c-like proteinase and the ires sequences. this fragment was then cloned into ncoi-digested pibv1b8 and pibv20, respectively, creating the two dicistronic constructs. plasmids pibv1b8 and pibv20, which cover the ibv sequences from nucleotides 15132 to 18495 and 15132 to 20506, respectively, were constructed by cloning ncoi-and bamhi-digested pcr fragments covering the relevant regions into ncoi-and bamhi-digested pkt0. replication and morphogenesis of avian coronavirus in vero cells and their inhibition by monensin impaired integrinmediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant ptp1b subcellular distribution of normal and mutant vitamin d receptors in living cells four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus comparison of the replication of positive-stranded rna viruses of plants and animals the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis molecular chaperones eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase protein folding in the cell identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e identification and subcellular localization of a 41 kda, polyprotein 1ab processing product in human coronavirus 229e-infected cells cleavage of structural proteins during the assembly of the head of bacteriophage t4 characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by orf 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products the missing link in coronavirus assembly: retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus characterisation and mutational analysis of an orf 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products identification, expression, and processing of an 87-kda polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products identification of a 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a the human coronavirus 229e superfamily 1 helicase has rna and dna duplexunwinding activities with 5ј-3ј polarity biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes characterization of endoplasmic reticulum by colocalization of b-p and dicarbocyanine dyes localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab virus encoded proteinases and proteolytic processing in the nidovirales acknowledgment this work was supported by the national science and technology board of singapore. key: cord-342354-j10m2dfh authors: chen, huijie; feng, rui; muhammad, ishfaq; abbas, ghulam; zhang, yue; ren, yudong; huang, xiaodan; zhang, ruili; diao, lei; wang, xiurong; li, guangxing title: protective effects of hypericin against infectious bronchitis virus induced apoptosis and reactive oxygen species in chicken embryo kidney cells date: 2019-12-31 journal: poultry science doi: 10.3382/ps/pez465 sha: doc_id: 342354 cord_uid: j10m2dfh abstract avian infectious bronchitis virus (ibv), a coronavirus, causes infectious bronchitis leading to enormous economic loss in the poultry industry worldwide. hypericin (hy) is an excellent compound that has been investigated in antiviral, antineoplastic, and antidepressant. to investigate the inhibition effect of hy on ibv infection in chicken embryo kidney (cek) cells, 3 different experimental designs: pre-treatment of cells prior to ibv infection, direct treatment of ibv-infected cells, and pre-treatment of ibv prior to cell infection were used. quantitative real-time pcr (qrt-pcr), immunofluorescence assay (ifa), flow cytometry, and fluorescence microscopy were performed and virus titer was determined by tcid50. the results revealed that hy had a good anti-ibv effect when hy directly treated the ibv-infected cells, and virus infectivity decreased in a dose-dependent manner. furthermore, hy inhibited ibv-induced apoptosis in cek cells, and significantly reduced the mrna expression levels of fas, fasl, jnk, bax, caspase 3, and caspase 8, and significantly increased bcl-2 mrna expression level in cek cells. in addition, hy treatment could decrease ibv-induced reactive oxygen species (ros) generation in cek cells. these results suggested that hy showed potential antiviral activities against ibv infection involving the inhibition of apoptosis and ros generation in cek cells. avian infectious bronchitis virus (ibv), a member of the coronaviridae family (cook et al., 2012) , causes mild-to-acute respiratory disease in chickens (benyeda et al., 2009; westerbeck and machamer, 2019) . ibv infects chickens via the respiratory tract, from which it can spread to other epithelial tissues. therefore, ibv not only injures the respiratory tract, but also causes damage to the digestive system and urogenital system, such as the proventriculus, kidney, ovary, and oviduct, resulting in respiratory disease, interstitial nephritis, and dysplasia of the oviduct (zhong et al., 2016) . ibv has led to severe economic losses in the poultry industry worldwide. it has been demonstrated that ibv not only causes direct loss due to high mortality, secondary bac-terial infection, poor meat production, and egg quality, but also indirect losses arises from increasing drug costs and vaccination in ibv prevention (liang et al., 2019) . there are multiple known strains of ibv, and increasing numbers of variants have emerged continuous recombinations, resulting in more diversified and complicated genotypes and serotypes (feng et al., 2015; laconi et al., 2019) . because of poor vaccine cross protection among different virus serotypes, it makes difficult to prevent and control ibv infection with the current precautionary measures (mo et al., 2013; yan et al., 2018) . thus, finding effective antiviral drugs or agents is imperative for alternative approach to prevent ibv infection. the chinese government has prohibited the use of antiviral drugs in food animals. thus, utilization of traditional herbs remains a major focus in antiviral research. several reports have confirmed that traditional chinese herbs could effectively inhibit the replication of various viruses (chen et al., 2018; dziewulska et al., 2018; lv et al., 2019; xie et al., 2017) . hypericin (hy), a natural 6367 polycyclic quinone, is mainly extracted from st john's wort (hypericum perforatum l.) (barnes et al., 2019) . hypericin has traditionally been used in a wide range of medical applications such as anti-virus (birt et al., 2009; marrelli et al., 2014; naesens et al., 2006; wolfle et al., 2014) , anti-tumor (mirmalek et al., 2015) , and anti-depressant (zhai et al., 2015) . hypericin has virucidal activity against both dna and rna viruses with lipid envelope (tang et al., 1990) , suggesting that hy has the potential to be developed and used as antiviral drugs. in particular, hy has been found to have significant inhibitory effect on hiv infection (taher et al., 2002) . recent studies on the use of hy for inhibiting hepatitis c virus (hcv) have attracted more attention (jacobson et al., 2001; shih et al., 2018) . hepatitis c virus infection could induce oxidative stress and endoplasmic reticulum stress, which enhanced the production of reactive oxygen species (ros) and promoted caspase 3 activity, and finally led to hepatocyte injury in vivo and in vitro (ríos-ocampo et al., 2019) . similar to hcv, ibv is also an enveloped singlestranded positive-sense rna virus, whether hy has the likely inhibitory effect on ibv has not been explored. the aim of this study is to elucidate the antiviral activity and related mechanism of hy inhibiting ibv replication in chicken embryo kidney (cek) cells, and provide alternative approach to prevent and treat ibv infection. the impact of hy on relative mrna expression levels of ibv, apoptosis-related genes (including fas, fasl, jnk, bax, bcl-2, caspase 3, and caspase 8), and ros production in ibv-infected cek cells were studied. our results clearly demonstrated hy had potential antiviral activities against ibv infection involving the inhibition of apoptosis and ros generation in cek cells. experimental procedures were approved by the institutional animal ethical committee of northeast agricultural university (number srm-08). all animal studies were complied with the animal experiment guidelines of the animal experimentation ethics committee of northeast agricultural university. the ibv m41 strain was kept at veterinary pathology lab, college of veterinary medicine, northeast agricultural university, and propagated in 10-day-old specific pathogen-free (spf) chicken embryos (harbin veterinary research institute, chinese academy of agricultural sciences). the allantoic fluid of infected embryos was harvested at 72 h post inoculation, and confirmed no other pathogens detected with reverse transcription polymerase chain reaction (rt-pcr) and bacterial culture, and finally stored at −80°c until use. primary cultures of cek cells from 18-day-old spf chicken embryos (harbin veterinary research institute, caas) were prepared according to standard techniques. the cells were cultured in m199 medium (thermo fisher scientific, waltham, ma, usa) supplemented with penicillin-streptomycin and 10% fetal bovine serum at 37°c with 5% co 2 to allow the formation of cell monolayers in cell culture plates. hypericin powder (sigma, st. louis, mo) with purity 99% was determined by high performance liquid chromatography. a total of 20 mg hy was pre-dissolved in 500 µl dimethylsulfoxide (dmso, sigma) and then added 4.5 ml m199 medium as hy stock solution, in which hy concentration is 4,000 µg/ml, and dmso concentration is 10% (v/v). the stock solution of hy (4.0 mg/ml) was stored at −20°c in the dark. ribavirin (rt, h51023508) was from sichuan biokin pharmaceutical co., ltd. (chengdu, china). n-acetyl cysteine (nac) and 2 7 -dichlorofluorescein diacetate (dcfh-da) were obtained from sigma (st. louis, mo, usa). annexin v-fitc kit was purchased from shanghai beyotime biotechnology co., ltd. (shanghai, china). the apoptosis promoter pac-1 (mce, brea, ca, usa) and anti-apoptotic z-vad-fmk (mce, brea, ca, usa) were prepared into 10 mm solution with dmso and stored at −20°c. fluorescein isothiocyanate (fitc)-conjugated affinipure goat anti-rabbit igg was purchased from zhongshan company (beijing, china). anti-ibv polyclonal antibody was prepared and kept in veterinary pathology laboratory, college of veterinary medicine of northeast agricultural university, which could specifically react with ibv. the cytotoxicity assays were performed according to published 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) protocols with minor modifications (sui et al., 2010) . cell monolayers plated onto 96-well culture plates were washed 3 times with d-hanks solution. hypericin at concentrations of 3. 125, 6.25, 12.5, 25, 50, 100, 200 , and 400 µg/ml were added onto the wells (3 wells per each dilution). control cells were incubated with the same corresponding concentrations of dmso solutions as negative controls. the cells were cultured at 37°c for 48 h, and the supernatant was taken out and washed 3 times with d-hanks solution, then 20 µl of mtt solution (5 mg/ml in m199 medium) was added to each well and incubated for 4 h at 37˚c. after washing with d-hanks solution, 200 µl of dmso was added to each well. the plates were shaken gently for 15 min to dissolve the formazan precipitate, and the od570 was recorded. cell survival rates were calculated from mean values according to the following equation: (od570 drug)/(od570 control) × 100%. cell viability was determined by mtt colorimetric assay. the survival rates of cek cells was given at different concentrations of hy, and cell survival rates of more than 50% (above the dotted line) was considered to be the maximum non-toxic concentration of hy. cek = chicken embryo kidney; hy = hypericin. data are expressed as mean ± sd of 3 independent experiments (t-test, *p < 0.05, **p < 0.01). to analyze the impact of hy on cell to inhibit ibv infection, the cek cells cultured on 96-well plates were treated with continuous multiple dilutions of hy solutions in serum-free m199 medium, at 37˚c for 1 h and then washed 3 times with d-hanks solution. the cells were subsequently infected with ibv (100 tcid 50 ) and cultured at 37˚c for 30 h. the cell samples were repeatedly frozen and thawed 3 times, extracted to obtain total rnas, and reverse transcribed into cdna. the relative mrna expression levels of ibv n gene was detected with quantitative real-time rt-pcr (qrt-pcr). in addition, the virus titer of the cell samples was determined by tcid 50 according to conventional figure 2 . the impact of hy on the relative mrna expression levels of ibv n gene and virus titer in cek cells. in 3 different hy treatment group of pre-treatment cells prior to ibv infection, direct treatment of ibv-infected cells, and pre-treatment ibv prior to cell infection, the relative mrna expression levels of ibv-n gene (2a) and the virus titer (2b) were detected, respectively. ibv-infected cek cells and 10 μg/ml rt-treated cek cells after ibv infection were included as controls. cek = chicken embryo kidney; hy = hypericin; rt = ribavirin. data are expressed as mean ± sd of 3 independent experiments (t-test, *p < 0.05, **p < 0.01). procedures. meanwhile, the 10 µg/ml rt-treated cells after ibv infection and ibv-infected cek cells were processed as controls. to determine the effect of hy on ibv-infected cells, the cek cells cultured on 96-well plates were infected with 100 tcid 50 ibv for 1 h at 37˚c, and then treated with continuous multiple dilutions of hy solutions at 37˚c for 30 h. according to the above description, the relative mrna expression levels of ibv n gene and virus titer were detected. pre-treatment of ibv prior to cell infection to investigate direct effects of hy on the virus, 100 tcid 50 ibv was incubated with continuous multiple dilutions of hy solutions at 37˚c for 1 h. the hy-treated ibv was subsequently used to infect cek cells at 37˚c for 30 h. as described above, the relative mrna expression levels of ibv n gene and virus titer were detected. in the above 3 experimental designs, the best way of antiviral effect of hy on ibv was selected, and ibv multiplication in cek cells was detected with indirect immunofluorescence assay (ifa). after washing with d-hanks solution, the 3.125, 6.25, and 12.5 µg/ml of hy directly treated ibv-infected cells were fixed with 4% paraformaldehyde in pbs followed by quenching with 0.1% glycine in pbs and 1% triton-x100 for 10 min. after washing, the cells were incubated with rabbit anti-ibv antibody (1:200) for 1 h followed by incubation with fluorescence-conjugated goat anti-rabbit igg (1:500) for 30 min in the dark. the fluorescent images were examined under a ti-s fluorescence microscope (nikon, tokyo, japan). additionally, 10 µg/ml rt-treated cek cells after ibv infection, ibv-infected cek cells, and mock cek cells were detected as controls. in total, 2 approaches were used to evaluate the effect of hy on cell apoptosis as described previously (singh et al., 2019) . in brief, 100 tcid50 ibv-infected cek cells treated with 12.5 µg/ml hy were cultured at 37°c for 30 h. then, the apoptotic cells were analyzed using an annexin v-fitc kit as described previously (leber et al., 2018) . the 20 µm pac-1-treated cek cells, 20 µm z-vad-fmk-treated cek cells followed with ibv infection, ibv-infected cek cells, and mock cek cells were prepared and assayed concurrently. moreover, flow cytometry was used to quantify the number of annexin v and propidium iodide-stained cells among different experimental groups. in brief, the ibv-infected cells were treated with 12.5 µg/ml hy as above and trypsinated. the viable cells determined with trypan blue staining were adjusted to a final density of 4 × 10 5 cells/ml. then the cells were incubated with fitc-conjugated anti-annexin v antibody and propidium iodide for 15 min in the dark prior to flow cytometry analysis. the ibv-infected cek cells and mock cek cells were tested as controls. the total rnas of different experimental groups were extracted using a universal rna extraction kit (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. all the concentrations and the a260/a280 ratios of extracted rna samples were measured using nanodrop 2000 spectrophotometer (thermo fisher scientific, figure 3 . inhibitory effects of hy on ibv infection detected by immunofluorescence assays. the cek cells were infected with 100 tcid50 ibv for 1 h at 37˚c followed by incubation with serially diluted hy at concentrations of 3.125, 6.25, and 12.5 µg/ml for 30 h. the ibv-infected cek cells, 10 μg/ml rt-treated cek cells after ibv infection and mock cek cells were included as controls, respectively. fluorescence intensity (20×) is proportional to viral infectivity and inversely related to the treatments with different concentrations of hy. cek = chicken embryo kidney; hy = hypericin; rt = ribavirin. waltham, ma, usa), and agarose gel electrophoresis was used to assay the integrity of the extracted rna. the total rnas were reverse transcribed into cdnas using moloney murine leukemia virus reverse transcriptase and oligo (dt) (takara bio inc., dalian, china) with pcr instrument (takara, japan) and stored at −20°c for the following qrt-pcr assay. the mrna expression levels of target genes were determined, including ibv n gene, fas, fasl, jnk, bax, bcl-2, caspase 3, and caspase 8 with an applied lightcycler 96 real-time pcr system (roche molecular systems, inc., schlieren, switzerland). all primers pairs used for qrt-pcr detection are listed in table 1 . these primers were synthesized by the genewiz biological technology co., ltd (suzhou, china). the relative expression ratios of target genes were calculated using the 2 −δδct method (yu et al., 2017) . the amplification reaction was conducted containing 2 µl of cdna, 0.6 µl of each of the forward and reverse primers, 10 µl universal sybr green (rox), and 3.4 µl nuclease free water for a final volume of 20 µl for each reaction. the following thermal profile for the qrt-pcr assays was used in all the primer sets: preincubation 1 cycles at 95°c for 600 s, followed by 2 step amplification: 42 cycles at 95°c for 15 s and 60°c for 30 s, followed by melting: 1 cycles at 95°c for 10 s, 65°c for 60 s and 97°c for 1 s, followed by cooling 1 cycles at 37°c for 30 s. 2 7 -dichlorofluorescein diacetate probe was used to measure the intracellular ros scavenging activity of hy in ibv-infected cells (chavez and pietras, 2018) . briefly, the cek cells were pre-incubated with ibv for 1 h, followed by incubation with 3.125, 6.25, and 12.5 µg/ml hy for 30 h. the medium was removed and the cells were washed with pbs. then, the medium containing 10 μm dcfh-da was added for 30 min at 37°c. after washing with pbs, cells were photographed using a ti-s fluorescence microscope (nikon, tokyo, japan). and the cek cells were pre-treated with 1.5 mm nac before ibv infection as control. the number of the experiment repetition in all the experiment was 3 times. the experimental data was analyzed with spss 17.0 software (spss inc., chicago, il). the results were expressed as the means ± standard deviation. differences between groups were evaluated using the one-way analysis of variance of two-tailed test. p-values of < 0.05 were considered as statistically significant, and p-values of < 0.01 were considered as highly significant. the cek cells were infected with 100 tcid50 ibv for 1 h at 37˚c followed by incubating 12.5 µg/ml of hy for 30 h. the 20 µm pac-1-treated cek cells, 20 µm z-vad-fmk pre-treated cek cells before ibv infection, ibv-infected cek cells and mock cek cells were included as control. the green fluorescence signals are designated as the index of early apoptosis in immunofluorescence analysis. fluorescence intensity (10×) is provided. (b) to quantify the cell apoptosis, the 12.5 µg/ml hy-treated cek cells after ibv infection, ibv-infected cek cells and mock cek cells were stained with annexin v-fitc and pi, and then analyzed by flow cytometry. data are presented as dual parameter consisting of annexin v versus pi. the early apoptosis (annexin v-fitc staining) and late apoptosis (pi staining) were reflected in q4 and q2 gates, respectively. (c) the percentages shown in the column chart are the proportion of apoptotic cells (right panel) in different experimental groups. the differences between means were considered highly significant at **p < 0.01 compared with the ibv-infected control group. cek = chicken embryo kidney; hy = hypericin; pi = propidium iodide. figure 5 . effects of hy on the mrna expression level of apoptosis-related genes in vitro. the cek cells were infected with 100 tcid50 ibv for 1 h at 37˚c followed by incubation with hy at the concentration of 12.5 µg/ml. the cek cells were simultaneously cultured with m199 medium as a mock control, and infected with 100 tcid50 ibv as a virus control, and treated with hy at a concentration of 12.5 µg/ml as a drug reference, respectively. total rna was subsequently extracted from cell lysates at 24, 30, and 36 h after treatment. the relative mrna expression of fas (a), fasl (b), jnk (c), bax (d), bcl-2 (e), caspase 3 (f), and caspase 8 (g) were assessed by rt-qpcr. cek = chicken embryo kidney; ibv = infectious bronchitis virus; hy = hypericin. the differences between the means were considered significant at *p < 0.05 and highly significant at **p < 0.01 as compared with the ibv-infected control group. figure 6 . inhibitory effects of hy on ros production. the cek cells were infected with 100 tcid50 ibv for 1 h at 37˚c followed by incubation with serially diluted hy at concentrations of 3.125, 6.25, and 12.5 µg/ml for 30 h. the cek cells were simultaneously cultured with m199 medium as a mock control, and infected with 100 tcid50 ibv as a virus control, and pre-incubated with 1.5 mm nac for 1 h before ibv infection as a positive control, respectively. fluorescence intensity (10×) is proportional to viral infectivity and inversely related to hy concentration. cek = chicken embryo kidney; hy = hypericin; nac = n-acetyl cysteine. the cell viability indicated by trypan blue staining was observed under an optical microscope, and further confirmed by mtt assays. the results showed that the maximum nontoxic concentration of hy was 12.5 μg/ml. trypan blue staining showed that cells survived below 12.5 μg/ml of hy ( figure 1a ). cell proliferation determined by mtt assay had no significant effect and the survival rate was nearly 100% below 12.5 μg/ml of hy ( figure 1b ). the antiviral effect of hy was analyzed by the relative mrna expression levels of ibv-n gene (figure 2a ) and the virus titer ( figure 2b ) in cek cells. from the figure 2a , it could be seen that in the 3 experimental designs, the viral mrna level significantly decreased compared with the virus control, and with the increase of hy concentration, the mrna level of ibv decreased in a dose-dependent manner. furthermore, at the same drug concentration, the relative expression levels of the viral mrna was the lowest when hy directly treated the ibv-infected cells, followed by the group of hy pretreatment of ibv prior to cell infection, and followed by the group of hy pre-treatment of cells prior to ibv infection. the virus titers at each group was detected and showed in figure 2b , with the increase of hy concen-tration, the titer of ibv decreased gradually. while, at the same drug concentration, the virus titer was the lowest when hy directly treated the infected virus cells, followed by the group of hy pre-treatment virus prior to infection, and followed by the group of hy pretreatment cells prior to infection. meanwhile, the antiviral effect of hy directly treated the ibv-infected cells at the concentration of 12.5 µg/ml, was similar to rt at the concentration of 10 µg/ml ( figure 2) . therefore, hy directly treated the ibv-infected cells was selected to conduct in followed experiments. to further confirm the inhibitory effect of hy on ibv-infected cells, the viral fluorescent signal was detected by ifa ( figure 3 ). as can be seen from figure 3 , ibv-infected cek cells generated strong fluorescence signals at 30 h post-infection. in contrast, the fluorescence signals in hy directly treated the ibv-infected cells decreased obviously in a dose-dependent manner. this further confirmed the inhibition effect of hy on ibv-infected cells. the immunofluorescence results showed that addition of hy to virus-infected cells resulted in fewer apoptotic cells compared with virus infection control and apoptosis promoter pac-1 (20 µm) control group, and similar to the apoptosis inhibitor z-vad-fmk (20 µm) control group ( figure 4a ). the number of apoptotic cells was quantified with flow cytometry. the results showed that there was ∼2.59-fold decrease in the percentage of apoptotic cells at 12.5 µg/ml of hy treatment group compared with virus infection group ( figures 4b and 4c ). the mrna expression levels of apoptosis-related genes, including fas, fasl, jnk, bax, bcl-2, caspase 3, and caspase 8 were determined. as can be seen from figure 5 , hy treatment significantly reduced the mrna levels of fas, fasl, jnk, bax, caspase 3, and caspase 8 genes ( figures 5a−d, 5f , and 5g) after ibv infection at 30 h and 36 h, and significantly increased the mrna expression levels of bcl-2 gene after ibv infection at 24 h, 30 h, and 36 h ( figure 5e ). these data demonstrated that hy inhibited apoptosis in ibvinfected cek cells by decreasing the expression of proapoptotic genes and increasing anti-apoptotic gene expression. however, no difference could be found in the above detected parameters in the only hy-treated cek cells compared with those in mock cells. we determined the ros generation in hy directly treated the ibv-infected cells using dcfh-da method. as shown in figure 6 , ibv could significantly induce the ros production, while hy could inhibit the ros production in ibv-infected cek cells. and with the increase of hy concentration, the fluorescence signal of ros decreased in a dose-dependent manner. the effect of inhibiting ros at 6.25, 12.5 µg/ml of hy treatment was similar to ros inhibitors nac. our results showed that hy could inhibit ibv infection and inhibit apoptosis and production of ros in cek cells. it has been reported that there were multiple known strains of ibv, ibv strain m41, is typical respiratory virus, which replicates primarily in the respiratory tract and subsequently spreads and replicates in a range of other tissues (de wit et al., 2019; hodgson et al., 2004) . the cek cells are the most susceptible cells of strain m41 in vitro (abdel-moneim et al., 2009; zhang et al., 2017) . therefore, we selected m41 strain for infection of cek cells to study the anti-ibv effect of hy. in the cytotoxicity assay, mtt assay was performed to measure cell viability. mtt method is a recognized and commonly used method to evaluate the metabolic activity of living cells (gao et al., 2019; şueki et al., 2019) . because hy is low levels of solubility in water (bianchini et al., 2019; montanha et al., 2017) , it is insoluble in m199 cell culture medium. in mtt assay, hy was pro-dissolved in dmso first, and diluted to a certain concentration with medium m199. in the process of dilution, the content of dmso is not more than 1% (hebling et al., 2015; şimşek et al., 2015) , in order to ensure that dmso is non-toxic to the cells and to obtain the maximum non-toxic concentration of hy accurately. by observing the cell morphology and calculating the cell survival rate, the maximum non-toxic concentration of hy was 12.50 µg/ml (figure 1) . a total of 3 different experimental designs: (1) pretreatment of cells prior to infection; (2) direct treatment of virus-infected cells; and (3) pre-treatment of virus prior to infection were used to analyze the antiviral activity of hy on cek cells infection by ibv (de la torre et al., 2011; yin et al., 2011) . our results showed that direct treatment of hy on virus-infected cells was the best among 3 different experimental designs ( figure 2 ). in the direct treatment of hy on virusinfected cells, it may be due to the effect of hy on infected cells, effectively inhibited the activity of viral transcriptase, and effectively reduced the replication of ibv in cek cells. the pre-treatment of hy on ibv prior to infection may be related to the photoperiod and oxygen supply of hy. under light conditions, hy could absorb photons, and then stimulate singlet oxygen to play an antiviral role. because this experiment was carried out under the condition of no oxygen and weak light, the direct attack effect of hy on virus particles was not brought into full play, and the killing effect of virus was weakened (miskovsky, 2002; xu et al., 2019) . in the pre-treatment of hy on cells prior to infection, hy blockaded on the adsorption of virus in cek cells was weak, or did not protect the cells, thus failed to effectively prevent ibv from entering the host cells (pu et al., 2009 (pu et al., , 2012 . therefore, in the following experiments, the cek cells were first infected ibv, and then incubated hy solution. to further confirm the inhibitory effect of direct treatment of hy on virusinfected cells, the viral fluorescent signal was detected by ifa (figure 3 ). our results confirmed the inhibition effect of direct treatment of hy on virus-infected cells. while apoptosis usually functions as a host defense mechanism that ensures killing of infected cells (elmore, 2007) , several viruses, including ibv, have been shown to induce apoptosis to enable efficient virus transmission, while avoiding overt inflammatory responses and activation of the immune system . our results showed that ibv could induce apoptosis in cek cells (figure 4) , and increase mrna expression levels of fas, fasl, jnk, bax, caspase 3, and caspase 8, with the exception of bcl-2, which significantly decreased after ibv infection ( figure 5) , which was consistent with other reports (chhabra et al., 2016; fung et al., 2014; liao et al., 2013) . it was found that ibv could increase the mrna expression levels of fas, fasl, and bax, and decrease the mrna expression level of bcl-2 in ibv-infected hd11 cells (han et al., 2017) . another research demonstrated that jnk served as a pro-apoptotic protein during ibv infection. interestingly, pro-apoptotic activity of jnk was not mediated via c-jun, but involved modulation of the anti-apoptotic protein b-cell lymphoma 2 (bcl2) (fung and liu, 2017) . moreover, our results showed that hy could inhibit apoptosis induced by ibv on cek cells (figure 4) , and reduced mrna expression levels of fas, fasl, jnk, bax, caspase 3, and caspase 8, but increased bcl-2 mrna expression level after ibv infection ( figure 5 ). this is similar with human umbilical vein endothelial cells, in which hy could inhibit cell apoptosis by affecting the expression of apoptosisrelated genes and inhibit production of ros (do and kim, 2017) . antioxidant activity of hypericum perforatum l. and its components have been reported (gioti et al., 2009 ). furthermore, the study showed that hy had an antioxidative effect (cakir et al., 2003) . therefore, we investigated whether hy ameliorated ibv-induced ros generation. our results revealed that hy could inhibit ros production in the absence of phototherapy ( figure 6) , which was consistent with other report (da silva et al., 2018) . in summary, the present study was the first time to show that hy inhibited ibv infection. furthermore, anti-ibv effect of hy may be associated with its inhibition of apoptosis and ros production. hy reduced the mrna levels of apoptosis-related genes such as fas, fasl, jnk, bax, caspase 3, and caspase 8, and upregulated anti-apoptotic gene (bcl-2) mrna expression. the data suggest the potential use of hy as antiviral agents against ibv, however, further studies are required to elucidate the molecular mechanism of hy on ibv infection. immunohistochemistry for detection of avian infectious bronchitis virus strain m41 in the proventriculus and nervous system of experimentally infected chicken embryos st john's wort (hypericum perforatum l.): botanical, chemical, pharmacological and clinical advances comparison of the pathogenicity of qx-like, m41 and 793/b infectious bronchitis strains from different pathological conditions hypericinapomyoglobin: an enhanced photosensitizer complex for the treatment of tumor cells hypericum in infection: identification of anti-viral and antiinflammatory constituents isolation and characterization of antioxidant phenolic compounds from the aerial parts of hypericum hyssopifolium l. by activity-guided fractionation hematopoietic stem cells rock around the clock: circadian fate control via tnf/ros signals antiviral effect of baicalin phospholipid complex against duck hepatitis a virus type 1 pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses the long view: 40 years of infectious bronchitis research mitochondrial bioenergy alterations in avian hd11 macrophages infected with infectious bronchitis virus. arch. virol action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus induction of ibv strain-specific neutralizing antibodies and broad spectrum protection in layer pullets primed with ibv massachusetts (mass) and 793b vaccines prior to injection of inactivated vaccine containing mass antigen hypericin, a naphthodianthrone derivative, prevents methylglyoxal-induced human endothelial cell dysfunction an evaluation of the impact of aloe vera and licorice extracts on the course of experimental pigeon paramyxovirus type 1 infection in pigeons apoptosis: a review of programmed cell death development and efficacy of a novel live-attenuated qx-like nephropathogenic infectious bronchitis virus vaccine in china the endoplasmic reticulum stress sensor ire1alpha protects cells from apoptosis induced by the coronavirus infectious bronchitis virus activation of the c-jun nh2-terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-jun effects of n-trans-feruloyltyramine isolated from laba garlic on antioxidant, cytotoxic activities and h2o2-induced oxidative damage in hepg2 and l02 cells antioxidant activity and bioactive components of the aerial parts of hypericum perforatum l. from epirus infectious bronchitis virus infection induces apoptosis during replication in chicken macrophage hd11 cells cytotoxicity of dimethyl sulfoxide (dmso) in direct contact with odontoblast-like cells recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity pharmacokinetics, safety, and antiviral effects of hypericin, a derivative of st. john's wort plant, in patients with chronic hepatitis c virus infection molecular characterization of whole genome sequence of infectious bronchitis virus 624i genotype confirms the close relationship with q1 genotype unleashing blocked apoptosis in cancer cells: new mcl1 inhibitors find their groove n-linked glycosylation of the membrane protein ectodomain regulates infectious bronchitis virus-induced er stress response, apoptosis and pathogenesis upregulation of chop/gadd153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway explore the potential effect of natural herbals to resist newcastle disease virus hypericum perforatum: influences of the habitat on chemical composition, photo-induced cytotoxicity, and antiradical activity cytotoxic and apoptogenic effect of hypericin, the bioactive component of hypericum perforatum on the mcf-7 human breast cancer cell line hypericin-a new antiviral and antitumor photosensitizer: mechanism of action and interaction with biological macromolecules molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china response surface method optimization of a novel hypericin formulation in p123 micelles for colorectal cancer and antimicrobial photodynamic therapy antiviral activity of diverse classes of broad-acting agents and natural compounds in hhv-6-infected lymphoblasts anti-influenza a virus effect of hypericum perforatum l. extract therapeutic efficacy of hypericum perforatum l. extract for mice infected with an influenza a virus. can hepatitis c virus core or ns3/4a protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress hypericin inhibits hepatitis c virus replication via deacetylation and down-regulation of heme oxygenase-1 dimethyl sulfoxide-caused changes in proand anti-angiogenic factor levels could contribute to an antiangiogenic response in hela cells regulation of apoptosis in health and disease: the balancing act of bcl-2 family proteins the effect of curcumin in antitumor photodynamic therapy: in vitro experiments with caco-2 and pc-3 cancer lines antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus mood-enhancing antidepressant st. john's wort inhibits the activation of human immunodeficiency virus gene expression by ultraviolet light virucidal activity of hypericin against enveloped and non-enveloped dna and rna viruses the infectious bronchitis coronavirus envelope protein alters golgi ph to protect spike protein and promote release of infectious virus topical application of st. john's wort (hypericum perforatum) effect of chinese herbal medicine treatment on plasma lipid profile and hepatic lipid metabolism in hetian broiler hypericin-photodynamic therapy inhibits the growth of adult t-cell leukemia cells through induction of apoptosis and suppression of viral transcription attenuation, safety, and efficacy of a qx-like infectious bronchitis virus serotype vaccine vitro and in vivo effects of houttuynia cordata on infectious bronchitis virus avian infectious bronchitis virus disrupts the melanoma differentiation associated gene 5 (mda5) signaling pathway by cleavage of the adaptor protein mavs lc-ms/ms based studies on the anti-depressant effect of hypericin in the chronic unpredictable mild stress rat model heat shock protein 70 in lung and kidney of specific-pathogenfree chickens is a receptor-associated protein that interacts with the binding domain of the spike protein of infectious bronchitis virus pathogenicity of virulent infectious bronchitis virus isolate yn on hen ovary and oviduct virus infection and death receptor-mediated apoptosis this research was supported by the national natural science foundation of china (31172295 and 31272569), and in part supported by grants from the key subject of traditional chinese medicine of jilin agricultural science and technology university. key: cord-349149-nqsohp9h authors: lounas, a.; oumouna-benachour, k.; medkour, h.; oumouna, m. title: the first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in algeria date: 2018-11-29 journal: vet world doi: 10.14202/vetworld.2018.1630-1636 sha: doc_id: 349149 cord_uid: nqsohp9h background and aim: avian infectious bronchitis virus (ibv) frequently infects broilers and is responsible for severe economic losses to the poultry industry worldwide. it has also been associated with kidney damage in the broiler flocks. the aim of the present study is to determine the presence of ibv and its possible involvement in kidney damage of broiler chicks. materials and methods: 14 clinically diseased broiler flocks from western and central algeria were sampled and analyzed by hemagglutination inhibition (hi) test and reverse transcriptase-polymerase chain reaction (rt-pcr) followed by phylogenic analysis. results: the qx (100%) and 4/91 (60%) ibv serotypes were the most prevalent in the kidney damaged broilers regardless of vaccination status. the molecular detection of avian ibv by rt-pcr identified six samples as positive, of which only two isolates were typable by sequencing. we identified a novel ibdz13a genotype which showed 93% sequence homology to the partial-s1 gene sequence of the ib 4/91 commercial vaccine strain. sequencing analysis characterized this virus as a novel and divergent ib 4/91 field virus with eight amino acid substitutions that might have resulted in altered immunogenicity. conclusion: the isolation of a new ibv strain (ibdz13a) from vaccinated broiler flocks may explain the failure of the vaccination programs against ibv field strains. combination of the hi test and rt-pcr indicated that the nephropathogenic ib outbreaks in broilers are related to this novel strain. poultry production in algeria faces many zootechnical and health constraints, such as viral infections like avian infectious bronchitis (ib). the avian ib virus (ibv), a member of the coronaviridae family (order nidovirales and genus coronavirus), frequently infects broilers and egg-laying hens and leads to severe economic losses to the poultry industry [1] . since its discovery in the 1930s [2] , the ibv has been identified as the major cause of respiratory infections and poor zootechnical performances. interestingly, it can also multiply in the renal tissue and cause nephritis [3] , a phenomenon first described in the united states [4] . more recently, ibv-associated nephritis has been accepted as the most pressing problem in broiler flocks in many countries [5] [6] [7] [8] [9] . the most effective method of protecting poultry from ibv infections is through live or killed vaccines [1] . however, nephritis associated with infectious bronchitis has been observed in several vaccinated flocks, suggesting that the current vaccination strategies against ibv may not provide adequate protection [3, 10] . in fact, outbreaks of ib are frequently caused due to the strains serologically different from those used for vaccination [11, 12] . since its discovery in 1931, a large number of serotypes or variants of ibv have emerged [5, 13, 14] , and little or no cross-protection occurs between these serotypes [15] . therefore, it is crucial to track epidemic-causing serotypes in each geographic region or country and produce new vaccines to control ib. in algeria, poultry flocks have been vaccinated against ib with the massachusetts (mass) strain combined with the ib 4/91 united kingdom variant strain or 793/b since the past few years. however, kidney damage with suspicion of ib has also been reported in recent years in spite of vaccination but has not been confirmed so far. this has led to the speculation of the possible emergence of variant strains against which conventional vaccines are not completely effective [16] . the aim of this study was to investigate the presence of ibv among algerian broiler flocks and its possible involvement in broiler kidney damage. clinically diseased broiler flocks were sampled and analyzed by the hemagglutination inhibition (hi) test and reverse transcriptase-polymerase chain reaction (rt-pcr) followed by phylogenic analysis. all experimental procedures were approved by the institutional animal care committee of the national administration of the algerian higher education and scientific research (ethical approval number: 98-11, law of august 22, 1998) and were conducted according to the recommendations of the "guide for the care and use of laboratory animals." fourteen commercial broiler flocks from western and central algeria in which the chickens were suffering from kidney damage and respiratory disease were sampled between january and december 2012. trachea and kidney specimens were taken from 10 diseased chickens from each flock and were directly applied to the fta ® cards (ge healthcare, uk) during the acute phase of the clinical outbreak. the samples were transported and analyzed at the laboratory of virology, instituto zooprofilattico sperimentale delle venezie, italy. in addition, blood samples were also collected from each bird to determine the prevalence of different ibv serotypes. the serum was collected from the samples by centrifugation (2000 rpm/min 5 min) and stored at -20°c for further analysis. hi test was performed at msd international laboratory, amman, jordan. three pieces of 2 mm discs from the spotted area of the filters were cut by a sterile scissor and placed in 1.5 ml eppendorf tubes. separate sterile scissors and pincers were used for each sample to prevent cross-contamination. each disc was placed in 750 µl trifast ® fl (peqlab, germany), homogenized, and incubated for 5 min at rt. the trifast fl kit (peq gold trifast tm fl, peqlab) was used to extract the rna from the fta ® paper according to the manufacturer's instructions (peqlab biotechnologie gmbh, erlangen, germany). rna purity and concentration were determined by nanodrop ® nd-1000 (peqlab biotechnologie gmbh, erlangen, germany). reverse transcription was performed using the improm-ii tm reverse transcription system (promega, madison, wi) with random primers according to the manufacturer's instructions. briefly, 0.1 µm lyophilized oligonucleotides provided with the kit were dissolved in 1 ml rnase-free water, and 1 µl of the random primer solution was mixed with 4 µl viral rna and incubated at 70°c for 5 min. after adding buffer 5× (56 µl), mgcl 2 (67.2 µl), dntp (14 µl), rnasin (7 µl), improm ii (14 µl), and h 2 o (51.8 µl), the mixture was incubated at 25°c for 5 min followed by 55°c for 60 min. the reaction was terminated by heating at 70°c for 15 min and cooling on ice. the primer sets (oligo5 [forward]: 5'-tga aaa ctg aac aaa aga ca-3' and ck2 [reverse]: 5'-ctc gaa ttc cng trt trt ayt grc a-3') were designed for the ibv s1 gene, which contains the hypervariable region 1 and most of the hypervariable region 2 [17] . the s1 gene was amplified with the superscript ® iii one-step rt-pcr platinum ® taq hifi (invitrogen tm life technologies tm , carlsbad, ca), which generated a pcr product of 344 bp. for the pcr reaction, the following reaction mix was made: 35 µl buffer 10×, 14 µl dntp, 14 µl ck2, 14 µl oligo5, 243.25 µl h 2 o, 1.75 µl taq polymerase (taq hifi, invitrogen tm life technologies tm , carlsbad, ca), and 2 µl cdna to a final volume of 25 µl. the pcr parameters were as follows: 45 cycles of denaturation (94°c, 20 s), annealing (51°c, 1 min), and polymerization (72°c, 1 min). the pre-denaturation step was done at 94°c for 5 min and post-polymerization step at 70°c for 10 min. a 20 µl aliquot of each pcr product was electrophoresed at 120 v for 40 min in 1.5% agarose gel, which was then stained with gelred tm (genaxxon bioscience, ulm, germany, gmbh). the amplified cdna was purified using the nucleospin ® (macherey-nagel gmbh, germany) gel and pcr clean-up kit according to the manufacturer's instructions and sent for sequencing to x-ovo limited (x-ovo limited laboratories, thomson cooper, united kingdom). the nucleotide and amino acid sequences of the s1 gene of the ibv isolates were assembled, aligned, and compared with reference ibv strains using basic blast program in dnastar. phylogenetic analysis of the s1 amino acid sequences was performed with the neighbor-joining method using the dnastar software [18] . the partial s1 nucleotide sequences from the ibv isolates have been submitted to the genbank database and assigned the accession number mg252734. the nucleotide and amino acid sequences of the s1 gene of ibv strains used for the comparison are available in genbank under the following accession numbers: cr 88 (km067900), 4/91 (jn600614), qx (af193423), ma5 (ay561713), h120 (m21970), m41 (gq219712), d274 (kr106999), italy 02 (aj457137), and qx d388 (dq674739). the specific ibv strains that were used in this study were m-41 (a us strain), 4/91 (european vaccine strain), d274 (a dutch field isolate), and qx (chinese strain). hi test was performed using the available at www.veterinaryworld.org/vol.11/november-2018/17.pdf flokscreen ib-hi test kit according to the manufacturer's instructions. briefly, the sera were serially diluted and mixed with an equivalent volume of the antigen. after 30 min of incubation at 20°c, the red blood cells of spf chickens (anticoagulant solution) were added and incubated further for 40 min at 20°c. the plates were then tilted to observe inhibition of hemagglutination. statistical package for the social sciences version 20 (ibm, usa) was used for statistical analysis. the geometric mean titer (gmt) was calculated by counting the number of wells from all serum samples within one flock showing hi activity, and the average number was then cross-checked against gmt values given in brug's table. the results were reported as the gmt for a given flock against a particular ibv antigen. descriptive statistics including percentages, means, and frequency distribution were calculated for each of the variables. information regarding the sampling date, biosecurity status, region of sampling, vaccination programs, necropsic and clinical signs, the age of the broilers, and mortality rates are summarized in table1. we collected 14 samples from broiler farms located in western and central algeria from the flocks showing kidney damage and high mortality rates. eight of these farms had not vaccinated their flocks against ibv, 2 farms conducted the prototype vaccination program but with a short interval between the first and second vaccinations, and 4 farms vaccinated their broilers only against the mass serotype. all vaccinations were performed through the drinking water route, and all the sampled broiler farms showed poor zootechnical performance with rare period elongation (until 71 days in farm 2) and high mortality rates (up to 50% in farms 11 and 13). the seroprevalence of various ibv strains in broiler flocks suffering from kidney damage in algeria is summarized in table2. all the broiler flocks (100%) tested in this study were seropositive for the qx variant strain, with high mean titer levels (mt=11 log 2 ) regardless of the vaccination status. furthermore, 60% and 50% of the broiler flocks were seropositive for antibodies against the 4/91 and m-41 strains, respectively, although with low mean titer levels. the seroprevalence of ibv d-274 was the lowest with only 7% of the broiler flocks testing positive. six of the 14 broiler samples were positive for the ibv s1 gene, but only two isolates were typable by sequencing and showed a high load of ib viral rna (ct 1 =22.15 and ct 2 =31.4). one of the two sequences was identified as the ibdz13a genotype. furthermore, ibv was only detected in the kidney tissues and not in the trachea. all samples showed 93% homology to the partial-s1 sequence acquired from (figure-1) . the ibdz13a strain was characterized as an ib 4/91 variant ibv with 29 nucleic acid substitutions differing from the known ib 4/91 vaccine reference strain sequence, which corresponded to eight amino acid substitutions relative to this reference strain (table-3 ). three of these amino acid substitutions were from a hydrophobic to a hydrophilic amino acid. a change in hydrophilicity altered the epitopes on the s1 protein of the ib dz13a virus. the predicted epitopes with differences between the field virus and ib 4/91 vaccine strain are shown in figure1. infectious bronchitis has had a devastating effect on broiler farms since the worldwide emergence and increased prevalence of ibv [11, 13] . due to the high rate of mutation and recombination in coronaviruses, new serotypes and genotypes of ibv frequently emerge and have been reported in different regions around the world [11] . more recently, ibv has been associated with nephritis which is now a major problem in broiler flocks worldwide [19] [20] [21] . similar concerns have been reported in algeria, but no study has been performed so far on ib-associated nephritis [22] . figure-1: predicted epitope alignment of deduced amino sequences of hvr s1 gene. new field algerian isolate and selected infectious bronchitis virus vaccine vial reference strains. the present study was carried out to investigate the presence of ibv in broiler flocks and its possible involvement in kidney damage. we also monitored the seroprevalence of different ibv serotypes by the hi test and analyzed the phylogenetic characteristics of the new ibv strains isolated in algeria in 2013 by sequencing. the hi test and virus neutralization test (vnt) are the most commonly used tests for detecting ibv antibodies [9, 23] . studies show extensive cross-reactions between the ibv serotypes detected by the hi test and similar antigenicity detected by the vnt and hi test [24] [25] [26] . however, since vnts are not cost-effective for routine examination, hi tests are commonly used for routine serology [27] . the most frequently detected ibv serotypes in the broiler flocks with nephritis were ib-qx (100%) and ib 4/91 (60%), which have been detected for the 1 st time in algeria by our group. after reporting the nephropathogenic form of ib [3] , the above serotypes have been reported in china, europe, and africa and have been seen to be associated with severe kidney damage, high morbidity, and mortality in the vaccinated flocks. at present, these serotypes are the predominant field strains in most poultry farms worldwide [13, 28] . the serological analysis showed significantly lower titers of ib 4/91 compared to ib-qx, which could be related to the vaccination of some flocks with the ib 4/91 strain. de wit [23] reported that many factors influence the level of ibv antibodies, of which the most critical is the presence of immunity at the time of infection. this is consistent with the lower anti-ib 4/91 antibody titers seen in the vaccinated flocks compared to the unvaccinated ones. another factor that can reduce the sensitivity of hi and thus affect the interpretation of the results is the cross-reaction between the different serotypes, especially when multiple serotypes of ibv are involved. we detected cross-reaction between the qx and 4/91 serotypes in many flocks, and 4 flocks showed mixed infection of three serotypes (qx, 4/91, and m41). therefore, the serotype specificity of hi is reduced after reinfection or vaccination, especially when the subsequent serotype is heterologous [23] . the s1 protein of ibv determines the genotype, serotype, and phenotype of ibvs [29] . the s1 gene of the ibdz13a field strain was most closely related to that of the ib 4/91-type strain (figure-2) as determined by the criterion of worthington et al. [13] and kannan et al. [30] , who classified ibv sequences with <99% homology to vaccine strains as variants. our findings indicate the circulation of virulent strains of ib 4/91, which have become the predominant genotype in africa, asia, and europe [28] . however, we did not see a strong correlation between the hi results which showed the circulation of the qx and 4/91 serotypes and rt-pcr which showed the emergence of a new genotype of the 4/91 strain. this is consistent with the findings of kusters et al. [31] who also reported that rt-pcr and hi results were not exactly correlated, wherein different serotypes showed considerable differences (20-50%) in the amino acid sequences of the s1 subunit. hi is used for routine testing of sera from young growing chickens to determine the serotype-specific antibody status of a flock [32] . however, the presence of multiple cross-reactive antibodies against antigenically unrelated strains, as is often seen during ib outbreaks, reduces the serodiagnostic value of hi. therefore, a combination of serotyping and genotyping is recommended to identify field strains. based on the results of hi and rt-pcr, we conclude that the ibdz13a variant of ib4/91 is involved in kidney damage of broiler flocks. when live-attenuated and inactivated vaccines of ibv were first introduced, they were effective in controlling ib among poultry flocks [33] . however, vaccinated flocks in many regions of algeria have shown considerable mortality and production losses in recent years. the isolation of a new ibv strain from the vaccinated and unvaccinated flocks can be explained by two hypotheses. the first hypothesis, based on the mutations and amino acid alterations in the s1 epitope, is that the ibv strain evolved to escape the host immune response elicited by the current vaccines [34] . however, this hypothesis is weak since amino acid alterations do not always translate into differences in antigenicity or biological function. furthermore, the sequence data only provide information regarding the primary structure of the protein and not the secondary and tertiary structures which are vital for its biological function and antigenicity [23] . the second hypothesis is the failure to adopt the vaccination programs to the ibv field strains circulating in the farms. in our study, only two flocks received the combined vaccine of ib ma5 (at day 7) and ib 4/91 (at day 14) . the failure to protect broilers against nephritis could also be related to the shorter inter-vaccination interval of 7 days. terregino et al. [35] demonstrated improved protection against several ib serotypes by vaccinating with two antigenically different ib vaccines given at the interval of 14 days. this approach obviates the need to develop a new ib vaccine for each new emerging serotype. one flock was vaccinated with the mass vaccine type h120 strain, which, however, did not provide sufficient protection against ibv infection [33, 36] . we identified ibdz13a, a new nephropathogenic variant of ib 4/91, for the 1 st time in algerian broiler flocks. its isolation from the vaccinated flocks indicates poor protection afforded by the vaccination programs to the broilers against infection with new ib variants. further studies are needed to explain the failure of the current vaccines against the new ib4/91 strain and to investigate the cross-protection and pathogenicity of the new genotype. al and mo planned and designed the whole study. al and hm collected samples, performed technique, and analyzed the data. ko and mo supervised the project and helped during manuscript writing, crosschecking, and revision. all authors read and approved the final manuscript. infectious bronchitis an apparently new respiratory disease of baby chicks a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china etiology of an infectious nephritis-nephrosis syndrome of chickens avian infectious bronchitis: characterization of new isolates from italy isolation and identification of four isolates of infectious bronchitis strains in china and analysis of their si protein gene isolation of 4/91 type of infectious bronchitis virus as a new variant in japan and efficacy of vaccination against 4/91 type field isolate an overview of infectious bronchitis virus in chickens phylogenetic analysis of avian infectious bronchitis virus s1 glycoprotein regions reveals emergence of a new genotype in moroccan broiler chicken flocks s1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during a reverse transcriptase polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from molecular epizootiology of avian infectious bronchitis virus in russia coronavirus avian infectious bronchitis virus evidence of natural recombination within the s1 gene of infectious bronchitis virus gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel characterization and analysis of an infectious bronchitis virus strain isolated from southern china in 2013 genetic grouping of nephropathogenic avian infectious bronchitis virus isolated in morocco virulent avian infectious bronchitis virus, people's republic of china ) isolation, identification and molecular characterization of ibv variant from outbreak of visceral gout in commercial descriptive study of an outbreak of avian urolithiasis in a large commercial egg complex in algeria detection of infectious bronchitis virus serological comparison and antigenic relationships of seven serotypes of infectious bronchitis virus using the haemagglutination-inhibition test application of the haemagglutination inhibition test to typing of infectious bronchitis virus comparison of the haemagglutination inhibition test and the serum neutralization test in tracheal organ cultures for typing infectious bronchitis virus strains manual of diagnostic tests and vaccines for terrestrial animals. world organisation for animal health infectious bronchitis virus variants history, current situation and control measures genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between genotypes of infectious bronchitis viruses circulating in the middle east between phylogeny of antigenic variants of avian coronavirus ibv a standard technique for haemagglutination inhibition tests for antibodies to avian infectious bronchitis virus protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus review of infectious bronchitis virus around the world pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen-free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma5 and 4/91 serotypes efficacy of different vaccination programs against thai qx-like infectious bronchitis virus the authors gratefully acknowledge the msd international laboratory, amman, jordan, and msd, algeria, for providing the facilitation and equipment throughout the study. the authors are also thankful to entisar and husam bakri for technical assistance and help with the hi test analysis. the authors had used the facility of msd international laboratory, jordan but no person from msd was involved at any stage of the experiment or publication. also, authors have not received any fund from msd algeria. key: cord-329468-vjsurl60 authors: okino, cintia hiromi; mores, marcos antônio zanella; trevisol, iara maria; coldebella, arlei; montassier, hélio josé; brentano, liana title: early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles date: 2017-02-15 journal: plos one doi: 10.1371/journal.pone.0172275 sha: doc_id: 329468 cord_uid: vjsurl60 avian infectious bronchitis virus (ibv) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. however, the association between the local immune responses induced by ibv and the mechanisms of pathogenesis has not yet been completely elucidated. this study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two brazilian field isolates (a and b) of ibv from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. we detected a suppressive effect on the early activation of tlr7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the ibv b isolate when compared to the challenge with to the ibv a isolate. cell-mediated immune (cmi) related genes presented also lower levels of expression in tracheal samples from birds challenged with b isolate at 1dpi. increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with ibv b isolate. this differential pattern of early immune responses developed after challenge with ibv b isolate, related to the downregulation of tlr7, leading to insufficient pro-inflammatory response and lower cmi responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken. avian infectious bronchitis virus (ibv) is a highly infectious causative agent of avian infectious bronchitis (ib), a disease of high economic impact, which affects poultry worldwide. ibv replicates primarily in tracheal epithelial cells, inducing several mucosal pathological changes, including ciliary loss, degeneration and necrosis of epithelial cells, glandular degeneration, inflammatory cell infiltration and epithelial hyperplasia [1, 2] . after replication at primary site in tracheal mucosa, viraemia and ibv secondary replication are also found in other respiratory a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 tissues (nose, lungs and air sacs) and in many non-respiratory epithelial tissues (kidneys, testes, oviduct, and gastrointestinal tract). nephritis is commonly observed, mainly in broilers, and depending on the pathotype of the ibv strain and on the bird age, it may cause high mortality, and microscopic lesions of degeneration and necrosis of the renal tubular cells and interstitial inflammatory cell infiltrate [3, 4, 5, 6] . prevention of ibv infection is currently achieved through vaccination, especially by attenuated viral vaccines, suggesting that local mucosal immunity is essential for induction of effective protection against disease [7, 8, 9] . in a previous study, we demonstrated that the dose of attenuated vaccine administered by local route is closely related to humoral and cellular immune responses at tracheal mucosal sites and their ability to confer effective protection against disease [8] . the continuous emergence of new ibv variants in several countries [3, 10, 11, 12, 13] are routinely pointed out as a cause of outbreaks in vaccinated flocks, leading to significant economic losses to the poultry industry [14] . despite the great number of ibv strains and variants described over the last years [14] , most of the studies have been limited to the classification and differentiation of ibv strains in genotypes, pathotypes, protectotypes, and/or serotypes, while, the specific immune mechanisms involved in pathogenesis of this virus remains poorly elucidated. a recent study has observed differential early immune response after infection with ibv when comparing ibv-susceptible and ibv-resistant chicken lines. even before infection, specific genes, including genes related to innate immune response, were found to be differentially expressed in each chicken line. despite these differences, viral loads were similar in tracheal samples of both chicken lines, indicating that ibv resistance might be associated with how the birds respond to the virus and not how they can prevent an initial infection [15] . the innate immune response is the first line of host defense against infections, and several immune and non-immune cells on mucosal surfaces are involved on recognition of pathogen associated molecular patterns (pamp) of microorganisms. pamps are recognized through pattern recognition receptors (prr), among which toll-like receptors (tlr) are an important group. during a rna-virus infection, activation of innate immune responses depends on the interaction of endosome-associated (tlr3 and tlr7) and/or cytosol-located (rig-i like receptors) prrs. tlr3 and tlr7 interact with double-stranded (ds) rna or single-strand (ss) rna, respectively, and the subsequent activation pathways for tlr3 and tlr7 are mediated by trif (tir-domain-containing adapter-inducing interferon-β) and myd88 (myeloid differentiation primary response gene 88), respectively, which lead to the production of anti-viral type i interferons (ifnα and ifnβ) and pro-inflammatory cytokines (il1β and il6) [16] . expression of genes related to the tlr pathway activation and type i ifn were already found to be upregulated in tracheal samples from birds challenged with reference strains of ibv [7, 16, 17] . the inducible nitric oxide synthase (inos or nos2), responsible for the production of nitric oxide (no) by macrophages in response against microbial infections, is often associated with synergic effects combined with acute phase proteins and cytokines, as ifns and tnfs, leading to enhanced phagocytosis. alternatively, an excessive production of inos and consequently no may have negative effects by promoting excessive inflammation or apoptosis [18, 19] . although a previous report did not identify a change in inos transcripts in both tracheal and lung samples during the early phase of ibv infection [16] , increased levels of this enzyme was associated with high degree of disease severity in chickens infected with avian influenza h5n1 [18] , and virulent newcastle disease [20] . the important role of pro-inflammatory cytokines on ibv pathogenicity was demonstrated in a previous evaluation of the kinetics of the inflammatory and cell-mediated immune responses in tracheal mucosa of birds infected with the m41 strain of ibv. tnfsf15 and tgfβ transcripts were found to peak at 1 day post infection (dpi), followed by higher upregulation of il6, il1β and ifnγ. these increased levels of pro-inflammatory cytokines were associated to highest scores of lesions and viral load at 3dpi, whereas later, at 7dpi, highest increases in cd8 and granzyme homolog a mrna expression were detected and associated to a significant decrease in lesions and viral load [2] . it has been recognized that il6 is associated with development of tissue damage in chickens challenged with ibv, including nephritis [21] . levels 20 times higher of il6 mrna after ibv challenge were observed in an ibv-susceptible chicken line compared to the disease-resistant chicken line [22] . high level of il6 gene expression were detected in renal samples of chickens challenged with a nephropathogenic ibv strain and it has been well correlated with viral load and influx of inflammatory cells in this organ [6] . during the development of adaptive immunity against ibv infection, the local cell-mediated immune (cmi) response was associated with cd3+, cd8+ and cd4+ cells influx in the trachea from 3 to 7 dpi with ibv [1, 9] . furthermore, cd8 and other cmi related genes were markedly increased during secondary response related to memory protection induced by the first antigen ibv exposition at 1dpi (8) . conversely, the cmi genes were found most upregulated later, at 5dpi, in the primary adaptive immune response of non-vaccinated chickens and correlated with high virus load in trachea, indicating this response might be more involved in the ibv pathogenesis in this post-infection period of non-immune chickens [8] . mechanisms underlying immune-pathogenesis caused by ibv and those relating to viral clearance have begun to be elucidated [2, 4, 7, 8, 16, 17, 22, 23] . however, there are no studies regarding the existence of differential pathological and immunological profiles induced by ibv strains or isolates differing in virulence for chickens. the aim of this study was to investigate and compare the pathogenesis and the innate and adaptive immune responses induced by the infection with two brazilian field isolates of ibv classified in the br-i genotype. two brazilian field isolates bi/br/embrapa/331/2000 (accession number: ku727196, identified as ibv a isolate) and bi/br/embrapa/127/2006 (accession number: ku727200, identified as ibv b isolate), were isolated from clinical cases of avian infectious bronchitis in poultry flocks located in southern brazil. virus isolation and titration were carried out by inoculation of five 10-day-old specific pathogen-free (spf) embryonated chicken eggs/sample via the allantoic sac route [24] and virus titers were expressed as 50% embryo infectious doses (eid 50 ) according to the reed & muench method [25] . ibv field strains were genotyped by automated dna sequencing (abi3130xl, applied biosystems) of a 1059 bp portion of the s1 gene amplified as described [26] . the deduced amino acid sequences of s1 protein of ibv a and b isolates have 93.16% of identity, and with regard h120 strain (massachusetts serotype) of ibv they have 69.4% and 70.0% identity, respectively. three groups of specific pathogen free (spf) chicks (white leghorn lineage) obtained from valo biomedia (uberlândia, sp, brazil) were housed in separate positive pressure isolators. at 28 days of age, groups a and b were respectively challenged with 10 4.0 eid 50 /bird of a strain of ibv and 10 4.0 eid 50 /bird of b strain of ibv by intra-ocular and intra-nasal routes. one group remained mock infected (negative control group, referred as nc). during whole experimentation, chickens were monitored twice a day. if animals have presented severe depression and lethargy, they were separated to be euthanized, though, there were no animals in these conditions during experiment. the birds from groups a, b and nc were randomly euthanized by cervical dislocation, at 1, 5 and 8 day post-infection (dpi). tracheal and renal samples were collected from each group; a portion of the proximal third from each tracheal sample and a fragment of distal left kidney were immediately frozen in liquid nitrogen and kept at -70˚c until processing for rna extraction. another portion of tracheal and renal samples were subjected to histopathological analysis. throughout the experimental period, birds received water and feed "ad libitum", the room temperature was adapted according with the bird´s age, and the monitored ammonia concentration levels remained below 2 parts per million (ppm). tracheal samples were divided into three fragments (proximal, medial and distal), and a portion of each fragment was prepared for histopathology. the severity of observed lesions scores were determined as previously described [8] . absence of injury was classified as 0, while mild, moderate and severe were scored as grades 1 to 3. morphological characteristics observed by histopathology included loss of cilia and epithelial cells, degeneration or necrosis of epithelial cells, degeneration of mucous glands, mucosal inflammatory infiltrate and epithelial hyperplasia. a sum of all scores of microscopic lesions from all evaluated fragments was amounted to final score per bird, ranging from 0 to 45. birds presenting score values ! 2 in two or more evaluated parameters or presenting marked heterophilic infiltration were considered as relevant lesions induced by ibv. the scores of microscopic lesions in the kidney samples were determined as previously described [6] . the parameters evaluated included tubular degeneration and necrosis, and presence of inflammatory infiltrate, and the scores ranged from 0 to 3 according to the severity of lesions. rna extractions from the proximal third of tracheal and renal samples of experimentally infected chickens were performed using trizol reagent (invitrogen) followed by rna purification using rneasy mini kit (qiagen). the extracted rna purity and quantification were estimated by 260nm ultraviolet absorbance and readings at 260/280nm, respectively. rna quality was verified with agilent rna 6000 nano kit (agilent technologies) in an agilent 2100 bioanalyzer instrument (agilent technologies) for determination of rin (rna integrity number) or by rna analysis in 1% gel electrophoresis. all tracheal and renal samples were tested for ibv viral load by rt-qpcr (hydrolysis probe system) using agpath-id one step rt-pcr kit (ambion), primers and lna-probe (5´fam-3 bhq1, idt) for amplification of the 3´utr genome region of ibv, as described [27] . cq (cycle quantification) results were used to calculate the log of rna copies (log10) using the linear equation from a standard curve. samples presenting cq 36 were classified as positive for ibv. two-step rt-qpcr was used for the relative quantification of gene expression in the tracheal samples. cdnas were synthesised according to instructions provided with high capacity cdna reverse transcription kit (applied biosystems) and 1μl (500 ng/μl) of oligo(dt) primers (idt). the pcr reactions contained 20 ng of cdna, 7.5 μl of 2x quantifast sybr green master mix (qiagen), and 3 μm of each primer (table 1 ) in a final volume of 15 μl. amplification included a pre-incubation step at 95˚c for 5 min, followed by 40 cycles of 95˚c for 15 seconds and 60.0˚c for 35 seconds. after amplification, a melting curve analysis was performed by raising the incubation temperature from 65˚c to 95˚c in 0.2˚c increments with a hold step of 1 sec at each increment. all oligonucleotides used were designed using primer3 [http://frodo.wi.mit.edu] software, spanning exons according to gene sequences from ensembl [http://ensembl.org] and mrna sequences deposited in genbank. except for ifnα and ifnβ which are intron-less mrnas, then, residual genomic dna was digested with 2 μl dnase i (promega) before cdna synthesis. efficiency of each specific real-time pcr was calculated table 1 using two-fold serial dilutions of cdna pooled from all tested animals, and cq values plotted into the linear equation. the relative expression of all tested genes (table 1) in tracheal samples of ibv-infected chickens was quantified as the fold change relative to the non-infected group (negative control), and the gene expression from each sample were standardised using the cq value of the top2b/hprt1 constitutive reference genes for the same sample [28] . the stability of the reference genes was tested using four candidates (top2b, hprt1, gapdh and histone h3) and the best genes were selected using bestkeeper and normfinder softwares. the comparisons of relative changes in gene expression, viral load and microscopic lesions between the experimental groups were performed using the kruskal-wallis test followed by wilcoxon test. all analyses were conducted using the sas 9.4 software (2012), and the probability level for significance was set as p 0.05. the tracheal lesions observed in groups challenged with a or b ibv isolates at 1 dpi were characterized by mild acute tracheitis, consisting of the presence of heterophils and mucus exudation in the tracheal lumen with congestion and heterophilic infiltration in the lamina propria and epithelial cell desquamation (fig 1a) . the most prominent lesions were observed at 5 dpi, with lesion scores ranging from 20 to 30, consisting of tracheitis. the microscopic pathological [1] , congestion [2] , heterophilic cell infiltrate [3] . (b) proximal third of trachea from the group challenged with ibv a strain, at 5dpi, showing ciliary loss [1] , degeneration and necrosis of some epithelial cells [2] , lymphoid cell infiltrate [3] and epithelial hyperplasia [4] . (c) proximal third of trachea from nc group. changes consisted mainly of lymphoplasmacytic inflammatory infiltrate in the mucosa, ciliary loss and epithelial hyperplasia (7 1b). degeneration of the mucous glands and of epithelial cells was also observed at lower frequency and intensity. at 8 dpi, lesions were mild, tending to tissue recovery. scores between 7 and 15 were found mainly in the challenged groups. the main histopathological changes were degeneration of the mucous glands, lymphocytic infiltration in the mucosa and epithelial hyperplasia. no relevant microscopic tracheal alterations were observed in the nc group at all post-infection intervals analyzed (fig 1c) . marked lymphoplasmacytic interstitial nephritis (lesion score 3) was only observed in the challenged groups (a and b), at 8dpi (fig 2a) . tubular necrosis was not observed in any renal sample from all experimental groups. a higher percentage of birds challenged with ibv b isolate showed relevant tracheal (at 1dpi and 5dpi) and renal (at 8dpi) microscopic lesions ( table 2 ) compared to the birds challenge with ibv a isolate. no positive samples were detected for the presence of ibv genome in tracheal and renal samples from the negative control (nc) group in any of the post-infection intervals analysed (fig 3 and table 3 ). high percentages (87.5% and 100%) of tracheal samples were positive at 1 dpi for the presence of ibv genome from chickens challenged with ibv a and b strains, and differed significantly from tracheal samples from nc group (fig 3a and table 3 ). at 5dpi, all chickens from the challenged groups were ibv positive, with similar virus loads to those detected at 1dpi, differing also significantly different from nc group. the renal samples from chickens of a group showed 88.89% of positivity for ibv genome in kidney samples while all the renal samples from chickens of b group were positive for ibv genome, (fig 3 and table 3 ). the presence and quantity of ibv genome in renal samples differed significantly from those of nc group. the viral load decreased at 8dpi in tracheal samples from all ibv challenged groups (a and b) (fig 4b) . despite of lower virus levels, all challenged groups maintained 100% of positive birds (table 3) . additionally, all the chickens from group challenged with ibv b isolate were positive for the presence of ibv genome in renal samples and only 16.67% were positive for the a challenged group, a significant higher viral load was found in b group when compared to other experimental groups (fig 3b and table 3 ). significantly higher means of ibv genome load (10 to 1000 times higher) were detected in group challenged with ibv b isolate compared to the group challenged with ibv a isolate, at 5dpi interval in tracheal samples (p = 0.0041) and at 8dpi in renal samples (p = 0.0041). (fig 3) . the mrna expression data for tlr3, tlr7, myd88, inos, ifnα and ifnβ in tracheal samples are illustrated in fig 4. tlr3 was significantly upregulated and peaked at 1dpi in ibv ibv positive birds at 1dpi ibv positive birds at 5dpi ibv positive birds at 8dpi trachea trachea kidney trachea kidney nc 0/8 (0%)* 0/9 (0%) 0/9 (0%) 0/5 (0%) 0/5 (0%) a 7/8 (87.5%)* 9/9 (100%) 8/9 (88.89%) 6/6 (100%) 1/6 (16.67%) b 9/9 (100%) 9/9 (100%) 9/9 (100%) 5/5 (100%) 5/5 (100%) nc = negative control group; a = group challenged with ibv a isolate; b = group challenged with ibv b isolate. * one sample was lost during processing. challenged groups. at 5dpi, tlr3 transcripts also remained upregulated in challenged groups, although the levels were lower compared to values found at 1dpi. at 8dpi, tlr3 mrna dropped markedly in challenged groups, but still remained significantly upregulated (fig 4a) . no significant differences were detected in the expression of tlr3 gene between the tracheal samples of chickens infected with a and b ibv isolates, and significant differences were seen by comparing the birds from groups a and b with those from control mock-infected birds. tlr7 transcripts were downregulated (5 times) at 1 dpi only in the tracheal samples from b-challenged group, but their levels of expression rose at 5dpi and maintained at high levels at 8dpi in all challenged groups (a and b) (fig 4b) . myd88 mrna transcripts were upregulated in tracheal samples from all challenged groups (a and b) and at all post-infection intervals. at 8dpi, the samples from a group presented higher levels than those from b group (p = 0.0176) (fig 4f) . ifnα was upregulated at 5dpi and 8dpi in tracheal samples from chickens of b group, and only at 8dpi in the samples from a group chickens. ifnβ transcripts were upregulated in chickens from b group at 1dpi and 5dpi, while in chickens from the a group, only at 5dpi (fig 4c and 4d) . the inos gene showed a significant increase in mrna expression at 1dpi in challenged groups. at 5dpi and 8dpi, these transcripts dropped to basal levels ( fig 4e) . il1β transcripts were significantly upregulated in tracheal samples from all challenged groups at 1, 5 and 8 dpi and the highest levels of il1β expression were reached at 8dpi. (fig 5a) . il6 mrna transcripts were significantly upregulated in tracheal samples from challenged groups at 1 and 5 dpi. at 8 dpi, only the samples from group a showed upregulation in the expression of this cytokine gene (fig 5b) . the tnfsf15 mrna was only significantly upregulated at 8dpi, in the group challenged with ibv a isolate (fig 5c) . the cd3 and cd4 mrna transcripts were downregulated at 1dpi only in the tracheal samples from the b group. at 5dpi and 8dpi, all tracheal samples from challenged groups showed upregulation of cd3 and cd4 expression, and the highest levels of expression of these genes were observed at 5dpi (fig 6a and 6b) . at 5dpi and 8dpi, all challenged groups presented upregulation of cd8β transcripts, and the highest levels of expression of this gene were detected at 5dpi (fig 6c) . the levels of ifnγ gene expression started to increase at 1dpi in group challenged with ibv a isolate. all challenged groups, presented upregulation of ifnγ transcripts, at 5dpi and 8dpi and the highest levels were found 5dpi (fig 6d) . the transcripts of granzyme homolog a mrna were upregulated in all challenged groups at the three post-infection intervals evaluated, except for the group challenged with ibv b isolate, at 1dpi (fig 6e) . the host-virus interactions that result in more severe pathogenesis induced by different pathotypes of ibv are not fully elucidate, but the ibv pathogenesis has been associated with high virus replication rates, induction of exacerbated inflammatory responses and delayed activation of anti-viral effector mechanism of innate and adaptive immune responses [2, 16, 31] . in this study, we found a suppressive effect on expression of some early innate and adaptive cell-mediated immune genes in the primary site of virus replication (trachea) from chickens infected with one of the tested ibv isolates (b). this possibly contributed to an exacerbated pathogenicity of this ibv isolate, especially for kidney tissues, but not for a isolate. ibv b isolate demonstrated an enhanced ability for viral replication in chicken host, as we observed significantly higher ibv genome loads in tracheal samples (at 5dpi), and in kidneys (at 8dpi) of birds challenged with this isolate, compared to the group infected with ibv a isolate. furthermore, a higher percentage of positive birds for ibv genome and the presence of pathological alterations were observed in the ibv b infected group, especially in renal samples, that were ibv positive in birds infected with this isolate at 8dpi, while only 17% were positive in the group infected with ibv a isolate. this enhanced viral replication of ibv b isolate compared to a isolate, may have played a role as one of the determinants for the higher percentage of chickens with relevant microscopic tracheal and renal lesions in birds from the ibv b challenged group. this could result in increased pathology of the ibv b isolate. however, the reason for the differences observed in tissue tropism and pathogenicity of different ibv strains remains an open question and cannot be associated in the present study to differences in s1 protein sequences, as a and b ibv isolates have an identity of xx % for this protein. similarly, a recent study demonstrated that despite the same efficiency of s1 protein binding to the epithelial cells of oviduct, in a sialicacid dependent manner, observed for qx ibv strain (associated with nephropathogenicity and reproductive tract disorders) and b1648 ibv strain (associated with nephropathogenicity), a high susceptibility to infection was observed for qx strain in contrast to the resistance to infection with b1648 strain [29] . the authors concluded that other cellular receptors and post-virus binding activation steps could be involved and play a role in the development of ib disease, although the attachment of ibv to host cells is considered as the first important step in virus infection and for determining the tissue tropism for this virus [29] . in view of the differences observed here for these two brazilian ibv isolates (a and b) in terms of viral replication and pathological alterations induced, the current study investigated how innate and adaptive immune responses are developed after challenge with each one of these viruses, aiming to better understand the virus-host interactions and the putative immune-related mechanisms that might determine the course of pathogenicity induced by ibv isolates with different virulence profiles. several studies on innate immune responses elicited by viruses and mediated by prrs have demonstrated that the levels of expression of tlr genes are usually upregulated, in order to activate intra-cellular pathways involved in the production of type i anti-viral ifns and proinflammatory cytokines [30, 7, 16, 17] . tlr7 was reported to be upregulated at 1dpi in trachea from chickens experimentally infected with a connecticut strain of ibv, but then the expression of this gene decayed to basal levels and remained unaltered at 2dpi and 3dpi [16] . in contrast, in the current study an unexpected suppressive effect in the tlr7 gene expression was observed only during the early phase of ibv b isolate infection (1dpi), as the transcripts of this gene were found 5 times downregulated. moreover, at 8dpi, other genes related to activation of tlr7 pathway were also affected, including the adapter myd88 and the pro-inflammatory cytokine gene (tnfsf15), which showed lower levels of expression in the birds from the group challenged with b isolate compared to the group challenged with isolate a. thus, these results demonstrate a differential profile of early type of innate immune responses induced by ibv b isolate compared to the ibv-induced innate immune responses of another study [16] . in the later study, an upregulation in the transcription of tlr7 and myd88 genes was observed at 1dpi in tracheal samples from chickens infected with a connecticut strain of ibv, that has a typical respiratory and pathogenicity for chicken respiratory tract. although other genes related to innate immune responses were also found to be differentially expressed in response to infection with the brazilian ibv isolates, including tlr3, type i ifns (ifnα and ifnβ) and inos, no significant differences were observed between the group infected with ibv b isolate and that infected with ibv a isolate. these results suggested that the identified differences between the two ibv isolates are not related to the biological activities associated with these innate response genes. however, tlr3 and inos genes were significantly upregulated and peaked at 1dpi, while type i ifns were significantly upregulated and peaked at 5dpi. these results contradict other findings for tlr3, ifnα, ifnβ and inos gene expression in the respiratory tract of chickens challenged with a connecticut strain of ibv [16] , since no significant differences were observed for inos and ifnα gene expression, and tlr3 was unaltered at 1dpi and ifnβ was upregulated at 1dpi. this indicates the likely existence of others factors and pathways in the innate immune responses that are triggered by the interaction of host and different pathotypes of ibv as well possible differences in structural and non-structural proteins of ibv strains involved in virus evasion from the immune responses [31] . as memory immune responses induced by ibv vaccination could have some delay until reach effective anti-viral activities, the innate immune responses might be crucial to reduce the viral replication and pathological alterations induced by a virus pathogen as well exert a relevant role in the activation and shaping of the anti-viral adaptive immune responses. thus, we hypothesize that the suppressive effect on tlr7 pathway could be associated with an enhanced ability for viral replication and induction of lesions for both tracheal and renal tissues. however, further analyses of these assumptions are necessary. in addition, a reduction of expression of cmi-genes for adaptive immune responses was also observed only in the group challenged with ibv b isolate, as the transcript levels of cd3 and cd4 were significantly downregulated at 1dpi only in this group. granzyme homolog a and ifnγ transcripts were also differentially expressed between the challenged groups in this interval, as significant upregulation was observed only in a group. therefore the downregulation of cell-mediated immune related genes could be a consequence of a primary virus challenge effect and could have negative consequences for the chicken host. in a previous study, we were able to detect an increase in the expression of cell-mediated immune related genes at 1 dpi in tracheal samples from birds vaccinated with massachusetts attenuated ibv strain and challenged with a virulent homologous strain. this early cmi response, was associated with the level of cellular memory immune responses conferred by immunization, and this effect was dependent on the vaccine dose administered and was negative correlated with the tracheal pathological changes induced by m41 strain of ibv [8] . although we have not evaluated in this study the gene expression and/or the biological activity of mhc i and ii pathways, or other genes related to antigen presentation by dendritic cells, we can speculate that ibv b isolate might have some differentiated properties that confer to this virus a greater ability to decrease antigen presentation and to evade the host adaptive immune responses against this virus. this assumption is supported by the findings that all cellmediated immune related genes analyzed here have unaltered expression or were significantly downregulated in the group infected by b isolate of ibv at 1dpi, as shown the significantly downregulated levels of expression observed for cd4 and cd3 transcripts, and unaltered expression for ifnγ and granzyme homolog a genes in birds of this group. in contrast, at 1dpi, the ugroup challenged with ibv a isolate showed unaltered levels of cd3 and cd4 mrna, while ifnγ and granzyme homolog a transcripts were significantly upregulated. natural killer (nk) cells have important roles in the initial mechanisms of innate immunity against ibv, and were also important source of granzyme homolog a and ifnγ. although some authors have associated ifnγ production with the activity of nk cells after challenge with ibv [32] , in our experiment, the profile of expression of ifnγ and granzyme homolog a may as well be associated with t cd8+ activation, since cd8β and cd3 were also found to be upregulated in coincident time-points post-challenge in which ifnγ and granzyme homolog a genes were upregulated, especially when considering that nk cells are unable to express cd8β and cd3 genes. in summary, this study has contributed to the better understanding part of host-ibv interactions, and to our knowledge, it is the first study that demonstrate that expression of distinct innate and cell-mediated adaptive immune genes are induced by two ibv isolates that were classified in the same genotype but differing in their virulence activities. overall, the results point out to the relevance of the involvement of the tlr7 pathway and the associate factors related to the suppression of early innate immune responses as well the early cell-mediated adaptive immune responses and the implications on the pathogenesis of infection by a more virulent ibv isolate. however, further studies are required to confirm this association, and to verify other cellular response pathways that may also be affected by this virus. it remains also to be further investigated the differences in the immune responses triggered by the ibv infection with more distinct genotypes of ibv strains that can differ in pathogenicity for the respiratory, urinary or other tissue targets for ibv infection in chickens. moreover, it is important for poultry industry and indirectly for human health to better understand how coronaviruses 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nephropathogenic infectious bronchitis virus strains of the massachusetts genotype in beijing lna probe-based real-time rt-pcr for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus ibv strains qx and b1648 are not related to the sialic acid binding properties of their spike proteins pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses recent progress in studies of arterivirus-and coronavirus-host interactions rapid nk-cell activation in chicken after infection with infectious bronchitis virus m41 the authors thank tania alvina potter klein, dejalmo alexandre da silva, altair althaus and gláucio mata mattos (in memorian) for support provided during experimental infection in chickens. the authors also thank to francielle ianiski for processing of tissue samples for histology.validation: cho lb. writing -review & editing: cho hjm. key: cord-339259-4oi7slk9 authors: naguib, mahmoud m.; höper, dirk; arafa, abdel-satar; setta, ahmed m.; abed, mohamed; monne, isabella; beer, martin; harder, timm c. title: full genome sequence analysis of a newly emerged qx-like infectious bronchitis virus from sudan reveals distinct spots of recombination date: 2016-10-26 journal: infect genet evol doi: 10.1016/j.meegid.2016.10.017 sha: doc_id: 339259 cord_uid: 4oi7slk9 infectious bronchitis virus (ibv) infection continues to cause economically important diseases in poultry while different genoand serotypes continue to circulate globally. two infectious bronchitis viruses (ibv) were isolated from chickens with respiratory disease in sudan. sequence analysis of the hypervariable regions of the s1 gene revealed a close relation to the qx-like genotype which has not been detected in sudan before. whole genome analysis of ibv/ck/sudan/ar251–15/2014 isolate by next generation sequencing revealed a genome size of 27,646 nucleotides harbouring 13 open reading frames: 5′-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′. highest nucleotide sequence identity of 93% for the whole genome was found with the chinese ibv strain ck/ch/lhlj/140906, the italian ibv isolate ita/90254/2005 and the 4/91 vaccine strain. phylogenetic analysis of the s1 gene revealed that the ibv/ck/sudan/ar251–15/2014 isolate clustered together with viruses of the gi-19 lineage. recombination analysis gave evidence for distinct patterns of origin of rna in the sudanese isolate in multiple genes. several sites of recombination were scattered throughout the genome suggesting that the sudan-qx-like strain emerged as a unique recombinant from multiple recombination events of parental viruses from 4/91, h120 and ita/90254/2005 genotypes. the sudanese qx-like isolate is plausibly genetically different from ibv strains previously reported in africa and elsewhere. gammacoronavirus in the coronaviridae family (king et al., 2011) . globally distributed ibv induces an acute, highly contagious infectious disease affecting chickens and results in huge annual losses in the poultry industry. ibv was first reported in north dakota, usa, by schalk and hawn 1931 (schalk & hawn, 1931 ) as a novel respiratory disease affecting chickens. only chickens and pheasants act as the natural hosts for ibv (ignjatovic & sapats, 2000) . ibv initially infects the respiratory tract; for some ibv strains further virus spread may involve kidneys and oviduct causing reduction of growth rate, decreased performance and reduction of egg quality and quantity (cavanagh, 2003) . also another shift of tissue tropism causing proventriculitis has been recorded (yu et al., 2001a) . the infection spreads by aerosols, direct contact and indirectly through contaminated fomites (ignjatovic & sapats, 2000) . ibv harbors a monopartite rna genome of positive polarity which is approximately 27.6 kb in size and codes for four structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope (e) protein (spaan et al., 1988) . the n protein is a major structural protein, which is highly conserved among different ibv serotypes. the spike (s) glycoprotein, an integral membrane protein, is another major structural protein of the ibv; it is post translationally cleaved into the s1 (n terminal part) and s2 fragments. in their matured forms the s1 constitutes trimers of the globular head while the s2 forms the trimerized stalk domain of the peplomer spikes in the viral lipid envelop (cavanagh, 2007; belouzard et al., 2012) . the s1 protein carries the receptors binding site and thus plays an important role in tissue tropism and induction of protective immunity (belouzard et al., 2012; wickramasinghe et al., 2011) . along the s1 gene three hypervariable regions (hvrs) are distinguishable that are targets of neutralizing and serotype specific antibodies (moore et al., 1997; cavanagh et al., 1988) . variation in these epitopes has been infection, genetics and evolution 46 (2016) 42-49 implicated in escape from vaccine-induced immunity (belouzard et al., 2012) . numerous distinct serotypes have been described which are based on the variability of the s1 protein sequence that differs by 20-25%, and sometimes up to 50% between serotypes (adzhar et al., 1997) . consequently, cross protection between these different serotypes is limited (jackwood, 2012; wickramasinghe et al., 2014; cavanagh, 2005; kuo et al., 2010) . new s1 genotypes of ibv that are often also showing antigenic variation and, hence, define new serotypes, appear to emerge frequently in different parts of the world (jackwood, 2012) . frequent mutations have been made accountable for the emergence and evolution of multiple s1 variants (jackwood, 2012; cavanagh et al., 1992) including point mutations, insertions, deletions, and also recombination between different strains (adzhar et al., 1997; hewson et al., 2014) . recombination tends to be not a rare event during ibv replication and the emergence of chimeric viruses harbouring sequences from two or more viruses were reported previously (jia et al., 1995; abro et al., 2012) . the qx ibv genotype, recorded for the first time in china in 1996, is associated with renal infections, proventriculitis, or impaired egg production (yu et al., 2001b; liu & kong, 2004) . this genotype has spread from asia to europe (monne et al., 2008) , and recently was reported from the southern part of the african continent, namely zimbabwe and south africa (toffan et al., 2011; abolnik, 2015) . qx ibv has become the predominant field strain in many asian and european countries de sjaak et al., 2011) . in the middle east, qx-like strains have been detected in kurdistan-iraq in 2011 (amin et al., 2012) . qx ibv was shown to be antigenically different from both classical vaccine type and other variant strains. (ducatez et al., 2009 ). however, a previous study has reported that vaccination using the ma5 (mass type) and 4/91 (793b) can reduce the clinical impact of qx ib virus infections in spf layers and in commercial broiler chickens (terregino et al., 2008) . in sudan, ibv was recorded for the first time in 1981 (elamin et al., 1986) . serosurveillance studies reported widespread ibv infection in sudan associated with the 4/91 virus strain in 2000 -2001 (ballal et al., 2005a ballal et al., 2005b) and again in 2013. widespread occurrence in non-vaccinated poultry of different sectors is assumed (selma & ballal, 2013) . little is known to date on ibv from sudan; in particular, genetic and virological data are missing. in the current study, an ibv isolate from chickens in sudan is characterized as a qx-like ibv recombinant genotype. two ibv viruses (ck/sudan/ar251-15/2014 and ck/sudan/ar252-15/2014) were isolated from field samples obtained at the national laboratory for veterinary quality control on poultry production (nlqp, ministry of agriculture) from chickens which showed severe respiratory disease in a holding in sudan. viruses were submitted to the friedrich-loeffler-institut, germany, for further molecular and genetic characterization. virus passaging was performed in 10-day old spf chicken eggs (oie, 2013). viral rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. rna was eluted in 70 μl nuclease-free water, and stored at − 80°c until use. presence of ibv rna was confirmed using rt-qpcr and conventional rt-pcrs specific for hvrs of the s1 gene of ibv and sanger sequencing of these regions was performed. primer sequences, amplification and sequencing conditions are available from the author upon request (callison et al., 2006) . spike gene-specific rt-pcr amplicons were size-separated by agarose gel electrophoresis, excised and purified from gels using the qiaquick gel extraction kit (qiagen). purified pcr products were used directly for cycle sequencing reactions (bigdye terminator v3.1 cycle sequencing kit, applied biosystems, darmstadt, germany). the reaction products were purified using nucleoseq columns (macherey-nagel gmbh & co, düren, germany) and sequenced on an abi prism® 3100 genetic analyzer (life technologies, darmstadt, germany). thereafter, megablast (http://blast.ncbi.nlm.nih.gov/blast.cgi) analyses using the s1 hvrs sequences were carried out. the obtained hvrs sequences of the s1 gene were assembled and edited using the geneious software, version 9.0.5 (kearse et al., 1647 (kearse et al., -1649 . alignment and identity matrix analyses were performed using mafft (katoh & standley, 2013) and bioedit (hall, 1999) . sequences generated in this study were deposited in the genbank database, and assigned accession numbers are shown in table 1 . sequences of other viruses required for further analyses were retrieved from public databases. for maximum likelihood analysis of phylogenetic relationships, a best fit model was chosen first on which further calculations and an ultrafast bootstrap equivalent analysis was based, using the models and algorithms implemented in the iq-tree software version 1.1.3 (minh et al., 2013; nguyen et al., 2014) . trees were finally viewed and edited using figtree v1.4.2 software (http://tree.bio.ed.ac.uk/software/ figtree/). for full-genome sequencing of ck/sudan/ar251-15/2014, rna was extracted using trizol ls reagent (life technologies) and an rneasy minikit (qiagen) with on-column dnase digestion according to the manufacturer's instructions. rna conversion into double-stranded dna was done using a cdna synthesis system (roche, mannheim, germany) according to the genome sequencer rapid rna library preparation manual (roche, mannheim, germany). library preparation was done as previously described (juozapaitis et al., 2014) . sequencing was performed with an illumina miseq instrument using the miseq reagent kit version 3 (illumina, san diego, ca, usa). the raw sequencing reads were assembled into a single contig representing the complete ibv genome using the genome sequencer software suite (v3.0, roche). the sequence was analyzed with blastn (blastn; http://blast.ncbi. nlm.nih.gov/blast.cgi) and orfs were detected and the genome annotation was carried out using geneious software, version 9.0.5. the s-orf encoding the spike protein was analyzed and compared using programs implemented in geneious to those of previously reported qx and qxlike strains from asia, europe and africa focusing in particular on the highly variable regions (hvrs). in addition, n-glycosylation sites in the spike protein were predicted by services available on http://www.cbs. dtu.dk/services/netnglyc. phylogenetic trees were also generated based on the obtained full genome sequence and the s1 gene sequence separately according to methods described in section 2.3. programs embedded in the recombination detection program 4 (rdp4) software suite (martin et al., 2015) were used to identify recombination events in the full-length ibv genome sequence of isolate ck/ sudan/ar251-15/2014 through detection of breaking points using specific algorithms implemented in rdp4: rdp, genecov, bootscan, maxchi, chimaera, siscan and 3seq with the highest acceptable p-value adjusted to 0.05. for this purpose, an alignment was produced, as mentioned in the previous section, featuring other complete ibv genomes from north america (usa), asia (china, korea and taiwan), africa (nigeria) and europe (sweden, ukraine and italy) that are relevant as vaccine strains and/or representatives of major ibv lineages. the same alignment was also used to examine nucleotide and amino acid identity for each orf between the ck/sudan/ar251-15/2014 and other ibv strains. two virus isolates generated from clinical samples collected from diseased chickens in a holding in sudan tested strongly positive in ibv specific rt-qpcrs targeting 5′-utr. conventional rt-pcr specific for the hvrs of the s1 gene amplified fragments of approximately 450 (hvr1 and 2) and 380 (hvr3) nucleotides, respectively. sanger sequencing of the amplicons confirmed the identity of these isolates as ibv (genbank accession number kx272466-7). nucleotide sequences of the s1 hvrs were used to conduct a blast search for further characterization. highest identities for the assembled sequences of hvr1 and −2 (amino acids 52 to 177) were found at the nucleotide level with the ibv isolates slo/305/08 (slovenia) and kr/ d42/05 (korea) (97%). at the amino acid level, the highest similarities were detected with kr/354/03 (korea) and rf/28/2011 (russia) (95%). for the hvr3 amplicon (amino acids 253 to 364), the highest identity (n98%) was found with ita/90254/2005 (italy) and az-40/05 (italy), both at the nucleotide and protein levels. these data revealed that the s1 gene of the two sudanese ibv isolates clusters with qx and qx-like ibv viruses. in order to confirm these findings, phylogenetic analysis was conducted using a maximum likelihood method (iqtree). the hvr1-2 and hvr3 nucleotide sequences of the s1 gene of ibv ck/sudan/ ar251-14/2014 and ck/sudan/ar252-14/2014 clustered together with qx and qx-like viruses reported previously in asia, europe, middle east and west and south africa (fig. 1a, b) . since the hvr sequences of the two viruses were very similar to each other (99.2% and 100% identity for hvr1-2 and hvr3, respectively), only one virus, ck/sudan/ar251-15/2014, was selected for further analysis by full genome sequencing. the complete genome of the ck/sudan/ ar251-15/2014 strain as obtained by next generation sequencing was found to be 27,646 nucleotides (nt) in length, including both utr 5′ and the poly (a) tail. the complete genome sequence of the ck/ sudan/ar251-15/2014 isolate has been assigned into the genbank sequence database in the national center for biotechnology information (ncbi) accession number kx272465. the sequence obtained showed a classical ibv genome organization with 13 open reading frames (orfs) in the order 5′-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′ (table 1, fig. 2 ). across the whole genome the highest identities were seen with the chinese ibv strain ck/ch/lhlj/140906 (93%; accession kp036502), an italian ibv isolate (ita/90254/2005; 93%; accession fn430414), and the 4/91 vaccine strain (93%; accession kf377577). overall nucleotide identities of 92% and 90% across the whole genome were obtained compared to the most commonly used ibv vaccine strains, h120 and m41, respectively. however, the s1 gene revealed identities of only 76.6 and 76.7% to these latter strains. similarity searches using each orf of the ibv/ck/sudan/ar251-15/ 2014 separately produced variable rankings with other ibv reference and vaccine strains: ibv/ck/sudan/ar251-15/2014 revealed higher sequence identities of the 1b, s, 3a, 3b, e, m, orfs with qx and qx-like viruses. on the contrary, the 1a, 4c, 5a, 5b and n orfs showed higher sequence identities with the h120, 4/91 and ibvukr27-11 (4/91 like) strains (table 2) . orf 4b revealed overall low identities with all genotypes as shown in table 2 . orf 6b showed mixed identity values with either qx-like viruses or with 4/91. these findings suggested a possible recombination between different ibv genotypes shaping the genome of the sudanese isolate. phylogenetic analysis based on the full genome sequence revealed a close relation with the qx-like viruses from italy, sweden and south africa (fig. s1) . by phylogenetic analysis based on the whole s1-gene according to (valastro et al., 2016) it appeared that the ibv/ck/sudan/ar251-15/2014 is closely related to ita/90254/ 2005 and the previously reported recombinant strains detected in south africa (ck/za/3665/11) and sweden (ck/swe/0658946/10) which are clustered together within gi-19 lineage (fig. 3) . the s-orf of the sudanese isolate has a length of 3498 nucleotides giving rise to 1165 amino acids. the precursor protein harbors an endoproteolytical cleavage site 537 rrrr/s 541 which divides the s-protein precursor into the s1 and s2 fragments of 540 amino-terminal and 625 amino acids at the carboxyl terminus, respectively. a total of 33 n-linked glycosylation sites was predicted in the s protein of ck/ sudan/ar251/qx strain (s1 = 20 and s2 = 13) similar to the chinese qx which are possessing seven additional glycosylation sites than the vaccine stain h120 in positions 141, 200, 279, 457, 545, 982 and 1061, and lost two sites at positions 283 and 714. compared to the chinese qx strain, the sudan isolate ar251 gained an additional glycosylation site at position 267, but lost a glycosylation site at position 5. the employed recombination detection methods embedded in rdp4 revealed that ck/sudan/ar251-15/2014 has undergone genetic recombination. three long recombined sequence stretches were identified with high reliability by at least five programs embedded in rdp4: the first and second recombination regions were observed at positions 1-6468 and 9988-12498 in the orf1a and orf1b genes, and the third recombination region was located in position 18369-23219 involving the or1b and s gene. further recombination events in different positions were identified as shown in fig. 4 albeit with lower reliability according to rdp4. the results showed that the sudanese isolate was a recombinant virus which probably emerged from at least three different genotypes, including the 4/91 genotype as a major parent and the h120 vaccine strain as well as italy/90254/2005-like viruses as minor parents (fig. 4) . different ibv variants are in circulation around the world; some of them show a geographic restriction, others are globally distributed (de sjaak et al., 2011) . different genotypes of ibv are classified based on the genetic variation of the spike protein, in particular its s1 fragment (cavanagh, 2007; belouzard et al., 2012; valastro et al., 2016) . the hvr1-2 and hvr3 regions of the s1 gene encode serotype specific determinants of ibv and harbor antigenic epitopes important for induction of protection (promkuntod et al., 2014) . little is known about the epidemiology of avian respiratory diseases in poultry in sudan although respiratory disease continues to threaten commercial poultry in the country. in this study, two ibvs were isolated from commercial broiler farms showing respiratory signs with an increased mortality. flocks were vaccinated with either mass type alone (h120 or ma5) or with ma5 and 4/91. partial s1 gene analysis of the three hvrs has demonstrated that ibv strains ck/sudan/ar251-15/2014 and ck/sudan/ar252-15/2014 are related to the qx-like serotype. qx-like ibv had never been reported from sudan. qx variant ibv apparently emerged in china in 1998 and reported thereafter in europe as qx-like viruses (monne et al., 2008; wang et al., 1998) . recombination in coronaviruses is likely to be occurred by their unique mechanism of rna synthesis involving polymerase jumping fig. 4 . examination of putative recombination events in the genome of the ibv isolate sudan ar251-15/2014 (query sequence) (a). the analysis was conducted using the recombination detection program v4. maximum likelihood trees of the selected recombinant regions were estimated using algorithms embedded in rdp4 (b, c, d). and discontinuous transcription (lai, 1996) . different recombination evidences were reported among different ibv field strains through recombination between two or more strains resulting in the emergence of new variants (abro et al., 2012; abolnik, 2015; ammayappan et al., 2008) . further, distinct ibv recombination has been experimentally illustrated in vitro, in ovo and in vivo (wang et al., 1997) . here, the full-length genome of the ck/sudan/ar251-15/2014 strain determined by next generation sequencing (ngs) revealed thirteen open reading frames orfs (5′utr-1a-1b-s-3a-3b-e-m-4b-4c-5a-5b-n-6b-3′utr) showing different spots of variations and recombination with other genotype located in multiple genes orf1a, orf1band s. the start of the orf1a gene revealed a high frequency of recombination events with 4/91. other genes exhibited recombination with the h120 and italy/90254/2005 type ibvs, indicating that recombination might involve more than two strains. taken together, phylogenetic and recombination analyses performed on the complete genome of the sudan/ar251-15/2014 virus showed that this strain is a mosaic of different parental lineages never described so far. in particular, the virus most likely resulted from a natural recombination event involving at least three distinct ibv variants namely the qx-like, 4/91 and h120 strains. in conclusion, it is noteworthy that there are no records of the presence of a qx-like variant in sudan prior to this study. whatever the way in which this virus has reached sudan, identification of this chimeric qx like virus highlights the need to improve monitoring programs for ibv in sudan and neighboring countries for a better understanding of its epidemiology. in addition, updating of vaccines may be required with further experimental studies to demonstrate the efficacy of the currently used vaccine. supplementary data to this article can be found online at http://dx. doi:10.1016/j.meegid.2016.10.017. mahmoud m. naguib, timm harder, abdel-satar a. arafa, conceived the study. dirk höper conducted and interpreted the ngs sequencing. mahmoud m. naguib and timm harder produced, analyzed and interpreted genetic and phylogenetic data. ahmed setta provided and analyzed epidemiological data. timm harder and mahmoud m. naguib drafted the manuscript. all co-authors critically analyzed, revised and finally approved the manuscript. the authors declare no conflict of interest. genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus characterization and analysis of the full-length genome of a strain of the european qx-like genotype of 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binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity the avian coronavirus spike protein a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia the authors thank diana wessler and aline maksimov, fli, germany, for excellent technical support. we are grateful to colleagues and coworkers at nlqp, cairo, egypt, contributing to sample preparation. m. naguib is recipient of a doctoral scholarship from the german academic exchange service (daad grant no. a/13/92967). key: cord-348660-qnbgywgy authors: yilmaz, huseyin; faburay, bonto; turan, nuri; cotton-caballero, maira; cetinkaya, burhan; gurel, aydin; yilmaz, aysun; cizmecigil, utku y.; aydin, ozge; tarakci, eda altan; bayraktar, erhan; richt, juergen a. title: production of recombinant n protein of infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic antigen date: 2018-07-09 journal: appl biochem biotechnol doi: 10.1007/s12010-018-2815-2 sha: doc_id: 348660 cord_uid: qnbgywgy the avian coronavirus-infectious bronchitis virus (avcov-ibv) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. sensitive diagnostics and efficacious vaccines are necessary to combat ibv infections in chickens. the aim of this study was to produce recombinant n protein of ibv in the baculovirus system to use in elisa diagnostic tests in order to enable the assessment of the sero-prevalence and risk of ibv infections in chickens in turkey. for this, the gene encoding the n protein of the beaudette strain of ibv was expressed using a recombinant baculovirus expression system. the recombinant n protein was purified using ni-nta affinity chromatography. an estimated 50-kda recombinant protein corresponding to the expected molecular weight of ibv n including the 6xhis tag was detected using an anti-his monoclonal antibody. specific immunoreactivity of the recombinant protein was confirmed by western blot using antiserum obtained from vaccinated and naturally infected chicken from turkey as well as using a monoclonal antibody raised against the n protein of the ibv massachusetts strain. the results obtained with the in-house elisa had high agreement with a commercial elisa. immunoreactivity analysis using antisera in western blotting and the in-house elisa suggests that the recombinant ibv n protein could be broadly cross-reactive with antisera produced against different ibv strains. we conclude that the recombinant baculovirus expressed ibv n protein could serve as a useful diagnostic antigen for detection of ibv infections in chickens by elisa. infectious bronchitis virus (ibv) is the causal agent of infectious bronchitis (ib), an acute and highly contagious disease, threatening the poultry industry worldwide [5, 11, 13, 17, 19] . avian coronavirus-infectious bronchitis virus (avcov-ibv) is an enveloped, positive single-stranded rna virus, and belongs to the genus gamma-coronavirus within the family of coronaviridae in the order nidovirales [2] . the virus is spread mainly by aerosol, consumption of contaminated feed and water, and contact with infected feces or equipment. ib is characterized by various clinical signs in broilers and layer hens: coughing, sneezing, and decreased weight gain [4, 13] . specifically, in layers, egg production can drop up to 70% with eggs that have shells that are wrinkled, thin, and soft. in young chicks, ibv infection can lead to oviduct cysts and reduced laying potential [2, 4, 9] . the 27 to 28 kb genome of ibv encodes nine genes, which includes spike (s), membrane (m), envelope (e), and nucleocapsid (n) [2] . the spike protein, a viral surface glycoprotein, was shown to induce neutralizing antibody response, and the nucleocpasid protein was shown to elicit strong antibody responses [2, 5] . despite the presence and application of ibv vaccines in poultry, there is a high rate of emergence of antigenic variants and recombinant strains, and the lack of cross-protection between different viral genotypes, making disease control difficult and vaccine development rather challenging [15, 16] . therefore, genetic characterization of circulating strains of ibv, appropriate vaccination programs, and application of sensitive diagnostic tests to detect and assess disease risk are important regional, national, and international strategies to control ibv infections [2, 8, 10, 21, 23] . several elisas have been developed for detection of antibodies to ibv in chickens. recombinant antigens used in these elisas were either based on the s1 protein [20] , which is highly variable, or the n protein and often produced in an e. coli expression system [15] . e. coli is a common flora or pathogen in chickens, thus creating the possibility of serological cross-reactivity and detection of false positives in these diagnostic assays. thus, it is necessary to assess the suitability of other expression platforms for production of diagnostic antigens for use in ibv serology. the objectives of this study were to clone and express ibv n protein using the recombinant baculovirus expression system and assess its use as diagnostic antigen for serological diagnosis of ibv infection in chickens in turkey. the complete coding sequence (1230 bp) of the ibv n gene of beaudette strain (genbank accession no. m28565.1) was initially amplified by pcr using primers, jar170f: 5′-cac cat ggc ttc cgg taa ggc tg-3′ and jar171r: 5′-cag ctc gtt ctc acc cag agc agc-3′. the pcr product was cloned into pfastbac vector (life technologies), to create a recombinant donor plasmid, pfastbac-n, which was transformed into one shot mach1 t1 chemically competent e. coli (life technologies). the donor plasmid was double digested with restriction enzymes bamhi and psti to determine the presence of the correct insert in the right orientation. the accuracy of the sequences was confirmed by dna sequencing. the donor plasmid was transformed into max efficiency dh10bac competent e. coli to construct a recombinant bacmid via site-specific transpositioning. to rescue recombinant baculoviruses encoding the ibv n gene, recombinant bacmids were purified using highpure miniprep kit (life technologies) and used to transfect spodoptera frugiperda (sf9) cells grown in sf-900 ii serum-free medium (sfm) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as manufacturer's instruction (life technologies). transfection was carried out using cellfectin ii reagent as previously described [8] and according to the manufacturer's instructions (invitrogen-life technologies). recombinant ibv n protein was expressed using passage 2 or higher passage recombinant baculovirus stocks (> 10 7 pfu/ml). the protein was expressed with a carboxy-terminal 6xhis tag, and purification using ni-nta superflow resin (qiagen inc., valencia, ca) was performed as described previously [7] . concentration of the purified protein was measured by the method of bicinchoninic acid (bca) assay (thermo scientific, rockford, il) at an absorbance of 562 nm, using bovine serum albumin (sigma-aldrich, st. louis, mo) as the protein standard. aliquots of the protein were stored at − 80 c until used. western blot analysis was performed to verify specific protein expression. for this, approximately 5 μg of purified recombinant ibv n protein was resolved in 12% bis-tris polyacrylamide gel (life technologies) as described previously [7] . following electroblotting of the proteins onto pvdf membrane, the blots were probed with mouse anti-his (c-terminal)-hrp monoclonal antibodies (1.2 mg/ml) (life technologies) diluted 1:5000 to determine expression of the correct molecular size protein. further confirmation of expression was performed using a mouse anti-ibv n (massachusetts strain) monoclonal antibody (6 mg/ml) diluted 1: 2000 (cat no. mbs312776; mybiosource, san diego, ca) and antiserum (at 1:50 dilution) collected from chickens naturally infected with local turkish wild-type ibv strains. the membrane was then incubated with anti-species igg-hrp secondary antibody conjugate (santa cruz biotechnology, dallas, tx) at 1: 5000 dilution, and specific signals were detected using enhanced chemiluminescent (ecl) detection system. to assess the induction of specific ibv n-specific antibodies in the immunized chickens (described below), the antisera were tested in immunoblot assays. approximately, 100 μl of sample containing approximately 5 μg of recombinant ibv n protein was diluted in 50-μl laemmli sample dilution buffer (santa cruz biotechnology, dallas, tx) and resolved in 12% bis-tris polyacrylamide gel as described above. following transfer, the blot was probed with ibv n antisera obtained from the chicks immunized with the recombinant ibv n protein at a dilution of 1:50 and with antisera from wild-type ibv-infected chicken at 1:50 dilution [19] . ibv negative chicken sera, at a dilution of 1:50, and mouse anti-ibv n monoclonal antibody (2 mg/ml) (thermo fisher, cat. no. 7500891) at a dilution of 1:500 were used as negative and positive controls, respectively. thereafter, the blots were probed using goat anti-chicken hrp-conjugated secondary antibody (200 μg/0.5 ml) (cat. no. sc2428; santa cruz biotechnology, dallas, tx) at a dilution 1:5000, and goat anti-mouse hrp conjugated secondary antibody (200 μg/0.5 ml) diluted 1:5000 (cat. no. sc2005; santa cruz biotechnology, dallas, tx). reactivity was detected using 3′-3′-diamino benzidine peroxidase substrate system (sigma cat no: d4418-5) as recommended by the manufacturer. to assess immunogenicity of recombinant baculovirus-expressed ibv n protein, embryonated spf chicken eggs were obtained from the bornova veterinary control institute (izmir, turkey). the eggs were incubated in an incubator for hatching. after hatching, 10-day-old ibv antibody negative chicks (determined by commercial elisa-biochek, ibv elisa, cat no: ck119ibv) were immunized with the recombinant ibv-n protein produced in this study. four chicks were immunized intramuscularly with 15 μg each of the recombinant ibv-n protein mixed with isa 71vg adjuvant (seppic, france) at a ratio of 70:30 (adjuvant/ibv-n solution), and four chicks were given placebo as mock-immunized controls. on day 16, immunization was repeated using the same amount of adjuvant and antigen. chicks were bled on day 0 (pre-immunization) and then on days 10, 16, and 23 to test for presence of antibodies to recombinant ibv-n protein. optimization of elisa an indirect elisa protocol was first optimized by checkerboard titration following the methods as described previously [16, 18, 22] . for this, two-fold dilutions of ibv-n protein were made starting from 400 to 25 ng in a carbonate bi-carbonate coating buffer (sigma, c-3041). the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (biochek). positive control was from chickens either vaccinated or had field infection tested ibv-positive by pcr. the ibv positive and negative sera were also diluted two-fold (from 1:50 to 1:1600), and the conjugate (hrp-conjugated goat anti-chicken antibody, santa cruz, sc2428) was diluted 1:5000, 1:10,000, 1:20,000, and 1:40,000, in blocking solution. per the results of the optimization, the optimal amount of the recombinant ibv protein for use in elisa ranged from 50 to 100 ng, whereas the optimal dilution of ibv positive chicken serum ranged from 1:100 to 1:800, that of the conjugate at 1:5000 dilution. following optimization of the elisa protocol, 18 test sera were obtained from broiler chickens exposed to natural wild-type ibv infection, 18 test sera from broiler chickens vaccinated with a live-attenuated commercial ibv vaccine, and sera obtained at different time-points from chicks immunized with recombinant ibv n protein (described above) were analyzed to detect ibv n-specific antibodies. for this, a 96-well plate (nunc maxisorb, thermo fisher scientific) was coated overnight at 4°c with 100 ng ibv-n protein/per well in 100-μl carbonate-bicarbonate coating buffer (sigma, cat. no. c-3041). the plate was then blocked for 1 h at room temperature with a blocking buffer (pbs containing 3% skimmed milk and 0.05% tween 20) . a volume of 100 μl of test sera, diluted 1:100 in blocking solution, was added and incubated for 1 h at 37°c. each serum sample was tested in duplicate, and each test plate included duplicate positive and negative control sera. the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (bochek). positive control serum was from chickens either vaccinated or had field infection and tested ibv positive by pcr. goat anti-chicken hrp-conjugated antibody (santa cruz biotechnology, cat. no. sc2428), diluted 1: 5000, was added to the wells and incubated at 37°c for 1 h. between each step, the plate was washed four times by using the wash buffer (pbs containing 0.05% tween 20) . hundred microliters of tmb substrate solution was added to each well and incubated at room temperature for 25 min. the reaction was stopped by adding 100 μl of 2 m h 2 so 4 and absorbance (optical density) was measured at 450 nm using a microplate reader . (slt-spectra, slt lab instruments, germany). the elisa cut-off value was determined by addition of 3 standard deviations to measurements of the mean od value of the negative control sera in each plate. od values above cut-off value were taken as positive. thus, a cut-off od value of 0.2 was determined for the in-house indirect elisa. to assess the reliability of the performance of our in-house indirect ibv n elisa, a panel of sera was obtained from chickens naturally infected with local wild-type ibv strains, chickens vaccinated with live-attenuated commercial ibv vaccine, and chickens immunized with recombinant ibv n protein (expressed in a baculovirus expression system). the serum samples were tested simultaneously in the in-house indirect elisa (using the procedure described above) and a commercial elisa (biocheck, san francisco, ca) per manufacturer's instruction. the cut-off value for the commercial elisa was defined according to the manufacturer and determined as samples with a sample to positive ratio (s/p ratio) of 0.2 or greater. a 1203 bp fragment representing the expected molecular size of the ibv n gene was successfully amplified by high fidelity pcr (fig. 1) . the pcr amplicon was successfully cloned into pfastbac/ct plasmid resulting in the creation of a donor plasmid pfastbac-n (5985 bp), restriction enzyme analysis of the recombinant donor plasmid confirmed the presence of the correct insert in the correct orientation by release of two restriction fragments of the expected size (5090 and 895 bp) (data not shown). the donor plasmid with the correct sequences was used to create recombinant bacmid for subsequent expression of the target protein. in order to express the recombinant n protein of ibv, sf9 cells were infected with a recombinant baculovirus encoding the n gene of ibv. at 72 h post-infection, cells were harvested and the recombinant protein was purified via affinity chromatography. an estimated 50 kda recombinant protein was overexpressed, which corresponded to the expected molecular size of ibv n protein as determined by coomassie staining (fig. 2a) and western blot analysis using anti-his (c-terminal)-hrp monoclonal antibody (fig. 2b) . to confirm specificity of the target protein, an immunoblot analysis was performed using monoclonal antibodies raised against the n protein of the massachusetts ibv strain (fig. 3a) and as well as antiserum obtained from local chickens naturally infected with turkish wild-type ibv strain (fig. 3b) . in both cases, specific reactivity was confirmed by detection of a single band (50 kda) of the expected molecular size. to assess the ability of the recombinant ibv n protein to induce host specific antibody responses, four chicks (10-day old) were immunized with the recombinant protein and the sera examined for specific reactivity via immunoblot analysis. specific reactivity was detected with serum samples obtained from 2 of the ibv n-immunized chickens at day 23 post-immunization (example see fig. 4 seen with the other two chicken sera. this result was supported by detection of specific ibv n-reactivity with the ibv-specific monoclonal antibody (fig. 4 , lane 1) and with antisera from a chicken exposed to a natural field infection (fig. 4, lane 3) . no specific bands were detected in sera from mock-immunized chickens or sera taken from chicks before immunization (fig. 4, lanes 2, 4, 5, 6, 8, 9, 10) . overall, sera from vaccinated and naturally infected (field-exposed) chickens that tested positive in the immunoblot assay were found to be correspondingly positive in the in-house indirect ibv n elisa (data not shown). to further assess the immunogenicity of the recombinant n protein and the reliability of our results, a panel of antisera from naturally infected (18 samples plus negative and positive controls), from chickens vaccinated with live-attenuated ibv (18 samples plus negative and positive controls) and from chickens immunized with recombinant ibv n protein (4 samples), were tested using both the in-house indirect elisa and a commercial elisa. with the in-house elisa, cut-off value was set at od of 0.2. there was relatively good agreement in performance between the two elisas in detecting ibv n-specific antibodies in the sera from naturally infected chickens (fig. 5a) and live-attenuated vaccinated chickens (fig. 5b) . of the 18 serum samples obtained from the naturally infected chickens, 72% (13/18) tested positive in the in-house indirect elisa, whereas 83% (15/18) tested positive in the commercial elisa detected (fig. 5a) . all 18 samples collected from vaccinated chickens tested positive in both the in-house and commercial indirect elisas (fig. 5b) . of the 15 commercial elisa-positive field serum samples, 13 tested positive in the in-house indirect elisa, indicating a sensitivity of 87% (using the commercial elisa as reference test). none of the sera obtained from preimmunized or naïve chicks tested positive in either elisas, indicating 100% specificity (n = 4). of the four chicks immunized with the recombinant ibv n protein, two chicks seroconverted at day 16 after immunization. all four chicks seroconverted at 23 days post-immunization in both elisa tests, indicating a 100% agreement between the two assays. the od values of chicks immunized with recombinant ibv n protein were 0.3, 0.4, 0.41, and 0.44. production of immunogenic recombinant proteins derived from pathogenic organisms represents a good strategy for identification of antigenic proteins that may serve as targets for sero-diagnostic and vaccine development [12, 14] . this represents the first study in turkey that expressed recombinant ibv n protein in baculovirus and examined its reactivity against antisera obtained from turkish chickens for potential use as antigen fig. 5 detection of ibv n specific antibodies in sera obtained from naturally infected chicken (a) and vaccinated chickens (b) using an in-house ibv-n elisa and a commercial elisa. the cut-off value for inhouse indirect elisa is set at od 0.2, and for the commercial elisa at s/p (sample to positive) ratio of 0.2 in serological investigation of ibv infection in domestic poultry. we have demonstrated that ibv n produced in a recombinant baculovirus expression system is immunogenic in local chickens and could detect ibv n-specific antibodies in sera obtained from chickens exposed to natural infection. the recombinant n protein was reactive in both immunoblot and indirect elisa assays. the performance of the in-house indirect elisa was comparable to the commercial elisa tested in this study, indicating the potential suitability of recombinant baculovirus ibv n as a diagnostic antigen that could be used for serological risk assessment of ibv in domestic chickens in turkey. indeed, the n protein of ibv, in contrast to the s1 protein, has been associated with high stability and immunogenicity and has been used by others as a diagnostic antigen [1, 12] . the recombinant n protein used in the current study was based on nucleotide sequences of the beaudette ibv strain. the reactivity of the recombinant ibv n protein with a monoclonal antibody (b819m) produced against the heterologous ibv massachusetts strain, and with sera from local turkish chicken indicate the conserved, cross-reacting nature of the n protein sequence and suggest potential broad cross-reactivity of the target protein among different ibv isolates and its suitability as a diagnostic antigen to detect ibv infection in local turkish poultry. other researchers have utilized recombinant ibv n expressed in various host systems in serological assays. for example, in a study performed by pradhan and others [14] , ibv-n protein was produced using a prokaryotic expression system. the protein was used for diagnostic purposes and similar to this study, compared the performance with a commercial elisa kit (idexx). the authors reported a 96.8% sensitivity and 95.8% specificity for the in-house elisa based on the recombinant ibv n antigen and they concluded that an indirect elisa based on the recombinant antigen is suitable for use in development of serodiagnostic tools [14] . in another study, the ibv-n protein was produced in both e. coli and baculovirus expression systems [3] , and the recombinant antigens were used in elisas to analyze chicken sera collected from farms [3] . their data from screening of sera for the presence of ibv antibodies indicated that using the recombinant n protein as coating antigen could achieve equivalent performance to an elisa kit based on extracts from infected material as coating antigen [3] . in a study performed by ding and others [6] utilizing a multi-fragment antigen composed of s (spike), m (matrix), and n proteins produced in e. coli [7] , a good reactivity with an anti-ibv chicken serum was demonstrated. an in-house elisa using the multi-fragment protein exhibited a 95.4% concordance with a commercial elisa [6] . in the present study, recombinant ibv-n protein was produced using a baculovirus expression system and utilized as a diagnostic antigen in an indirect elisa. the chicken sera from farms and the sera from immunized chickens were reactive with the recombinant antigen with comparable performance to a commercial elisa test kit. this study describes the successful cloning and recombinant baculovirus expression of ibv nucleoprotein and its evaluation as a potential serodiagnostic antigen. the recombinant antigen was immunoreactive with hyperimmune sera from local turkish chickens and the performance of an in-house indirect elisa based on the antigen was comparable to a commercial elisa, suggesting the potential utility for serological detection of ibv infections in chickens in turkey. further studies to validate the assay using a larger sample size including comprehensive assessment of assay specificity will be performed. decreased neutralizing antigenicity in ibv s1 protein expressed from mammalian cells coronavirus avian infectious bronchitis virus evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus the long view: 40 years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures development of an elisa based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection rift valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep. vector borne and zoonotic diseases a glycoprotein subunit vaccine elicits a strong rift valley fever virus neutralizing antibody response in sheep s1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel avian infectious bronchitis virus review of infectious bronchitis virus around the world expression and purification of the 26 kda periplasmic protein of brucella abortus: a reagent for the diagnosis of bovine brucellosis cleavage of structural proteins during the assembly of the head of bacteriophage t4 expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of brucella melitensis m5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay immune responses to mucosal vaccination by the recombinant a1 and n proteins of infectious bronchitis virus molecular epidemiology and evolution of avian infectious bronchitis virus recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification an elisa for antibodies against infectious bronchitis virus using an s1 spike polypeptide a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from detection of antibodies to equine arteritis virus in horse sera using recombinant chimaeric n/g(l) protein. the veterinary record phylogeny and s1 gene variation of infectious bronchitis virus detected in broilers and layers in turkey compliance with ethical standards national and international ethical rules were followed during this study. the authors declare that they have no conflicts of interest. key: cord-334090-66d8c75g authors: seger, waleed; ghalyanchilangeroudi, arash; karimi, vahid; madadgar, omid; marandi, mehdi vasfi; hashemzadeh, masoud title: genotyping of infectious bronchitis viruses from broiler farms in iraq during 2014-2015 date: 2016-02-18 journal: arch virol doi: 10.1007/s00705-016-2790-2 sha: doc_id: 334090 cord_uid: 66d8c75g infectious bronchitis virus (ibv) is one of the most critical pathogens in the poultry industry, causing serious economic losses in all countries including iraq. ibv has many genotypes that do not confer any cross-protection. this virus has been genotyped by sequence analysis of the s1 glycoprotein gene. a total of 100 tracheal and kidney tissue specimens from different commercial broiler flocks in the middle and south of iraq were collected from september 2013 to september 2014. thirty-two ibv-positive samples were selected from among the total and were further characterized by nested pcr. phylogenetic analysis revealed that isolates belong to four groups (group i, variant 2 [is/1494-like]; group ii, 793/b-like; group iii, qx-like; group iv, dy12-2-like). sequence analysis revealed nucleotide sequence identities within groups i, ii, and iii of 99.68 %-100 %, 99.36 %-100 %, and 96.42 %-100 %, respectively. group i (variant 2) was the dominant ibv genotype. one chinese-like recombinant virus (dy12-2-like) that had not been reported in the middle east was detected. in addition, the presence of qx on broiler chicken farms in the area studied was confirmed. this is the first comprehensive study on the genotyping of ibv in iraq with useful information regarding the molecular epidemiology of ibv. the phylogenetic relationship of the strains with respect to different time sequences and geographical regions displayed complexity and diversity. further studies are needed and should include the isolation and full-length molecular characterization of ibv in this region. coronaviruses are an important group of viruses that cause highly contagious respiratory and enteric diseases in animals and humans [13] . avian corona viruses can infect a wide range of avian species, particularly those reared in close proximity to domesticated poultry, such as fowl, partridge, geese, pigeons, guinea fowl, teal, ducks, and pea fowl [8] . infectious bronchitis virus (ibv) causes enormous problems, causing a highly contagious disease in chickens. respiratory, reproductive, digestive, and renal infections are the primary clinically important types of ibv infections in domestic chickens [15] . ibv belongs to the genus gammacoronavirus, along with other avian coronaviruses. it is an enveloped, positive-stranded rna virus with a genome of about 27 kb containing 5' and 3' untranslated regions (utrs) and a poly(a) tail [5] . a major part of the genome is composed of two overlapping open reading frames (orfs), 1a and 1b, which are translated into large polyproteins 1a and 1ab, respectively, through a ribosomal frameshift mechanism. the primary structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) are encoded in the remaining part of the rna genome [20, 24] . the glycoprotein s consists of two subunits (s1 and s2) and contains a wide variety of antigenic determinants that may induce the production of specific neutralizing antibodies. mutations within this genomic region may result in the emergence of new viral variants [19] . in recent years, genotyping has been performed using s1 gene sequencing [17] , which has become the procedure of choice for differentiating between vaccine and field viruses. more than 20 ibv serotypes have been recognized worldwide [30] . ib still causes serious problems in the iraqi poultry industry due to the inability of vaccines to provide cross-protection between different genotypes. due to the limited network of poultry diagnostic laboratories in iraq, differential diagnosis is can only be done based on clinical signs and gross lesions. the characterization of ibv has raised additional problems in terms of both epidemiology and control. although ibv on the poultry farms in iraq (with h120 and 4/91 strains) is presently controlled by inactivated and live attenuated vaccines, outbreaks of ib have nevertheless been observed on broiler farms [1, 21] . thus, the spread of viral respiratory diseases has become the most commonly reported condition in commercial broiler flocks in iraq; however, there have been no studies on the detection and genotyping of these viruses using molecular techniques and sequencing. in this study, genotyping of ibv isolates from broiler farms in the south and middle of iraq was carried out based on partial s1 sequencing. samples were collected from 100 broiler chicken farms in the south and middle (five governorates) of iraq (al-kut, al-najaf, thi-qar, al-muthanna and al-basrah; 20 farms per governorate; fig. 1 ) from september 2014 to december 2015. the samples (trachea and kidney) were taken from chickens that showed clinical signs suggesting ib (respiratory problems such as gasping, sneezing and bronchial rales, and nephritis lesions such as enlargement, and congestion in kidneys). the samples were collected with aseptic technique and frozen at -70°c. the details of positive samples are shown in table 1 . rna was extracted from tissue samples using cinnapure rna extraction kit (sinaclone, iran). for cdna synthesis, 1 ll (0.2 lg) of random hexamer primer (sinaclon, iran) was added to 5 ll of extracted rna, and the mixture was heated at 65°c for 5 minutes. fourteen ll of cdna master mix containing 7.25 ll of depc-treated water (sinaclon, iran), 2 ll of dntp mix (sinaclon, iran), 0.25 ll of ribolock rnase inhibitor (thermo fisher scientific, usa), 0.5 ll of revert aid reverse transcriptase (thermo fisher scientific, usa), and 4 ll of 5x rt reaction buffer was added to each tube, resulting in a final volume of 20 ll. then, the mixture was incubated at 25°c for 5 min, 42°c for 60 min, 95°c for 5 min, and 4°c for 1 min. the cdna was stored at -20°c until use. sampling, rna extraction, and cdna synthesis were conducted in iraq, and the remainder of the work was carried out in iran. real-time pcr for ibv detection based on the 5 0 utr was chosen for this study. the amplification was performed by using an amplification kit (bioneer, south korea) with the forward primer 5 0 gcttttgagcctagcgtt3 0 , reverse primer 5 0 gccatgttgtcactgtctattg3 0 and taqman ò dual-labeled probe fam-caccaccagaac ctgtcacctc-bhq1 as described by callison et al. [7] . nested pcr was performed using spike gene primers that were designed to amplify a *390-bp fragment of the gene [16] . first-round amplification (494 bp) was performed in a final volume of 20 ll containing 2 ll of distilled water, 13 ll of sinaclon 2 9 pcr master mix (sinaclon, iran), 2 ll of sx1 (5 0 cacctagaggtttgytwgcatg3 0 ) and sx2 (5 0 tccacctctataaacaccyttac3 0 ) primers (10 lm), and 3 ll of cdna . the amplification was performed with a 35-cycle thermal profile (94°c for 2 min, 94°c for 30 s, 58°c for 30 s, 72°c for 30 s, 72°c for 10 min). in the second round of the nested pcr, sx3 (5 0 taatactggyaatttttcagatgg3 0 ) and sx4 (5 0 aatacagattgcttacaaccacc3 0 ) primers were used. the second round of amplification was performed in a volume of 20 ll (4.5 ll of distilled water, 13 ll of sinaclon 2 9 pcr master mix (sinaclon, iran), 2 ll of sx3 and sx4 primers (10 lm), and 0.5 ll of the first-round pcr product). the reaction was carried out under the same cycling conditions. the pcr product was analyzed by electrophoresis on a 1.5 % agarose gel and visualized under uv light. an accuprep ò pcr purification kit (bioneer co., korea) was used for the purification of the pcr products. sequencing was performed with the primers (both directions) that were used in the second step of nested pcr (bioneer co., korea). chromatograms were evaluated with cromaspro (cromaspro version 1.5). a phylogenetic tree was constructed by the neighbor-joining method, using mega 5.1 software, and each tree was produced using a consensus of 1000 bootstrap replicates [31] . the nucleotide sequences of a partial segment of the s1 gene were compared with several s1 sequences from genbank, including h120 ( table 3 ). the s1 gene sequences of the ibvs were submitted to the ncbi genbank database with the accession numbers ku143883-ku143914. real-time pcr for ibv detection (ibv incidence rate) real-time pcr for ibv detection based on the 5 0 utr was used in this study. of the 100 samples tested, 32 were positive for ibv by real-time pcr. the number of positive samples from each governorate is presented together with the sample size from each governorate. the total incidence rate for the middle and south iraqi governorates was 32 %, where the highest incidence rate of ibv in broiler chickens was detected in al-najaf governorate 10/20 (50 %), while the lowest rate 4/20 (20 %) was detected in al-muthana governorate ( table 2) . to carry out ibv genotyping, all 32 positive samples were subjected to amplification of a portion of the s1 gene for sequencing. all samples that were positive in real-time pcr were positive in nested pcr. four genotypes were detected after sequencing. the percentage of is/1494, 793/b, qx, and dy12-2 genotypes in five iraqi governorates was 46.87 %, 40.62 %, 9.37 % and 3.12 %, respectively ( table 2 and fig. 2 ). the qx genotype was detected in al-kut and al-najaf governorates, while the dy12-2 genotype was only detected in al-najaf governorate. is/1494 and 793/b genotypes were detected in all governorates ( table 2) . the nucleotide sequences of the s1 gene from 32 iraqi ibv isolates obtained in this study were aligned and compared with those of previously identified isolates from iraq, neighboring countries, and worldwide reference ibv strains, as shown in table 3 . the analysis revealed that all sequences obtained in this study were genetically different. phylogenetic analysis of the 32 strains (fig. 2) revealed that iraqi ibv strains could be classified into four genetic groups or genotypes: group i, variant 2 is/1494-like viruses, including 15 field isolates (46.87 %); group ii, 793/blike viruses, including 13 field strains (40.62 %); group iii, qx-like viruses, including three field strains (9.37); and caused by bacteria and mycoplasmas have already been detected on broiler farms located in the central and southern parts of iraq. however, the molecular detection studies (infection rate and genotyping) of respiratory viral diseases such as ib in broilers in iraq are limited (personal communication with veterinary organizations). we conducted the first study to determine the infection rate and ibv genotypes on broiler farms, using molecular and phylogenetic techniques. the approved vaccine strains used in iraq are based on serotypes ma5, h120, and 793/b [21] . ib still causes serious problems in the iraqi poultry industry due to the inability of the vaccines to provide cross-protection between different genotypes [1] . the genotyping of ibv is necessary not only for understanding virus evolution but also for effective modification of the vaccination programs [10] . in this study, the infection rate of ibv was 32 %, which indicated widespread distribution of ibv in the southern and middle parts of iraq. the highest infection rate of ibv was observed in al-najaf (50 %) governorate, while the lowest (20 %) was observed in al-muthana (fig. 3) . [32] . the infection rate was lower than the rate previously reported on commercial farms within neighboring countries: 58.8 % and 42.8 % in jordan and iran, respectively [2] . even where there were no poultry farms in iraq, the infection rate was as high as 32 %. this may be attributed to poor biosecurity on the farms, resulting in the spread of infection between and within the flocks [22] . phylogenetic analysis showed that the iraqi isolates clustered into four genetic groups (group i, variant 2 (is/ 1494)-like; group ii, 793/b-like; group iii, qx-like; and group iv, dy12-2-like). variant 2 (is/1494-like) viruses were the dominant ibv genotype infecting broiler chickens, with an overall prevalence of 46.87 % in ibvpositive samples. they shared high nucleotide sequence identity with ibv isolates from iran, israel, egypt, turkey, and kurdistan. the isolation of is/1494/06 (eu780077) ibv, one of two israeli variant strains, was first reported by meier and mahar in israel [23] . ibv variants have been recognized in iraq since 2012 (iraqi veterinary directorate/ministry of agriculture). the present work is similar to another study in which three ibv genotypes were found in slemani-kurdistan, iraq, including group a (very similar to iranian isolates), group b (closely related to chinese isolates) and group c (similar to is/1494 and egypt/beni-seuf/01 isolates) [26] . these findings are in agreement with those of kanan et al., who reported that is/1494/06 ibv was the most common genotype in egypt (2010), lebanon (2010-2012), jordan (2011-2012), kuwait (2012), and oman (2013) [12] . the current study is similar to another study on an is/1494 strain in turkey [18] . the second group consisted of 793/b-like viruses that were detected in different iraqi governorates at the high prevalence of 40.62 %. the present study revealed the presence of 793/b on the broiler farms. the ibv strains of the 793/b serotype were first identified in france in 1985, followed by great britain in 1991. subsequently, these strains spread to other countries in europe, asia (particularly to iran, turkey), and north america and are some of the most common ibv serotypes in some countries [9] . our result were in line with those of mahmood et al., who conducted the first study of identification and genotyping of ibv isolates and indicated that the 4/91-like virus is circulating on vaccinated broiler farms of the kurdistan region of iraq [21] . the detection of 793/b in commercial flocks was previously reported in iran, jordan, and israel [23, 27, 29] . however, because all flocks have been vaccinated, we concluded that the detected viruses probably belonged to the vaccine strains. therefore, the full-length s1 gene of the isolates should be determined to differentiate field and vaccine isolates. in this study, another important ibv variant (qx-like) known to cause respiratory, renal, and reproductive problems [12] was detected with a prevalence of 9.37 %. in 1996, qx was first described and identified in china, after which the prevalence of the so-called qx-like ibv genotype was reported, and it became one of the most dominant genotypes in many countries [28] . the genotype, designated as qx ibv, has spread from asia, where it was described for the first time in 1996 in china, and spread to europe and recently to the southern part of the african continent [4] . the qx-like genotype was also previously detected in central iraq by the iraqi veterinary directorate (official report). this finding was in line with another study in the kurdistan region of iraq, which showed that the phylogenetic aspects of the kurdistan viruses were closely related (98.9 %) to the qx strain reported in china from 2009 to 2010 [4] . qx-type virus was isolated in israel in 2004 [4] . the pcr lab/06/2012 (jx477827) strain was c fig. 3 phylogenetic tree of 32 iraqi isolates and 24 reference strains of infectious bronchitis virus based on the nucleotide sequences of the s1 gene. the phylogenetic tree was constructed, using mega version 5, by the neighbor-joining method with 1000 bootstrap replicates (bootstrap values are shown on the tree). the isolates from this study are indicated by black circles. white dots indicate iranian reference strains, and black triangles indicate iraqi reference strains isolated in iran, which is genetically (99.36 %-99.68 %) related to strains of the present study [6] . this finding revealed that the qx strain has been widely transferred from other countries (probably iran) to iraq. although no information is currently available about the introduction of the qx strain from china into other countries, it has been hypothesized that wild birds may be the source of introduction based on the evidence that ibv may replicate in members of the order anseriformes [25] . it is of interest that that dyi2-2-like ibv genotype was detected in the present study. this is the first report of this genotype in iraq, and it has not yet been reported in other countries in the middle east. the virus appeared following the recombination of ck/ch/gd/lz09 and ta09 ibv in china and is currently a circulating ibv genotype in china [11] . it is also fair to say that the origin of this virus and the means by which it was introduced to iraq are not clear. based on the persistence of the dy12-2 like genotype in china, it could be predicted that this genotype might spread in iraq and the middle east in the future. in this survey, some ibv genotypes, such as is/720, massachusetts, d274, and q1, were not detected. these ibv genotypes were detected previously and reported in iraq, iran, the united arab emirates and saudi arabia [3, 12, 14, 21] . new serotypes or variant strains may emerge because of only a few changes in the amino acid sequence of the s1 protein. these changes could be due to immunological pressure caused by the extensive use of vaccines, recombination as the outcome of mixed infections, or a decrease in the prevalence of dominant serotypes as a result of vaccination, allowing other field strains to emerge [25] . not much is known about the mode of spread of ibv between the countries in the middle east. however, crossborder movements of poultry and poultry-related products are likely to be important factors [21] . in summary, our study demonstrated a high rate of infection with ibv (32%) in central and southern iraq. this is the first report indicating the presence of an ibv dy12-2like strain in the middle east. it is an updated and comprehensive study of genotyping of ibv in iraq and completes the ibv puzzle in the region. phylogenetic analysis showed that the detected strains are closely related to other ibv strains infecting broilers, pullets, and layers in the region. finally, it could be suggested that 1) work be done on whole-genome sequencing, 2) cross-protection studies be conducted for designing the best vaccination program in the iraqi poultry industry, 3) molecular surveillance in other governorates in iraq be continued, and 4) the pathogenesis of different iraqi ibv isolates be studied. molecular detection of infectious bronchitis virus and it is relation with avian influenza virus (h9) and mycoplasma gallisepticum from different geographical regions in iraq molecular detection of infectious bronchitis and avian metapneumoviruses in oman backyard poultry molecular detection of infectious bronchitis and avian metapneumoviruses in oman backyard poultry circulation of qx-like infectious bronchitis virus in the middle east completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus detection of the chinese genotype of infectious bronchitis virus (qx-type) in iran development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronaviruses in poultry and other birds variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs factors influencing the outcome of infectious bronchitis vaccination and challenge experiments analysis of s1 gene of avian infectious bronchitis virus isolated in southern china during genotypes of infectious bronchitis viruses circulating in the middle east between turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review phylogenetic study of iranian infectious bronchitis virus isolates during 2010-2011 using glycoprotein s1 gene review of infectious bronchitis virus around the world efficacy of live infectious bronchitis vaccines against a novel european genotype, italy 02 development and validation of rt-pcr tests for the detection and s1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples presence of is/1494/06 genotype-related infectious bronchitis virus in breeder and broiler flocks in turkey evolutionary and bioinformatics analysis of the spike glycoprotein gene of h120 vaccine strain protectotype of infectious bronchitis virus from india fenner's veterinary virology isolation and molecular characterization of sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east infectious bronchitis virus: a major cause of respiratory disease outbreaks in chickens in ghana identification of a novel nephropathogenic infectious bronchitis virus in israel complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand investigation and molecular characterization of avian infectious bronchitis virus in suspected broiler farms in slemani governorate. council of college of veterinary medicine university of sulaimani in partial fulfillment of the requirements for the degree of master in veterinary medicine/clinical pathology by hana sherzad rauf bvm &s molecular subtype of infectious bronchitis virus in broiler flocks in jordan the pathogenesis of a new variant genotype and qx-like infectious bronchitis virus isolated from chickens in thailand a survey of the prevalence of infectious bronchitis virus type 4/91 in iran infectious bronchitis virus variants: a review of the history, current situation and control measures mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods molecular survey and phylogenic analysis of infectious bronchitis virus (ibv) circulating among chicken flocks in riyadh province, saudi arabia acknowledgments the authors gratefully acknowledge dr. hamideh najafi, dr. iraj ashrafi, and mr. behrooz asadi for their extensive technical support. funding financial support for this study was provided by the ministry of higher education and scientific research, iraq, university of basrah (7.30.4603), and the research council of the university of tehran under grant no. 28692-6-2. ethical approval this article does not contain any studies of animals performed by any of the authors. key: cord-304278-0qy1nngs authors: raj, g. dhinakar; jones, r. c. title: infectious bronchitis virus: immunopathogenesis of infection in the chicken date: 2007-11-12 journal: avian pathol doi: 10.1080/03079459708419246 sha: doc_id: 304278 cord_uid: 0qy1nngs the immunopathogenesis of infectious bronchitis virus (ibv) infection in the chicken is reviewed. while infectious bronchitis (ib) is considered primarily a disease of the respiratory system, different ibv strains may show variable tissue tropisms and also affect the oviduct and the kidneys, with serious consequences. some strains replicate in the intestine but apparently without pathological changes. pectoral myopathy has been associated with an important recent variant. several factors can influence the course of infection with ibv, including the age, breed and nutrition of the chicken, the environment and intercurrent infection with other infectious agents. immunogenic components of the virus include the s (spike) proteins and the n nucleoprotein. the humoral, local and cellular responses of the chicken to ibv are reviewed, together with genetic resistance of the chicken. in long-term persistence of ibv, the caecal tonsil or kidney have been proposed as the sites of persistence. antigenic variation among ibv strains is related to relatively small differences in amino acid sequences in the s1 spike protein. however, antigenic studies alone do not adequately define immunological relationships between strains and cross-immunisation studies have been used to classify ibv isolates into ‘protectotypes’. it has been speculated that changes in the s1 protein may be related to differences in tissue tropisms shown by different strains. perhaps in the future, new strains of ibv may arise which affect organs or systems not normally associated with ib. the study of pathogenesis was formerly regarded as a messy, unsatisfactory area for virus research-a sad state of affairs that was lamented by early enthusiasts (mimms, 1964) . that view has changed now and there is an urgent need to know how viruses cause diseases. the study of mechanisms of pathogenicity was recently placed at the top of the research objectives proposed for priority treatment by an ad hoc group on vaccinology (bourne, 1996) . however, pathogenesis cannot be studied without also considering immunology and mechanisms of protection. this review attempts to concisely present the current state of knowl-edge on the immunopathogenesis of infection in chickens with infectious bronchitis virus (ibv). in 1931 a 'new respiratory disease of baby chicks' was identified in the usa by schalk & hawn and named infectious bronchitis (ib). following infection of chickens with ibv, falls in egg production and quality were reported for the first time by van roekel et al. (1951) and broadfoot & smith (1954) . in 1962, cumming found that ibv was the cause of nephrosis-nephritis syndrome seen in australia. in more recent years, ibv has also been isolated from cloacal swabs and gut tissues, but while infection has sometimes been associated with diarrhoea, no pathological changes have been described in the alimentary tract. swollen head syndrome (shs) in chickens has generally been associated with infection with the avian pneumovirus (apv), turkey rhinotracheitis virus (trtv) (picault et al, 1987; lu et al, 1994) . however, in the first description of shs, morley & thomson (1984) isolated a coronavirus, while in other reports, massachusetts strains of ibv have also been isolated (shirai et al., 1993; droual & woolcock, 1994) . recently, a variant strain of ibv, 793/b was isolated from broiler breeder flocks where the affected birds had bilateral myopathy affecting both superficial and deep pectoral muscles (gough et ah, 1992) . thus, it is clear that strains of ibv have wide and variable tissue tropisms, and the clinical manifestations of the disease can be diverse. although the name ib does not encompass the varied clinical manifestations observed with ibv infections, for the present this name has still been retained to avoid confusion. replication of ibv in the respiratory tissues causes characteristic, but not pathognomonic signs such as gasping, coughing, tracheal rales and nasal discharge. occasionally, puffy, inflamed eyes and swollen sinuses may be seen (parsons et al, 1992; capua et al, 1994) . in uncomplicated cases these signs last for only 5 to 7 days and disappear within 10 to 14 days. the affected chickens also appear depressed, and feed consumption and weight gain are significantly reduced from 3 days after infection (otsuki et al, 1990) . in uncomplicated cases, mortalities are generally low and have been attributed to asphyxiation due to blocking of the lower trachea or bronchi by plugs of mucus. the upper respiratory tract is the main site of ibv replication, following which a viraemia occurs and the virus gets widely disseminated to other tissues (mc-martin, 1993) . the virus is primarily epitheliotropic and enters the epithelial cells by viropexis (patterson & bingham, 1976) . studies using immunofluorescence (if) qones & jordan, 1972; yagyu & ohta, 1990) , immunoperoxidase (ip) (nakamura et al, 1991; owen et al, 1991) and electron microscopy (purcell & clarke, 1972; nakamura et al, 1991) have shown virus replication in ciliated epithelial and mucus-secreting cells. during the clinical phase of the disease, maximum virus titres are recorded in the trachea between 5 and 10 days post-infection (p.i.) (ambali & jones, 1990; otsuki et al, 1990 ), but occasionally virus may be present for up to 28 days p.i. cook, 1968) . virus also replicates in the epithelial cells of lungs (janse et al, 1994 ) and airsacs (nauwynck & pensaert, 1988) . high virus titres are seen in these tissues between 4 to 11 days p.i. (nauwynck & pensaert, 1988; otsuki et al, 1990) . infected chickens have mucosal thickening with serous or catarrhal exudate in the nasal passages, sinuses and trachea. the incidence of nasal exudate in experimentally-infected chickens has been used to assess the severity of disease in different inbred lines (parsons et al, 1992) and in bursectomised birds (cook et al, 1991a) . small areas of pneumonia may be observed in the lungs. the air sacs may appear cloudy or contain a yellow caseous exudate (king & cavanagh, 1991) . the progression of lesions in the trachea has been divided into three stages, degenerative, hyperplastic and recovery (nakamura et al, 1991; purcell & mc-ferran, 1972) . deciliation and desquamation of epithelial and mucous-secreting cells occur in the first 1 or 2 days with a mild infiltration of heterophils and lymphocytes in the lamina propria. heterophils are often seen infiltrating between ciliated epithelial cells and occasionally in the lumen of the trachea. during the hyperplastic stage, newly-formed epithelial cells are observed which usually have no cilia. by 4 to 6 days the reparative processes begin with complete recovery by 10 to 20 days (chen et al, 1996) . in affected airsacs, epithelial cell desquamation, oedema and some fibrinous exudate may be seen (king & cavanagh, 1991) . difficulty in quantifying severity of respiratory infection caused by ibv has always been a problem in studying its pathogenicity . the virus causes stasis of tracheal cilia both in vivo and in vitro, and this parameter has been used to assess severity of respiratory infection. otsuki et al. (1990) found that following infection with the m41 strain, the duration and severity of tracheal ciliary damage was longer in a susceptible line of chicken (151) than in a resistant line (c). the authors proposed that this method could be used to compare the pathogenicity of different strains or susceptibility of different breeds to ibv. cubillos et al. (1991) found that in unvaccinated chickens challenged with four ibv isolates from chile (one belonging to the massachusetts serotype and three variants), the tracheal damage in terms of ciliary activity was variable, suggesting differences in virulence of ibv strains for the trachea. while tracheal organ cultures (toc) have been used extensively for cultivation and assay of ibv, distinct differences in virulence among ibv strains have not been observed in this in vitro system. cook et al. (1976) , while standardizing toc for the isolation and assay of ibv, compared three strains of ibv on the basis of their effect on tracheal cilia, but found no marked differences. dhinakar raj & jones (1996c) also reported little difference among seven ibv strains using measurement of ciliary activity as a criterion for damage to the tracheal epithelium. of nine strains compared in toc by cubillos et al (1991) , the hyperplasia caused by two viruses was the only histological difference. ibv infections may not occur as a single entity in the field. with the presence of several respiratory diseases in chickens caused by bacteria and viruses, the role of these agents in increasing the severity of ibv or otherwise is an important contributory factor in influencing the pathogenesis of the disease (see later). in layers, ib can cause a severe decline in egg production, and later, a deterioration in shell and internal quality (mcdougall, 1968; sevoian & levine, 1957) . such effects may be accompanied by mild (muneer et al, 1986 (muneer et al, , 1988 or no respiratory signs (cook, 1984; . some strains produce only a loss in shell colour . the severity of production decline varies with the period of lay, the virulence of the virus involved and other non-specific factors. the response of individual hens varies greatly (mcmartin, 1968) . production may start to increase after 2 to 3 weeks, but reaches only sub-optimal levels. when laying is resumed, some eggs have soft-shells, while others are mis-shapen or rough-shelled. when laying hens were infected with ibv strain m41, viral antigen was demonstrated in the epithelium of the oviducts between 6 and 9 days p.i. (jones & jordan, 1971) . areas of glandular hypoplasia caused by ibv leads to reduction in the synthesis of albumen proteins especially ovomucin, lysozyme and other major proteins which constitute the structural matrix of the thick albumen (butler et al, 1972) . hence, there is a decrease in the proportion of both thick and inner thin albumen, and an increase in the amount of outer thin albumen causing 'watery-whites'. presence of blood or meat spots in the egg albumen has also been reported (mcdougall, 1968; muneer et al, 1987) . inspissated yolk material may be seen in the abdominal cavity of infected layers. microscopic changes in the oviduct include reduction in the height of the epithelial cells, reduction in number or complete absence of cilia, dilation of glands, lymphocytic foci and cellular infiltration in the lamina propria and inter tubular stroma (sevoian & levine, 1957) . the mechanisms whereby ibv infection causes egg production of some birds to cease and for varying periods of time have not been elucidated. ibv infection of female chicks of less than 2 weeks of age can cause permanent damage to the developing reproductive tract, resulting in 'false layers' that do not lay normally at sexual maturity (broadfoot et al, 1956; jones & jordan, 1970) . following infection of day-old chicks with ibv strain m41, virus was isolated between days 5 and 11 p.i. (jones & jordan, 1972) and was also found to replicate in the epithelium of the oviducts (crinion & hofstad, 1972a,b; jones & jordan, 1972) . gross changes in the oviduct caused by early infection may vary from the presence of a continuous patent but underdeveloped structure to a blind sac projecting forward from the cloaca (jones & jordan, 1970 . the middle third of the oviduct is the most severely affected with areas of localised hypoplasia seen between normal patent oviducts. caudal to the hypoplastic regions, macroscopic cysts filled with a clear serous fluid may be seen (crinion et al., 1971a) . the histopathological changes in the oviduct include decreased height and loss of cilia from epithelial cells, dilation of the tubular glands, infiltration of heterophils, lymphocytes and plasma cells, and oedema and fibroplasia of the lamina propria (crinion et al, 1971a,b; crinion & hofstad, 1972a,b) . the effect of ibv on the male reproductive tract has not been reported. variations in the ability of ibv strains to cause decreases in egg production and quality were reported by guittet et al. (1988) ; d274 was the least virulent while m41 and a variant strain pl84084 had the same degree of pathogenicity. found that some variant strains of ibv caused only small decreases in egg production, but had a marked effect on egg colour. in contrast, a more recent variant (parsons et al., 1992) caused a substantial decline in egg production with little loss of egg colour in the field, although no experimental work has been done with this virus. differences in virulence of ibv strains for the immature chicken oviduct were reported by crinion & hofstad (1972a) ; massachusetts and t strains were virulent, while connecticut and iowa 609 were not. embryo passage of ibv strain m41 reduced its virulence for the oviduct (crinion & hofstad, 1972b) . in vitro, oviduct organ cultures (ooc) were highly susceptible to the h52 strain of ibv regardless of the age of the chickens and no differences in susceptibility were seen between magnum and uterus regions . pradhan et al. (1984) showed that ibv strain m41 causes stasis of cilia in ooc prepared from precociously-induced oviducts in young chicks by oestrogen treatment. this work has been extrapolated to compare the virulence of seven strains of ibv in vitro using ciliostasis and a calmodulin assay to quantify the damage to oviduct epithelium (dhinakar raj & jones, 1996c ). an ibv isolate belonging to serotype d207 was the most virulent while an enterotropic variant strain g was the least. although even those strains of ibv considered primarily to affect the respiratory tract such as m41 can occasionally cause kidney damage (jones, 1974) , nephropathogenicity has been associated only with certain strains. greater virulence of the virus for the kidney was first reported in australia (cumming, 1962) . since then, nephropathogenic ibv (nibv) has been reported from usa and certain parts of europe (butcher et al., 1990; kinde et al., 1991; lambrechts et al, 1993; picault et al, 1991; zanella, 1988) . nibv strains initially cause respiratory symptoms followed by signs due to kidney damage which include increased water consumption and wet droppings (winterfield & hitchner, 1962; cumming, 1963) . mortalities occur and follow a consistent pattern (cumming & chubb, 1988) . the first deaths usually occur around 6 days p.i., increase rapidly to a peak around 10 days with the last deaths seen around 16 days p.i. however, the mortality rates depend on several intrinsic and extrinsic factors (see below). virus replication in the kidneys has been shown in the proximal convoluted tubules (chong and apostolov, 1982) , distal convoluted and collecting tubules (owen et al, 1991) and collecting ducts (chen et al, 1996) . structural alterations in the tubular epithelial cells (condron & marshall, 1986) caused impaired fluid and electrolyte transport leading to acute renal failure. an increase in urinary water losses in chickens infected with nibv was found to be associated with lower urine osmolality and higher rates of urinary excretion of sodium, potassium and calcium (afanador & roberts, 1994; condron & marshall, 1985; heath, 1970) . negative sodium balance was a direct effect of increased output of sodium in the urine, while negative potassium balance was due to decreased intake. the kidneys of chickens infected with nibv are swollen and pale, with tubules and ureters distended with urates (cumming, 1963) . the relative kidney weight and kidney asymmetry are increased. the haematocrit values of infected birds was decreased and plasma uric acid levels were increased (afanador & roberts, 1994) . despite lack of gross lesions microscopic changes of nephritis may still be present (winterfield & albassam, 1984) . chen et al., (1996) proposed that ibv-induced renal lesions can be considered to be a ductotubular interstitial nephritis. the virus causes granular degeneration, vacuolation and desquamation of the tubular epithelium with massive infiltration of heterophils in the interstitium in acute stages of the disease. the changes in the chronic phase were classified as being active or inactive types of interstitial lymphocytic nephritis (albassam et al., 1986) . the chronic inactive form of nephritis was indicative of ibv replication in the kidneys and subsequent clearance; while the chronic active type suggested a persistent viral replication and damage to epithelial cells of the kidney tubules. virus was isolated from 50% of the birds with chronic nephritis (chong & apostolov, 1982) . the histopathological changes in the kidneys following nibv infections have been described by several workers (albassam et al., 1986; chen et al., 1996; pohl, 1974; purcell et al, 1976; siller & cumming, 1974) . the type of kidney lesions produced by different nibv strains were similar but their severity varied (albassam et al., 1986; chandra, 1987) . the australian strain 't' induced the most rapid and severe lesions following both intra-venous (i.v.) (chandra, 1987) or intra-ocular (albassam et al, 1986) inoculations of susceptible chickens. the effect of ibv on the trachea is apparently independent of the effect on kidney (ratanasethakul & cumming, 1983) . a strain of nibv, 's' virus was less severe on the kidneys than 't', but more severe on the tracheal mucosa. using an i.v. inoculation model to titrate kidney infectivity, lambrechts et al. (1991) found no significant differences in the infectivity among belgian field nibv isolates, but the infectivity of egg-passaged virus was highly reduced. several strains of ibv have been isolated from cloacal swabs, faeces and caecal tonsils alexander et al, 1978; cook, 1984; lucio & fabricant, 1990) . in vitro explants of several gut tissues have been shown to support the growth of ibv (bhattacharjee, 1994; bhattacharjee & jones, 1997; darbyshire et al, 1976) . in studies where virus isolation was attempted from several tissues, maximum virus isolations were obtained from the oesophagus of chickens infected with ecv2, an enteric isolate of ibv (lucio & fabricant, 1990) and 793/b-like virus (dhinakar raj & jones, 1996a) . oesophageal swabs have also been used to identify ibv using the polymerase chain reaction (d. cavanagh, personal communication). however, it is not clear whether the virus actually multiplies in the oesophagus or whether virus is swallowed after being expelled from the trachea. ibv has also been isolated from proventriculus, duodenum and jejunum (ambali & jones, 1990; lucio & fabricant, 1990) . darbyshire et al, (1976) have shown that proventriculus was inferior only to respiratory tissues and oviduct in supporting virus multiplication in vitro. however, the site of virus multiplication in these tissues has not been confirmed, but presumably occurs in the epithelial cells. in contrast, in the tissues of the lower gut, ibv replication has been described (without photographs) in cells resembling histiocytes and lymphoid cells in the caecal tonsils (owen et al, 1991) and demonstrated by if in apical epithelial cells of the villi in ileum and rectum (ambali & jones, 1990; dhinakar raj & jones, 1996a) . although ibv has a wide tropism for gut tissues no gross or histological changes have been reported. a variant strain of ibv, strain g, was classified as being enterotropic by virtue of its prolonged persistence in the gut compared to the trachea (el houadfi et al, 1986) . recently, it was seen that a variant ibv strain 793/b was more enterotropic than pneumotropic and was even associated with diarrhoea in broilers (dhinakar raj & jones, 1996a) . it seems that studies on the replication of ibv in the gut have been neglected, probably because no ibv strain has been demonstrated to be enteropathogenic. macdonald et al. (1983) reported that following crop inoculation of chicks with h52 and h120 vaccines, virus was rarely isolated from the gut or any other visceral tissues and no antibody response occurred. however, the kidneys were found to be resistant to i.v. challenge. more intensive studies on oral infection with enterotropic ibv strains or dual infections with the virus and other enteric pathogens, such as salmonella, coccidia or rotavirus, might yield more information on the effects of ibv replication in the gut. the ability of ibv strains to survive in the presence of low ph, digestive enzymes and bile salts may be relevant to enteric replication. otsuki et al (1979a) and cowen & hitchner (1975) showed that some strains suffer a greater loss of titre than others when kept at ph 3.0 for 3 or 4 h, but there was no indication that any of the more resistant strains were isolated from the gut. ambali & jones (1991a) compared strain m41 with an enterotropic variant (g). both viruses had a similar sensitivity to trypsin, but the variant showed a 50-fold greater resistance to sodium tauroglycocholate, which might partly explain its ability to replicate in the gut. the important variant strain of ibv, 793/b, was recently isolated from a broiler breeder flock where the affected birds had bilateral myopathy affecting both deep and superficial pectoral muscles (gough et al, 1992) . there was marked swelling and pallor of deep pectoral muscles together with the presence of occasional fascial haemorrhages and a layer of gelatinous oedema over its surface. experimental infection of 6 week old broilers with an isolate of ibv belonging to this group resulted in mild oedematous separation of muscle fibres but with no corresponding increase in serum creatine kinase concentrations (dhinakar raj & jones, 1996a ). it has been clearly shown that the virus was not involved directly in the pathogenesis of the muscle lesions but formation and deposition of immune complexes, such as those found in the kidneys (dhinakar raj & jones, 1996a) , in the capillary walls of the muscle could be a possible reason for the development of this strange lesion. ibv has been isolated from the harderian gland (dhinakar raj & jones, 1996a; gelb et al, 1991b; toro et al., 1996a) and ibv-positive cells have been shown in the stroma of the gland by if staining (toro et al, 1996a) . ibv vaccination by eyedrop resulted in a massive infiltration of lymphocytes, increase in plasma cell numbers (davelaar & kouwenhoven, 1976; survashe et al, 1979; montgomery et al, 1994; toro et al, 1996a) and desquamation of tubular epithelium in the harderian gland with restoration from 14 days after vaccination (toro et al, 1996a) . increased numbers of plasma cells (survashe et al, 1979) and lymphoid tissue development (montgomery et al, 1994) were also seen in the lachrymal gland following ibv vaccination. ibv has also been isolated from the bursa of fabricius (el houadfi et al, 1986; ambali & jones, 1990) , and gross and histopathological lesions have been shown following experimental h52 and hi20 infections (macdonald & mcmartin, 1976) . although ibv has been isolated from a variety of other tissues such as liver ambali & jones, 1990 ) and spleen (otsuki et al, 1990) it has not been documented to be involved with any functional damage. ibv has been isolated from semen and eggs of infected chickens (cook, 1971 ), but vertical transmission appears to be of little importance. several intrinsic (age and breed of chickens) and extrinsic (nutrition, environment and intercurrent infections) factors influence the pathogenesis of ibv infections. all ages are susceptible, but the clinical disease is more severe in young chicks (animas et al., 1994b) although recovery of the virus from the trachea was similar compared to 6-week old infected chickens (animas et al., 1994a) . however, the duration of virus excretion in the faeces was longer in 2-week old infected chicks compared to 4-and 6-week-old chickens. as age increases chicks become more resistant to ibv-induced mortality (smith et al., 1985) , nephropathic effects (albassam et al., 1986) and oviduct lesions (crinion & hofstad, 1972a) . mac-donald et al. (1980) described fewer pathological changes in kidneys of chickens infected at 3 weeks of age than those infected at day-old or 10 weeks of age, although the virus was inoculated i.v., which is an unnatural route of infection. purchase et al. (1966) demonstrated variation in mortality following ibv inoculation of embryos from different inbred lines. bumstead et al. (1989) compared mortalities in several inbred lines of chickens following inoculation with a pool of strains of ibv and/or escherichia coli and found marked differences among them. on this basis, the lines of chickens were classified as being resistant or sensitive to ibv. however, these inbred lines did not show marked differences in susceptibility to the variant strain of ibv, 793/b (parsons et al, 1992) . genetic differences in susceptibility to nephritis are also marked, with light breeds being more susceptible than heavy breeds (cumming & chubb, 1988) . the mortalities in 4-week-old cockerels of 10 australian commercial egg-laying strains varied from 11 to 59%. nibv has been shown to cause higher mortalities in broilers than layers (ignjatovic, 1988; zanella, 1988) although the virus multiplies to the same extent in both types of birds (lambrechts et al., 1993) . male chicks are twice as susceptible as females to nephritis (cumming, 1969) . high protein diets increase mortality from ibv-induced nephrosis; chickens fed meat meal or poultry by-product meal-based diets experience higher mortality than those fed soybean based diets (cumming, 1969; cumming & chubb, 1988) . low temperatures have a dramatic effect on mortality due to nibv. reduction in temperature from 20°c to 16°c increased mortality from 8 to 50% (cumming, 1969) with greater severity of histopathological lesions in kidneys (ratanasethakul & cumming, 1983) . exposure to cold stress has been used to increase the severity of challenge for assessing protection afforded by ibv vaccines (klieve & cumming, 1990) . cold stress also increased the severity of ibv-induced tracheal lesions (ratanasethakul & cumming, 1983) and promoted more extensive air sacculitis after combined infections with ibv and mycoplasma synoviae (ms) (yoder et al, 1977) . mycoplasmas adler et al. (1962) found that intranasal inoculation of either the massachusetts strain of ibv or m. gallisepticum (mg) given alone produced little by way of clinical signs, but dual infections resulted in coryza, tracheitis and airsacculitis. the incidence of airsacculitis was highest in chickens infected with ibv 1 week after mg inoculation. in adult chickens combined infections with ibv and mg has been shown to cause a more severe effect on egg production and quality than inoculation with either agent alone (blake, 1962) . olson et al, (1964) exposed chickens to ibv and ms but found that dual infections were not synergistic. in contrast, later work by kleven et al, (1972) found that airsacculitis resulted when chickens were infected with ib and newcastle disease (nd) vaccines after ms infection. hopkins & yoder (1982) , in similar experiments with ms, gave different strains of ibv 2 to 5 days before the mycoplasma. they showed that different strains of ibv had differing abilities to induce airsacculitis. field isolates of ibv had the most severe effects. vaccines of high passage produced very mild lesions while those of low passage caused intermediate lesions. gross (1958) , in one of the first studies, infected 5-week-old chickens with ibv and gave pathogenic e. coli by aerosol at intervals afterwards. the most severe pericarditis was seen when the bacteria were given 11 to 22 days after the virus. smith et al. (1985) , using a combined inoculation of a pool of strains of ibv and e. coli, developed an experimental model for ibv which closely resembled natural outbreaks of the disease. the damage to tracheal epithelium caused by ibv facilitates e. coli invasion and multiplication leading to death or lesions in surviving chickens. using this model, several reports have described differences in virulence of ibv strains, differences in susceptibility between genetic lines of chickens and detailed interactions between the agents (avellanda et al., 1994; bumstead et al, 1989; cook et al, , 1991b cubillos et al, 1991; smith et al, 1985) . raggi et al. (1967b) showed that when ibv and haemophilus paragallinarum were given together intra-nasally, the incubation period was shorter, there was higher mortality and lesions were more severe. there is no reported evidence of dual infections with ibv and pasteurella multocida, the causative agent of fowl cholera. however, bisgaard (1977) , in a study of chickens in the field, found that where the bacterium p. haemolytica was present with ibv, there was no apparent exacerbation of the viral infection. ibv is known to interfere with the replication of ndv in eggs and raggi & lee (1964) have shown that interference also occurs in chickens, but this is only one way. they found that when ibv was given to chickens with ndb1 vaccine, there was subsequently no production of haemagglutination-inhibition (hi) antibodies to ndv, but ndv did not interfere with ibv infection. raggi et al. (1967a) found no synergism between ibv and infectious laryngotracheitis virus (iltv), but pattison et al. (1971) reported that aerosol vaccination with ibv increased the mortalities due to iltv. apv, the cause of trt and mild respiratory disease in chickens may also occur in respiratory outbreaks involving ibv, but the significance of these two agents being present together is yet to be reported. fabricant & levine (1962) examined triple infections involving ibv, mg and pathogenic e. coli. they reported that with regard to disease lesions the descending order of severity was ibv, mg and e. coli; mg and e. coli; ibv and e. coli; and e. coli alone. a similar study by nakamura et al. (1994) was performed with live ib and nd vaccines given to young chicks with mg and/or e. coli or both. again, the most severe combination was when vaccine, e. coli and mg were given together. thus, it is clear that several respiratory pathogens interact synergistically with ibv or ib vaccines and enhance the severity and duration of the disease. in many instances, the interval between infections is important, as are the challenge doses. however, with few exceptions (e.g. nakamura et al., 1992) , it is not known whether the synergisms are due to immunosuppression or simply that the epithelial damage caused by one agent permits greater penetration by others. the two classical examples of immunosuppressive viruses of chickens are infectious bursal disease (ii3d) which principally affects the b-cells and chicken anaemia virus (cav) which affects the t-cells. there is abundant evidence that ibdv infection impairs the humoral response to ibv. giambrone et al. (1977) infected chicks with ibdv at day-old and ibv at 14 days. compared to single ibv infection with both viruses, antibody titres were lower and airsac lesions greater. rosenberger & gelb (1978) also showed that ibdv at day-old affected the response to ib vaccines so that there was lowered resistance to challenge. furthermore, winterfield et al. (1978) showed that ibdv increased susceptibility to ibv, reduced ibv antibody levels and explained unsatisfactory immunity when replacement and broilers were vaccinated at an early age. there are no reports of dual infection of chickens with ibv and cav. goodwin et al. (1992) reported that there were no significant differences in ibv-specific antibody titres between chicken flocks positive or negative for cav antibodies. however, when chicks infected with ibv strain m41 were treated with the t-cell immunosuppressor drug cyclosporin a (csa), the clinical signs in these birds were more severe and virus titres in tissues, especially kidneys, were greater than in infected but untreated birds, although the persistence of the virus was not increased (dhinakar raj & jones, 1997a). ibv-induced mortality was also increased. it seems possible that infection with cav at a critical time might induce some of these effects on ib. although ibv has been isolated from lymphoid tissues such as bursa of fabricius and harderian gland, the evidence for a direct immunosuppression is limited. it is not clear whether ibv multiplies in the lymphocytes but cultured macrophages were found to be resistant (von bulow & klasen, 1983) . a virulent ibv has been shown to induce transitory reduction in proliferative responses of whole blood lymphocytes to t-cell mitogens, pha (wakenell et ah, 1995) and concanavalin a (dhinakar raj & jones, 1997a). ibv vaccines were found to depress the harderian gland responses to killed brucella abortus (montgomery et al., 1991 (montgomery et al., , 1994 . however, no correlation between histological responses and hi titres to ibv were found with depression of responses to br. abortus. ibv is a positive-stranded rna virus and is the prototype of the family coronaviradae. it has three structural proteins. the spike 's' glycoprotein is located at the surface of the virion, and consists of two subunits, si and s2, with molecular weights of 92 and 84 k, respectively. the membrane 'm' glycoprotein is partially exposed at the surface of the virion with molecular weights ranging from 27 to 36 k, and the nucleocapsid 'n' protein is internally located with a molecular weight of 52 k (wadey & westaway, 1981; cavanagh, 1983a,b,c) . the si glycoprotein of ibv induces virus neutralizing (vn) and hi antibodies (cavanagh et al, , 1986 kant et al, 1992; koch et al, 1990; mockett et al, 1984; niesters et al, 1987) and has been considered as the most likely inducer of protection (cavanagh et al, 1986; ignjatovic & galli, 1994) , but s2 and n proteins may also be important since they carried epitopes for induction of cross-reactive antibodies (ignjatovic & galli, 1995) . the time of appearance of si, s2 and n eljsa-antibodies have been shown to be similar, being detected 2 weeks after live ibv vaccination (ignjatovic & galli, 1995) . this coincides with the appearance of vn antibodies mockett & darbyshire, 1981; darbyshire and peters, 1984) . epitopes on n and s2 proteins that gave rise to cross-reactive antibodies showed the same degree of conservation while si epitopes were shown to be marginally less conserved (ignjatovic & galli, 1995) . a t-cell epitope has been identified in the ibv 'n' protein (boots et al, 1991) and has been shown to induce anti-viral responses (boots et al, 1992) . cellular immune responses elicited by a live ibv vaccine have also been found to be cross-reactive and the responses varied in magnitude with the serotype of ibv used for in vitro stimulation (dhinakar raj & jones, 1997b) . immunity can be considered to be either innate or acquired. innate immunity comprises a collection of factors which resist invasion by external agents, such as physical barriers provided by skin and mucous membranes, soluble factors like lysozyme, complement and acute phase proteins, and cells such as granulocytes, macrophages and natural killer (nk) cells. the main features of innate immunity are lack of specificity and immunological memory. heterophils (neutrophils) constitute the 'first line of defence' against infectious agents and are the first cells to be recruited to the site of infection, following initiation of an inflammatory response. in ibv-infected chickens, heterophils are the most numerous early inflammatory cells in respiratory lavage fluids (fulton et al, 1993) . using a heteropaenic chicken model (kogut et al, 1993) the importance of these cells in limiting ibv replication was studied (dhinakar raj et al., 1997c) . it was found that they had no effect on virus multiplication and, in fact, contributed to the damage in the tracheal epithelium. the role of macrophages in ibv infections is unknown, while no alterations in nk cell activity has been found following ibv infection (wakenell et al, 1995) . serum levels of an acute phase protein, °° i acid glycoprotein, have been found to peak on day 6 following ibv infection (nakamura et al, 1996) . acquired immunity results in the activation of antigen-specific effector mechanisms including b-cells (humoral), t-cells (cellular) and macrophages, and the production of memory cells. to ibv infections, measurable by enzyme-linked immunosorbent assay (elisa), haemagglutiation inhibition (hi) or vn tests (de wit et al, 1992; monreal et al, 1985; wilcox et al, 1983) . however, there is a lack of correlation between titres of circulating antibodies and resistance to infection (raggi & lee, 1965; winterfield & fadly, 1972; . immunoglobulin g (igg), the major circulating ig, is the antibody detected by hi and an elisa developed to measure it is more sensitive (mockett & darbyshire, 1981) . anti-ibv igg can be detected as soon as four days pi, reaches a peak at about 21 days but can remain in high titre in the serum for many weeks (mockett and darbyshire, 1981) . this is the antibody measured in conventional serological tests to monitor ibv infections or vaccine uptake. immunoglobulin m (igm), present only transitorily after infection, reaches peak concentrations about 8 days after ibv infection and levels then decline (mockett and cook, 1986) . although an igm-specific elisa has been shown to be useful in the diagnosis of recent infections (martins et al., 1991) , these antibodies needed to be separated either by sucrose density gradient centrifugation (gillette, 1974) or column chromatography (mockett & cook, 1986) before performing the elisa. the availability of an antibody-capture elisa for ibvspecific igm assays would facilitate ib diagnosis where demonstration of the virus is time-consuming. the importance of b-cells in ibv infections has been studied by depletion experiments using the hormone testosterone propionate (chubb, 1974) , the chemical cyclophosphamide (chandra, 1988; chubb, 1974) and surgical bursectomy (cook et al, 1991a) . cyclophosphamide-treated chickens showed increased clinical signs and more severe histopathological lesions in the kidney (chandra, 1988) attributable to the prolonged persistence of virus. ibv infection of a surgically bursectomised resistant chicken line (line c) resulted in increased severity and duration of clinical infection but not mortality (cook et al., 1991a) . however, humoral antibodies seemed to protect the tracheal epithelium following secondary challenge. presence of high titres of humoral antibodies correlate well with the absence of virus recovery from kidneys and genital tract macdonald et al, 1981; yachida et al, 1985) and protection against drop in egg production (box et al., 1988) . ibv-specific antibodies probably prevent the spread of virus by viraemia from the trachea to other susceptible organs such as the kidneys and oviduct. vaccination studies with ibv have always focussed on humoral immune responses in relation to protection. nevertheless, the lack of correlation between antibodies and resistance, discrepancies between in vitro strain differentiation by vn tests and in vivo cross-protection results (darbyshire, 1985) and re-excretion of virus in the presence of high titres of circulating antibodies (jones & ambali, 1987) all suggest that while humoral antibodies play a role in recovery from ibv infection, other immunological mechanisms are involved. maternally derived antibodies (mda) can provide protection against ibv, but they are short-lived (darbyshire & peters, 1985; cook et al, 1991b) . presence of mda has no adverse effect on the efficacy of live ibv vaccines administered at one-day of age (davelaar & kouwenhoven, 1977 , cook et al, 1981 . maternally-derived igg has been demonstrated in tracheal washes (mockett et al, 1987) . local immunity in the respiratory tract is of fundamental importance in protection against ibv (gomez & raggi, 1974; gillette, 1981; hawkes et al, 1983) . this has been exemplified by the use of an in vitro challenge model using toc from immunized chickens, for cross-protection studies . ibvspecific iga and igg have been demonstrated in tracheal washes of infected chickens (hawkes et al, 1983; dhinakar raj & jones, 1996b ) and antibody-secreting cells were shown in tracheal sections (nakamura et al, 1991) . local immunity at the oviduct level has been shown by demonstration of virus-specific igg and iga in the oviduct washes of infected hens (dhinakar raj & jones, 1996b) . it was found that in addition to local production, antibodies also transuded from the serum later in the course of infection, but their value in protection of the oviduct has not been determined. in young chicks, local antibodies in the oviduct were found to be less protective compared to those in the trachea using in vitro challenge of ooc prepared from vaccinated chickens (dhinakar raj & jones, 1996d) . although ibv has been shown to multiply in the gut (lucio & fabricant, 1990; ambali & jones, 1990) , lutticken et al. (1988) could not detect any antibodies in gut washings following vaccination of day-old chicks with hi20 vaccine and revaccination at 4 weeks of age with live h52 vaccine, inactivated m41 in an oil emulsion or inactivated m41 adjuvanted with avridine. in contrast, dhinakar raj & jones (1996b) demonstrated local antibody production in the duodenum and caecal tonsils of 16-week-old hens infected with an enterotropic strain of ibv (strain g). it is not clear whether the induction of these antibodies is strain-related and their role in limiting the replication of ibv in the gut needs to be investigated. the harderian gland of the chicken contains a large age-dependent population of plasma cells and is the source of immunoglobulins in the lachrymal fluid (baba et al, 1988) . it plays an important role in the development of vaccinal immunity since vaccines are generally given by spray or eye-drop. davelaar & kouwenhoven (1981) reported that the protection against ibv of day-old ocular-vaccinated chickens was localized mainly in the oculo-nasal mucosa and removal of the harderian gland caused a decreased level of protection (davelaar & kouwenhoven, 1980) . ibv-specific iga has been demonstrated in the lachrymal fluid davelaar et al, 1982; and its synthesis in the harderian gland has been shown (davelaar et al, 1982) . igg in tears was mainly serum-derived (davelaar et al, 1982; toro et al., 1993) . iga levels in tears appeared to be better correlated with resistance to ibv re-infection (toro & fernandez, 1994) than levels of serum antibody (yachida et al, 1985) and their measurement was recommended for antibody profiling of chicken flocks. cook et al. (1992) found more ibv-specific iga in the lachrymal fluids of chicken lines resistant to ibv while antibody titres in tracheal washes were similar. variation in ibv-specific igg levels in serum and iga levels in lachrymal fluids has also been demonstrated in different chicken lines after ocular vaccination with ibv (toro et al, 1996b) . it was found that light layer (white leghorn) chickens had a significantly higher and more homogenous serum igg response between days 5 and 9 p.i., and lachrymal iga response between days 5 and 14 p.i. than broiler or brown-egg (heavy) layer chicks. reports concerning a role for cell-mediated immunity in protection against ibv are limited. antigen-specific proliferation of t-lymphocytes in ibv-infected or vaccinated chickens has been demonstrated (timms et al, 1980; timms & bracewell, 1981 . in some chickens, a positive correlation between lymphoproliferative responses and resistance to challenge has been shown (timms & bracewell, 1981) . mouse monoclonal antibodies (mab) that distinguish between chicken tlymphocytes have been described (chan et al., 1988; iillehoj et al, 1988) . the cd4 and cd8 antigens are found on two main populations of t-cells, t-helper (t n ) and t-cytotoxic / suppresser (tc/ s ) cells, respectively. janse et al. (1994) contended that local immunity to ibv in the trachea is mediated by t-cells. cd4 and cd8 cells were shown in sections of trachea and lung of chickens infected with ibv (janse et al, 1994; dhinakar raj & jones, 1996a) . however, it is not clear which of these cells are more important in virus clearance, since janse et al. (1994) found an increase in cd4 cells, while dhinakar raj & jones (1996a) found higher proportions of cd8 cells. the differences may be related to the strains of ibv used. when chickens were treated with csa to suppress the t-cells, virus titres in the kidneys were 1 to 3 logio median ciliostatic doses (cd 50 ) higher than in intact birds (dhinakar raj & jones, 1997a) . thus, t-cells may also play an important role in limiting virus replication in the kidneys. chubb et al (1987) demonstrated the presence of cytotoxic lymphocytes (ctl) in the spleen and peripheral blood following ibv infection using adherent cells as target cells and neutral red as indicator of lysis. however, wakenell et al. (1995) could not demonstrate ctl using kidney cells as targets and the conven-tional chromium-release assay. delayed-type hypersensitivity (dth) responses were induced in response to live ibv and to affinity-purified si, s2, n and m proteins (igjnatovic & galli, 1995) . cytokines secreted in response to a general stimulus such as concanavalin a (con a) or by specific antigen are important mediators of cellular immunity. t-cell growth factor (tcgf) or il-2 and ifn-y are among the most important soluble factors produced by lymphocytes. only ifn-y has been studied in relation to ibv but results are conflicting. otsuki et al (1988) detected variable levels of ifn-y in chickens with various strains of ibv whereas other workers (lomniczi, 1974; holmes & darbyshire, 1978) could not detect ifn-y in serum or organ cultures of chickens infected with ibv. furthermore, whether ibv is susceptible to anti-viral effects of ifn-y is controversial (holmes & darbyshire, 1978; otsuki et al, 1979b) . otsuki et al. (1991) found no differences in ifn-y levels of ibv-resistant and ibv-sensitive lines of chickens. the identification of resistant and sensitive inbred lines of chickens to ibv (bumstead et al, 1989) has provided an excellent model to study immune mechanisms in relation to resistance of chickens to ibv. otsuki et al, (1990) found that although a resistant line (c) and a sensitive line (151) were equally susceptible to infection initially, recovery was more rapid in the resistant line. ultrastructural and histochemical studies showed that though the type of damage to the tracheal epithelium following ibv infection was similar in both lines of chickens, lesions were more severe and longer lasting in the sensitive line (nakamura et al, 1991) . the severity and duration of clinical infection in bursectomised line c chickens were similar to those seen in the sensitive line (cook et al, 1991a) . however, no increase in mortality was observed, in contrast to high mortality recorded in line 151 chickens. comparisons of secretory antibody responses between the two lines of chickens revealed increased local antibody secretion in the saliva and lachrymal fluid of the resistant line . suppression of t-cells by csa in a line of chickens resistant to ibv (brown leghorn; blh), induced them to behave like a sensitive line, in terms of mortality (dhinakar raj & jones, 1997a) . the mortality of intact blh chickens infected with a pool of ten ibv strains was 0% compared to 43% in csa-treated blh chickens. the virus titres in kidneys were much higher than in intact birds. the classes of t-cells involved in these effects need to be studied in further detail by analysing the kinetic changes in t-cell subsets following ibv infections using flow cytometry or by specific depletion in vivo by mabs against t-cell subsets. although ib is generally considered as an acute respiratory disease, prolonged virus excretion has been reported (cook, 1968; alexander et al, 1978; chong & apostolov, 1982) . when day-old chicks were infected with an enterotropic ibv, strain g, faecal excretion could not be detected beyond day 35 p.i., but when birds reached sexual maturity re-excretion occurred (jones & ambali, 1987) . virus re-excretion could not be induced earlier by hormone injections (ambali & jones, 1991b) , but it could after t-cell suppression by csa (bhattacharjee et al, 1995) . reactivation of virus was confirmed by appearance of ibv-specific igm in the serum (bhattacharjee et al, 1995) . mainly because of the prolonged or intermittent recovery of ibv from these tissues, 2 candidate sites have been proposed for virus persistence in the chicken, the kidneys (chong & apostolov, 1982) and/or caecal tonsils (cook, 1968; alexander et al, 1978) . however, recent work has suggested that the kidneys are the more likely site. when strain m41 was used to infect day-old chickens and t-cell immunosuppression by csa treatment was given from 5 weeks p.i., virus was recovered first from the kidneys and then from the trachea and lungs but never from the caecal tonsils (dhinakar raj & jones, 1997a) . the target cells of the virus in the kidneys are in the tubular epithelium (chong & apostolov, 1982; ambali & jones, 1990 ) which provides an ideal site for virus persistence because of its immunologically privileged nature (mimms, 1988) . occurrence of persistence was also found to be related to the age at infection (dhinakar raj & jones, 1997a) . when chicks were infected at 2 weeks of age and treated with csa from 5 weeks p.i. no virus re-excretion was observed, but when chicks were infected at day-old, virus re-excretion was successfully induced with csa. notably, virus re-excretion was not accompanied by clinical symptoms. the persistence of ibv may have practical implications in the epizootiology of the disease and perhaps a role in evolution of variant strains of the virus. furthermore, re-excretion of persistent virus may serve as an unnoticed source of infection to susceptible chickens. ibv does not constitute a single homogenous antigenic type. the prototype virus is massachusetts m41. since the first identification of a different serotype (named connecticut) by jungherr et al, (1956) several new antigenic types have been reported in various countries (see captua et al, 1994; cook, , 1984 gelb et al, 1991a; parsons et al, 1992; picault et al, 1986) . the major virus neutralizing antibody site of ibv, which defines serotype, resides in the si subunit of the spike protein (mockett et al, 1984; cavanagh et al, 1986) . differences in only a few amino acids in the si protein can result in different vn serotypes (cavanagh et al, 1992a) which accounts for the plethora of ibv strains which exists today. hence, different antigenic types identified by vn tests do not imply that the isolates have substantially different si proteins or overall antigenic properties or greatly different evolutionary lineages. as in other rna viruses, antigenic variation is probably facilitated by the high error rate during the transcription of rna template and the absence of a proof-reading mechanism. it has been shown that point mutations may lead to the generation of ibv variants in the field qia et al, 1995) . however, both circumstantial (cavanagh & davis, 1988; cavanagh et al, 1992b; jia et al, 1995; kusters et al, 1989 kusters et al, , 1990 wang et al, 1994) and experimental (kottier et al, 1995) evidence suggest that the main mechanism of generation of variant strains of ibv is by recombination. this could be promoted by the use of more than one strain of ibv for vaccination or by a mixture of vaccine and challenge viruses. isolates of ibv shown to be distinct by the vn test can still induce partial or complete cross-immunity (arvidson et al, 1990; darbyshire, 1980 darbyshire, , 1985 hitchner et al, 1964; raggi & lee, 1965; winterfield & fadly, 1972; winterfield et al, 1976) . for example, vaccination with h120 caused a 30,000-fold reduction in titres of the challenge virus, australian 't' strain (darbyshire, 1985) , while on the basis of vn tests in vitro, no evidence of a serological relationship between these two viruses could be demonstrated . hence, antigenic studies alone do not adequately define immunological relationships between strains. thus, it was suggested that cross-immunization studies could be used to classify ibv isolates into protectotypes (lohr, 1988) as this would reduce the large number of serotypes to a smaller number of protectotypes and provide more practical information to the field. cross-immunisation tests have been performed in experimental chickens (darbyshire, 1980 (darbyshire, , 1985 lambrechts et al, 1993) or in in vitro conditions using toc from immunized birds dhinakar raj & jones, 1996d) to determine protective immunological relationships between ibv strains. arvidson et al. (1990) have described a model to study immunogenic relationships between ibv strains based on the vaccinating dose required to prevent the multiplication of a standard challenge dose of a homologous strain in the lungs of chickens. these cross-immunization tests would help to determine whether an already existing vaccine could offer protection to a new variant. this information is critical before embarking on a long and cumbersome process of new vaccine production. since tissue affinities are a function of the viral peplomer-mediated attachment of virus to cells, changes in the spike protein might lead to altered tropism of the virus. six of ten differences in amino acid sequences of gray and jmk strains of ibv were found between residues 99 and 127; hence it was postulated that this region may play a role in differences in tissue tropism exhibited by these viruses (kwon & jackwood, 1995) . however, no correlation was found by sapats et al. (1996) between si amino acid sequences and nephropathogenicity of nine australian strains of ibv. nevertheless, it is not unreasonable to expect new variant strains of ibv to emerge with unusual tissue tropisms and such an example of this is strain 793/b. conclusions ibv has been effectively controlled by the extensive use of vaccines but it still remains a major economic problem some 65 years after it was first reported. the constant emergence of variant strains has challenged vaccination strategies. with the advent of molecular techniques much of the recent work on ibv has concentrated on improved diagnostic and strain differentiation methods. however, the appearance and spread of the economically-important variant strain 793/b has given us a reminder of the need for a more detailed understanding of the immunopathogenesis of the disease and to be prepared for emergence of variants with unusual tissue tropisms and disease manifestations. tionstraktes angesehen wird, können verschiedene ibv-stämme unterschiedliche gewebstropismen aufweisen und, mit ernsten folgen, auch den eileiter und die nieren befallen. manche stämme vermehren sich im darm, anscheinend aber ohne pathologische veränderungen. pektorale myopathie ist mit einer wichtigen neuen variante in verbindung gebracht worden. mehrere faktoren können den verlauf der infektion mit ibv beeinflussen und umfassen das alter, die rasse und die fütterung des huhnes, die umgebung und die interkurrente infektion mit anderen infektionserregern. die immunogenen bestandteile des virus sind die s (spike)-proteine und das n-nukleoprotein. die humoralen, lokalen und zellulären reaktionen des huhnes gegen ibv sowie die genetische resistenz des huhnes werden besprochen. bei der langfristigen ibv-persistenz sind die blinddarmtonsillen oder die nieren als orte der persistenz vermutet worden. die antigenvariation bei ibv-stämmen hängt mit relativ kleinen unterschieden der aminosäuresequenzen im spike-protein s1 zusammen. antigenuntersuchungen alleine reichen jedoch nicht aus, um immunologische beziehungen zwischen virusstämmen ausreichend zu definieren, und kreuzimmunisierungsversuche sind benutzt worden, um ibv-isolate in 'protektotypen' zu klassifizieren. es sind vermutungen darüber angestellt worden, daß veränderungen im spike-protein s1 eine beziehung zu unterschieden der gewebstropismen, die verschiedene stämme aufweisen, haben könnten. vielleicht könnten in der zukunft neue ibv-stämme auftauchen, die organe oder systeme befallen, welche normalerweise keinen zusammenhang mit der ib haben. virus de la bronquitis infecciosa: inmunopatogenia de la infeccion en la gallina se revisa la inmunopatogenia de la infeccion por el virus de la bronquitis infecciosa en la gallina. mientras que la bronquitis infecciosa (ib) es considerada primariamente una enfermedad del aparato respiratorio, distintas cepas de ibv pueden mostrar tropismos diversos y afectar tambien al oviducto y el riñón con consecuencias serias. algunas cepas se replican en el intestino pero aparentemente sin producir lesiones. una reciente variante de ibv ha sido relacionada con la miopatia pectoral. diversos factores pueden influir el euros de la infección por ibv incluyendo la edad, estirpe y estado nutritivo del ave, el medio ambiente e infecciones intercurrentes con otros agentes infecciosos. los componentes inmunogénicos del virus incluyen las proteinas s y la nucleoproteína n. se revisan las respuestas inmunes humorales y celulares de las gallinas frente a ibv asi como la resistencia genética de la gallina. en situaciones de persistencia de ibv se considera que las tonsilas cecales o el riñón son los lugares probables de acantonamiento. las variaciones antigénicas entre las distintas cepas de ibv están en relatión con diferencias aminoacidicas pequeñas en la proteína s1. no obstante, los estudios antigenicos por si solos no son capaces de definir adecuadamente las relaciones entre las cepas, y se 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histopathology of infectious bronchitis in the domestic fowl the histopathology of infectious bronchitis in fowls infected with a nephrotropic t strain of virus genetic differences among chicken embryos in response to inoculation with an isolate of infectious bronchitis virus lack of correlation between infectivity, serologic response and challenge results in immunisation with an avian infectious bronchitis vaccine co-existence of infectious bronchitis and infectious laryngotracheitis synergism between avian infectious bronchitis and haemophilus gallinarum effect of environmental temperature on the mortality in vaccinated chickens after challenge with australian infectious bronchitis virus response to several avian respiratory viruses as affected by infectious bursal disease virus sequence analysis of the s1 glycoprotein of infectious bronchitis viruses-identification of a novel genotypic group in australia an apparently new respiratory disease of baby chicks effects of infectious 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and chickens with maternal immunity after infectious bronchitis virus vaccination avian infectious bronchitis-viral persistence in the harderian gland and histological changes after eye-drop vaccination local and systemic antibody response of different chicken lines after ocular vaccination against infectious bronchitis infectious bronchitis effects of avian viruses on cultured chicken bone marrow-derived macrophages structural proteins and glycoproteins of infectious bronchitis virus particles labelled during growth in chick embryo cells embryo vaccination of chickens with infectious bronchitis virus-histologic and ultrastructural lesion response and immunological response to vaccination evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus comparison of a microneutralisation test with elisa and precipitin tests for detection of antibodies to infectious bronchitis virus in chickens nephropathogenicity of infectious bronchitis virus some characteristics of isolates of infectious bronchitis virus from commercial vaccines etiology of an infectious nephritis-nephrosis syndrome of chickens immunity to infectious bronchitis virus from spray vaccination with derivatives of holland strain vaccination against infectious bronchitis virus and immunosuppressive effects of infectious bursal disease relationship between several criteria of challenge-immunity and humoral immunity in chickens vaccinated with avian infectious bronchitis vaccines detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibodies influence of environment on airsacculitis: effects of relative humidity and air temperature on broilers infected with mycoplasma synoviae and infectious bronchitis avian infectious bronchitis: properties and application of attenuated vaccine prepared with nephropathogenic strain az-23/74 résumé virus de la bronchite infectieuse: immunopathogènie de l'infection chez le poulet bien que la bronchite infectieuse (ib) soit d'abord considérée comme une maladie du système respiratoire, différentes souches d'ibv peuvent montrer des tropismes tissulaires variables et affectent aussi l'oviducte et les reins avec des conséquences sérieuses. quelques souches se multiplient dans l'intestin mais apparemment sans troubles pathologiques en ce qui concerne la persistance à long terme de l'ibv, les amygdales caecales ou les reins ont été proposés pour être des sites de persistance. la variation antigénique parmi lès souches d'ibv est reliée à des différences relativement faibles au niveau de la séquence en acides aminés de la protéine de la spicule s1. néanmoins, les études antigéniques seules ne définissent pas de façon adéquat les relations immunologiques existant entre les souches, et des essais d'immunisation croisées ont été utilisés pour classer les isolats d'ibv en 'protectotypes'. il a été spéculé que les changements au niveau de la protéine s1 peuvent être reliés à des différences de tropismes tissulaires mises en évidence pour certaines souches key: cord-344745-sgkq1l93 authors: selim, karim; arafa, abdel satar; hussein, hussein a.; el-sanousi, ahmed a. title: molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 date: 2013-11-18 journal: int j vet sci med doi: 10.1016/j.ijvsm.2013.10.002 sha: doc_id: 344745 cord_uid: sgkq1l93 one of the major problems of avian infectious bronchitis virus (ibv) is the frequent emergence of new variants. in the present study 205 tracheal swabs and organs were collected from broilers and layers chicken farms during january to august 2012 from 19 governorates all over egypt. the chickens demonstrated respiratory signs and mortality. out of the examined samples, 130 of which (about 64%) of suspected farms were positive for ibv with real time rt-pcr. 13 ibv-positive samples were selected for further isolation and characterization. isolation in specific pathogen free (spf) embryos was carried out after studies three blind successive passages and the hypervariable region of spike protein1 (sp1) was amplified by rt-pcr and sequenced to study the genetic diversity between the isolated viruses. phylogenetic analysis of the obtained sequences of 13 isolates compared with other ibv strains from the middle east and worldwide reveled that 11 out of the 13 isolates had close relationship the israeli variants (is/885 and is/1494/06) with nucleotide homology reached up to 89.9% and 82.3%, respectively. only two isolates had close relationship with cr/88121 and 4/91 viruses with identities of 95% and 96%, respectively. this study indicates existence of two variant groups of ibv circulating in egypt during 2012. group i was similar but distinguishable from israeli variant is/885 and group ii was related to 4/91 and cr/88121 vaccine strains. there was no geographical link between the 2 groups as they were distributed all over the country. these findings necessitate the need to revise the vaccination programs and control measures for ibv. similar but distinguishable from israeli variant is/885 and group ii was related to 4/91 and cr/ 88121 vaccine strains. there was no geographical link between the 2 groups as they were distributed all over the country. these findings necessitate the need to revise the vaccination programs and control measures for ibv. ª 2013 production and hosting by elsevier b.v. on behalf of faculty of veterinary medicine, cairo university. infectious bronchitis (ib) is an acute and highly contagious respiratory disease of chickens. this disease is characterized by respiratory signs and in young chickens severe respiratory distress commonly occurs while in layers it causes decrease in egg production [1] . until recently, the chicken was considered the only natural host of ibv and in which the virus cause disease. pheasants are the other avian species that is now considered as a second natural host for ibv [2] . the disease is transmitted by the air-borne route, direct chicken to chicken contact and indirectly through mechanical spread [3] . the ib virus is a member of the genus coronavirus, family coronaviridae, order nidovirales. ibv and other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses, with mammalian coronaviruses comprising groups 1, 2 and 4. group 4 is the more recently identified severe acute respiratory syndrome (sars) coronavirus [4] . ibv is an envelope; positive sense single stranded rna virus containing an un segmented genome approximately 27.6 kb in size. the virion has four structural proteins: nucleocapsid protein (n), membrane glycoprotein (m) small envelope protein (e), and glycosylation spike glycoprotein (sp) [5] . many ibv serotypes have been described probably due to the frequent point mutations that occur in rna viruses and also and recombination events. for this reason, the characterization of virus isolates existing in the field is very important [6] . the spike (sp) protein is one of the major structure proteins of ibv proteins that is cleaved into two smaller proteins namely sp1 and sp2. sp1 gene contains two hypervariable regions that are responsible for the induction of neutralizing and serotype specific antibodies [7] . the existence of variable genotypes of ibv in egypt was recognized and strains related to the massachusetts d3128, d274, d-08880 and 4/91 genotypes have been detected at different poultry farms in egypt [4, [8] [9] [10] . accordingly, genotyping of ibv field strains is very important for screening the new variants as well as evaluating the vaccination programs. avian ibv was isolated from broiler chickens showing respiratory and renal lesions and characterized as beni-suef/01 with 89% and 84% amino acid sequence identity and 88% nucleotide sequence identity to the is/885 strains [11] . the aim of this study aimed to survey the egyptian chicken field for infectious bronchitis virus and study the genomic differentiation between isolated field samples seeking the new variant strain emerged in the egyptian field. in this study 205 samples were collected from chicken farms showing respiratory manifestations and mortalities during surveillance from jan to aug 2012, to study the prevalence of ibv in 19 egyptian governorates from 179 broiler farms and 26 layer farms. pooled 10 tracheal swabs/each case and organs (trachea, kidney, and lung) were collected from dead birds. from which thirteen samples were selected for virus isolation representing positive cases from different governorates (13 governorates were positive, 3 were positive by pcr but fail in isolation and 3 governorates were negative by pcr and isolation). as shown in table 1 . detection of ibv in collected samples and confirmation of the presence of virus after isolation in spf-ece has been conducted using real-time reverse transcription-polymerase chain reaction (rt-pcr) for un-translated region of ibv. rna for rt-pcr was extracted from the supernatants of 10% w/v sample suspensions and allantoic fluid. the extraction of viral rna was performed using a qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's instructions. amplification of the specific target genome was conducted using the forward primer ibv5_gu391, 5 0 -ac gtatgactacccgcagtattca-3 0 and reverse primer ibv5_gl533, 5 0 -agaccagccaccatgattgc-3 0 and probe ibv5_g, 5 0 -famcaccaccagaacctgtcacc tc-bhq1-3 0 [12] . real-time rt-pcr was performed using qiagen one step rt-pcr kit (qiagen, gmbh, hilden, germany) rt-pcr reactions were performed on stratagene thermal cycler machine. for virus isolation, the supernatants of ibv-positive selected 13 samples determined by rt-pcr were inoculated into five specific pathogen free embryonated chicken eggs (koumoshiem spf chicken farm, fayoum, egypt) 10-day-old for each sample. the eggs were inoculated with 0.2 ml of the sample into the allantoic cavity then incubated at 37°c with candling daily. allantoic fluids were harvested at 96 h post inoculation. three successive blind serial passages were performed. the allantoic fluids were harvested and stored at à70°c with examination of embryo for curling and dwarfism [13] . for the positive 13 isolates the hvr of sp1 gene were amplified using conventional pcr by qiagen one step rt-pcr kit (qiagen, gmbh, hilden, germany) using forward primer ibv-s1-f 5 0 -cactggtaatttttcagatgg-3 0 and reverse primer ibv-s1-r 5 0 -cagattgcttacaac-cacc-3 0 [14] , the amplicons were purified using the qia quick gel extraction kit (qiagen, gmbh, and hilden, germany). the sequence reactions were performed using genetic analyzer applied biosystems 3130 (abi, usa) by big dye terminator v3.1 cycle sequencing kit. (perkin, elmer, foster city, ca) using forward and reverse primers as previously mentioned. the sequence analysis of the sp1 gene of the egyptian ibv sequences used for comparison in this study were from genbank and were available from the national center for biotechnology information (ncbi) infectious bronchitis viruses resource (http://www.ncbi). sequence identities were calculated using dnastar software [15] and the phylogenetic tree of the nucleotides sequence were constructed using mega 5 [16] the sequences from the genbank used in this study included m41 (acc no.hf674411), ma5 (acc no.ay561713), h120 (acc no.jn600610), connecticut (acc no.af094818), cr/88 (acc no.jn592567), qxibv (acc no.gq253481), 4/ 91 (acc no.af093794), d41 (acc no.af036937), d274 (acc no.x15832), egypt-f-o3 (acc no.dq987085), ir/4/2010-s1 (acc no.jn792558), ibv-sul/01/09-s1 (acc no.gq281656), is/1366-sp1 (acc no.eu350550), is/236-s1 (acc no.ay135205), ibv-s1-1494 (acc no.hm131453) and is-885 s1 (acc no.ay279533). there were 205 farms tested for ibv, from which 117 broiler and 13 layer farms were positive. fayom, ismailia and benisuif governorates had the highest number of positives. however, port-said, north sinai, cairo and aswan were tested negative for ibv using real-time rt-pcr. the positive percent of the ibv in broiler were higher than layer farms as it reached up to 65.4% in broilers while it was 50% in layer farms. as shown in table 2 . there were 13 positive isolates were identified after egg inoculation after 3 blind passages. the inoculated embryos showed table 3 identity and diversity of ibv. curling and dwarfing with subcutaneous hemorrhages as shown in fig. 1 . the allantoic fluids were collected and tested for confirmation of ibv by rt-pcr and they were positive as indicated in fig. 2 , where the 13 selected isolates showed specific 400 bp of sp1 gene. we selected 6 isolates (numbered from 15 to 20 in table 3 table 3 . the genetic analysis of 100 amino acids sequence from position 263-362 of sp1 gene for the selected 13 egyptian viruses was done and the hypervariable region of sp1 gene showed multiple mutations as shown in table 4 in comparison with variant-2 strain. group i had 10 amino acid substitutions in table 4 amino acid substitution mutations in hvr of s1 gene of ibv. that part of hypervariable region of sp1 gene while group ii had much higher genetic diversity where they were 15 amino acid substitutions in comparison to variant-2 israeli strain table 4 . the phylogenetic analyses of the 13 selected isolates were constructed (fig. 3) and revealed that the egyptian viruses in this study can be classified into two distinct groups. group i consisted of eleven isolates which include eg/1219f, eg/1226b, eg/12164b, eg/12105b, eg/1290b, eg/12249f, eg/1284b, eg/1267f, eg/12103b, eg/12177f and eg/1236b, the isolates in group i belong to israeli ibv (is/885 strain). group ii consisted of two isolates eg/1260b and eg/12150b, which had a close relationship with, 4/91 and cr/88121 strains. in this study the prevalence of ibv infection in 205 examined farms by real-time rt-pcr indicated widespread distribution of infectious bronchitis virus all over egypt. the positive percent in beni-sueif, ismailia and fayoum governorates showed the highest incidence (83%, 78% and 76%) respectively. the percent of positive broiler farms were higher than in layers assuit was 65% in broilers while in layers it was 50%. samples for virus isolation commonly are inoculated into embryo chicken eggs from a specific-pathogen-free source. samples should receive at least 3 blind passages before being called negative based on failure to cause death or lesions in embryos [17, 18] . as mentioned in table 1 , there were 13 cases successfully isolated in chicken eggs. the thirteen isolates showed curling and dwarfing of embryos after the third passage. the sequencing of the sp1 gene is important for ibv genotyping. ibv identification and genotyping was performed by the sequencing of hyper variable region (hvr) of the sp1 gene [13] . in this study, the 13 isolates were sequenced for hvr of sp1 and the similarity among isolates was addressed in (fig. 3) . the similarity of egyptian isolates with others from the neighboring countries like israel was ranged between 82% and 90% in comparison to stains is/1494, variant-2 and is/885.this comparison is important due to uncontrolled movement of inhabitants and smuggling through borders [19, 20] . the ib variant (delaware de072) was first reported in usa in 1992 [21] was found to show little genomic relatedness in the s1 region of the s gene to other us variants; however it was related to the dutch variant, d1466. the de072 increased in incidence causing major disease problems in vaccinated flocks in usa [22] . ibv variants have been recognized in egypt since the 1950s [23] with the isolation of a variant shown by neutralization tests to be closely related to the dutch variant d3128. the emergence of israeli variant-2 and is/1494/06strains and the information of their sp1 gene sequence (genbank accession number: eu780077) had been previously reported [24] . the is/1494 has been reported to be a major variant involved in ibv infections in the middle east like in israel, and possibly in turkey. [25] . currently, ma5 and h120 ibv-based vaccination strategies have been applied for the control of ib in chickens in egypt. in addition recent variant vaccines like cr/88 and 4/91 have also been available for vaccination. however, there is persistence in problems related to ib suspected cases in spite of the use of different vaccine seeds. there is also a report, indicating the persistence of the israeli variant isolate (is/1494/06) related problems in spite of vaccinations of the broiler flocks with h120, which supports the argument of is/1494/06 ibv presence in the middle east countries. also a nephropathogenic ibv related problem other than is/1494/06 ibv has been reported in iraq as a neighboring country to turkey [20] . in this study, the phylogenetic analysis of 13 egyptian isolates in comparison with other variant and classic viruses classified egyptian viruses into two groups. there were11 viruses placed in group i and other two isolates belong to group ii. the circulating viruses in 19 egyptian governorates widely distributed without geographical limitation as shown in table 1 . it was found that, group i present in samples from luxor, menia, sharkia and fayuom. group ii was found in samples from beni-suif and qaluobia. sp1 gene sequencing is used for distinguishing between different ibv serotypes. diversity in s1 probably results from mutation, recombination and strong positive selection in vivo. the generation of genetic variants is thought to be resulted from few amino acid changes in the spike (s) glycoprotein of ibv [8] . analysis of the hypervariable region of the s1 spike glycoprotein gene of thirteen viruses in group i belong to israeli ibv (is/885) and group ii consisted of two isolates which had a close relationship with variant vaccines like (4/ 91&cr/88). in this study, the ibv circulating in egypt during 2012 can be classified into two groups of viruses. group i was clearly variant from is/885 and group ii was more closer to variant vaccine viruses like 4/91, cr/88121 that indicates independent evolution of ibv in egypt and persistence of divergent stains currently circulating in the country. it is very critical to complete genetic characterization of circulating ibv viruses to study the genetic relatedness among viruses and vaccine strains. this will guide us for best vaccines selection and improve our effort to control the disease. one of a series of the veterinary medicine-large animal clinical sciences department, florida cooperative extension service avian infectious bronchitis virus detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand severe acute respiratory syndrome vaccine development experiences of vaccination against avian infectious bronchitis coronavirus identification of five bronchitis virus (ibv) strains isolated in china and phylogenetic analysis of the s1gene complete genomic sequence analysis of infectious bronchitis virus ark dpi, strain and its evolution by recombination isolation and molecular characterization of infectious bronchitis virus isolate shiraz ibv, by rt pcr and restriction enzyme analysis s1 gene sequence analysis of a nephro-pathogenic strain of avian infectious bronchitis virus in egypt studies on infectious bronchitis in broiler chickens in el-menia governorate present status of infectious bronchitis in egypt emergence of a novel genotype of avian infectious bronchitis virus in egypt molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates molecular analysis of the 793/b serotype of ibv in great britain clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods detection of infectious bronchitis laboratory manual for the isolation and identification of avian pathogens molecular epizootiology of avian infectious bronchitis in russia isolation and molecular characterization of sul/01/09avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east antigenic and s-1 genomic characterization of the delaware variant serotype of infectious bronchitis virus spike gene analysis of the de072 strain of infectious bronchitis virus: origin and evolution isolation and characterization of infectious bronchitis virus strain 4/91 from commercial layer chickens in the sudan identification of a novel nephropathogenic infectious bronchitis virus in israel presence of is/1494/06 genotype-related infectious bronchitis virus in breeder and broiler flocks in turkey key: cord-330057-3vucm0s1 authors: franzo, giovanni; tucciarone, claudia maria; moreno, ana; legnardi, matteo; massi, paola; tosi, giovanni; trogu, tiziana; ceruti, raffaella; pesente, patrizia; ortali, giovanni; gavazzi, luigi; cecchinato, mattia title: phylodynamic analysis and evaluation of the balance between anthropic and environmental factors affecting ibv spreading among italian poultry farms date: 2020-04-29 journal: sci rep doi: 10.1038/s41598-020-64477-4 sha: doc_id: 330057 cord_uid: 3vucm0s1 infectious bronchitis virus (ibv) control is mainly based on wide vaccine administration. although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. despite this, the determinants of ibv epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. in the present study, 361 ibv qx (the most relevant field genotype in italy) sequences were obtained between 2012 and 2016 from the two main italian integrated poultry companies. several biostatistical and bioinformatics approaches were used to reconstruct the history of the qx genotype in italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third “ghost” deme, representative of minor industrial poultry companies and the rural sector. the obtained results suggest that the integration of the poultry companies is an effective barrier against ibv spreading, since the strains sampled from the two companies formed two essentially-independent clades. remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of northern italy. the inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in northern italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of northern italy. accordingly, when the effect of different environmental and urban parameters on ibv geographic spreading was investigated, no factor seems to contribute to ibv dispersal velocity, being poultry population density the only exception. finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on ibv epidemiology. overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale. a total of 361 qx sequences were included in the final dataset. of those, 135 belonged to "company a" and 226 to "company b". the sampled farm location is reported in fig. 1 . overall, farms were mainly located in the "pianura padana" region (central area of northern italy) and, to a lesser extent, in north-eastern, north-western, central and southern italy. although the two companies tend to operate in different italian regions, a clear overlapping was present in the high densely populated poultry area of northern italy (fig. 1 ). qx-population genetics parameter estimation. all considered field sequences formed a monophyletic group including only italian strains ( supplementary fig. 1 ). tempest investigation revealed that the positive correlation between genetic divergence and sampling time (i.e. r = 0.335) was suitable for phylogenetic molecular clock analysis 20 . the tmrca of the overall qx population in italy (i.e. qx genotype introduction) was estimated in 2003.52 [ 95hpd: 1999 95hpd: .73-2006 .76] using the structured coalescent approach. almost identical results were obtained including a third "ghost" deme (i.e. an estimated deme for which no sequences were available, representative of other unsampled companies and farms) in the analysis or using the "traditional" coalescent approach. when strains collected from integrated poultry companies were considered independently, the tmrca was predicted in 2003. 19 [95hpd: 1994 19 [95hpd: .11-2010 for company a and in 2010. 6 [2007.26-2011.99 ] for company b. the viral population dynamics evidenced a substantially constant ne*t (effective population size * generation time, or relative genetic diversity) with the remarkable exception of the period between mid-2014 and mid-2015, when a sudden fluctuation was observed. however, a quite different scenario was demonstrated between the two integrated poultry companies. in fact, company a was featured by a substantially constant population size, with a minor decrease affecting particularly the period 2013-2015. however, the ne*t 95hpd were relatively broad and at odds with the significance of the observed variations. on the contrary, a much more changeable pattern was observed in company b (fig. 2) . migration among companies. the structured coalescent model fitted with the two company, evidenced the presence of 2 separate clades (fig. 3a) for the 2 companies, with only 11 exceptions, represented by strains sampled in company b but clustering in the company a clade, thus suggesting the migration of strains from company a to company b. accordingly, the migration rate from company b to company a was 2.5*10 −2 [95hpd: 6.02*10 −2 -8.40*10 −2 ], while the one from company a to company b was 6.66*10 −2 [95hpd: 2.2*10 −2 -0.12]. the figure 1 . location of farms from which samples have been obtained. different companies have been color coded. samples collected in company b but clustering with company a clade have been colored in red (herein named "imported"). farm location has been jittered using an internal routine of ggplot library to guarantee anonymity. the map was generated in r (version 3.4.4), using the library ggmap 51 . phylogeographic analysis. all the samples phylogenetically belonging to company a but collected from company b originated from farms located in the high densely populated area of northern italy (fig. 1) . the continuous phylogeographic analysis reconstructed a spreading pattern originating from a single introduction in emilia romagna region (company a), followed by a progressive expansion and persistence at high level in the pianura padana region. more rarely, spreading episodes toward other italian regions were observed (fig. 4) . after qx introduction, the infection wave front increased slowly approximatively until 2009, when a rapid expansion led to the final distribution range by the middle of 2012 (fig. 5) . accordingly, the dispersal velocity progressively increased in the first years after qx genotype introduction, peaking in the period 2009-2011 and then remaining essentially constant, despite some fluctuations (fig. 5) . the presence of a high dispersal velocity after 2012, when no further increase in wave front was observed, suggests that ibv continued to circulate at high rate after its first establishment in a region. the analysis of the effect of different environmental factors on qx genotype dispersal velocity led essentially to negative results (i.e. absence of significant correlation). the only exception was represented by the poultry density www.nature.com/scientificreports www.nature.com/scientificreports/ sl model, which was positively and significantly correlated to viral dispersal velocity: d = 0.0225, percentage of d with p-value < 0.005 = 74%. despite the economic relevance, the epidemiology of ibv and the factors affecting its behavior have been only partially investigated. even if a huge amount of knowledge and literature has accumulated over time, most of the reports are anecdotal or based on the analysis of single clinical outbreaks 17, 21, 22 . although relevant pieces of information could be obtained, the risk of being biased by personal believes or the particular condition under www.nature.com/scientificreports www.nature.com/scientificreports/ investigation is high. a certain caution is thus required when inferring and extending the same conclusion on a broader/general scale. moreover, most of the available studies are focused on avian influenza and, to a lesser extent, newcastle disease and infectious laryngotracheitis 17, 23, 24 . the aim of the present study was to construct an objective and statistically sound framework to understand ibv field strains behavior, the effect of control measures and the factors conditioning their epidemiology. the field of phylodynamics, and all related extensions, provides an invaluable tool for the study of viruses and particularly of rapidly evolving ones, whose evolution can be measured in "real time", over the course of an epidemic 25 . ibv qx genotype is the most relevant field strain in italy 26, 27 , and despite a relatively long circulation and the efforts devoted to its control, it still remains one of the main menaces for poultry industry profitability. therefore, the understanding of the forces shaping its epidemiology would be of remarkable relevance in order to prevent the induced damages, rather than try to control them. remarkably, the italian ibv strains appear to originate from one introduction events only, as previously reported 27 . therefore, it was possible to reconstruct ibv italian strain evolution and epidemiological pattern without the biasing effect of strains recently introduced from other counties. the implemented approach allowed to reconstruct the migration history of the qx genotype over time. the estimated introduction, in emilia romagna region, shortly predates the first detection, posing in favor of the effectiveness of the italian monitoring and early detection systems. all the analyses, independently of the underlying statistical model, support that company a was the first introduction site (fig. 3) . thereafter, the virus circulation was limited to farms belonging to this company for years, until approximatively 2010, when company b became involved. contextually, a progressive increase in diffusion speed was noticed (fig. 5) , not unexpectedly considering the rising number of involved farms (especially at the border between veneto and lombardy regions, where most farms are located) and thus the increase in spreading potential and opportunity. the high farm density of this area has been described as a risk factor for different infectious diseases 22 , and ibv seems to be no exception. interestingly, the viral population size remained relatively constant in this time period, evidencing that, even if qx strains were able to effectively spread from farm to farm, their replication was adequately controlled, likely by effective vaccination strategies. actually, a certain slowdown in dispersal velocity was noticed in 2011-12, potentially because of a progressive decrease in naive populations availability. a dramatic change was observed in 2014, when a new spreading wave (fig. 4) and an increase in diffusion rate (fig. 5 ) and population size (fig. 2) were detected. a more detailed analysis demonstrated that this variation affected company b only (fig. 2) . a previous study has ascribed this episode to a change in the vaccination scheme adopted by this company, which moved from a heterologous mass+793b based vaccination to a mass only vaccination leading to an increased viral circulation and clinical outbreaks number 4 . moreover, experimental studies demonstrated a significant reduction in r 0 in vaccinated groups compared to unvaccinated ones 28 . it can therefore be speculated that the increase in infectious pressure within-farm and the higher flock susceptibility to infection could have enhanced the risk of ibv spreading to other farms and regions. in support of this hypothesis, the geographical spreading affected mainly northern italian farms (where company b is located). moreover, when a new double vaccination was implemented, the decrease in viral population size was mirrored by a reduction of dispersal velocity. www.nature.com/scientificreports www.nature.com/scientificreports/ continuous phylogeography showed that the areas interested by a more intensive viral circulation were those featured by a higher poultry density, and this evidence was confirmed by a statistically significant correlation between poultry density and dispersal velocity. the association between spatial proximity and farm infection is probably the most consistently reported risk factor for poultry infectious diseases 17, 23, 29 . although an airborne transmission has been proposed for ibv, its occurrence has rarely been demonstrated experimentally 30 . however, the spatial proximity likely increases the likelihood of a greater number of horizontal contacts between farms, including the movement of people, vehicles and fomites between farms, as well as sharing similar risk factors (e.g. environmental conditions, climate, presence of wild animals, etc.) 16, 23 . based on these premises, the presence of segregated poultry companies should represent an effective obstacle to viral shedding and the obtained results partially confirm these evidence. the strains from different poultry companies formed two independent clusters, which suggests the effectiveness of independent production flow/chain in protecting farms from exogenous introductions. additionally, the application of adequate biosecurity measures, enforced also by the italian legislation, likely contributed in limiting new strain introduction. the exceptions to this general rule were farms located in the high densely populated poultry area of northern italy, where an overlap between the two companies occurs. the unidirectionality of the viral flux from company a to company b implies that other factors, besides spatial proximity, must be in place. a detailed survey could shed some insights into relevant factors like different biosecurity measures, structural factors, vaccination strategy etc.. the mediation of other "actors" cannot also be excluded. in fact, the analysis of just two companies, however predominant they are on the italian poultry sector, cannot be considered an accurate depiction of the italian situation. remarkably, the inclusion of a third deme (representative of other unsampled companies and farms) in the analysis model highlighted that several transmission events could be mediated by smaller entities operating in the same region. actually, the high migration rate estimated between company b and this ghost deme poses in favor of its pivotal role in maintaining an active ibv circulation. even if the idea of modeling demes for which no sequences are available could seem counterintuitive, previous studies showed that the structured coalescent can provide meaningful estimates even in absence of samples from one population 31 and this approach has already been applied and proven effective for other diseases, including ebola 32 . since also company a was evaluated in the same analysis run, the absence of relevant links between this company and the ghost deme further supports the analysis reliability, posing in favor of an actual interaction between company b and the ghost deme rather than a mere low specificity of the method. a less effective control of ibv infection could be speculated for small companies, whose management capability and resources are limited compared to big-integrated companies. in fact, all italian farms have to follow national legislation 33 dictating the minimum biosecurity measures to be applied. however, integrated poultry farms, part of major companies, enforce additional managerial practices to increase biosecurity levels. personnel and veterinarian formation, internal audits and periodic controls guarantee a higher level of application of the required standards, compared to most of small non-integrated farms. the higher spatial overlap and the likely sharing of some infrastructures (e.g. streets, accessory personnel, services and infrastructures) could nevertheless have a negative indirect effect on the major companies, especially in northern italy where company b is located. however, differences between company a and company b in the application of biosecurity measures and production flow management could also explain the different ibv epidemiology, as demonstrated by the dissimilar patterns in viral population fluctuations in the two companies (fig. 2) . a further risk factor that would deserve further investigation is the presence of the rural sector, which is highly concentrated in the densely populated poultry area of northern italy. this sector is characterized by a complex mix of growers, dealers and backyards flocks, often applying poor biosecurity measures and linked together by a poorly traceable contact network 34 . although interactions with industrial poultry farming is hardly discouraged, illegal/indirect interactions have been documented and multiple epidemiological connections could result in a bidirectional transmission between the two sectors, as demonstrated in the italian low pathogenicity avian influenza (ai) outbreaks occurred in 2007-2009 34 . after these episodes, a stricter legislation has been developed, imposing limits to animal movements and more active surveillance in the rural sector. nevertheless, no measures were taken for the monitoring and control of ibv in these enterprises, and therefore their role as sources of encroachment in intensive farming cannot be excluded. other environmental factors do not seem to play a relevant role in affecting viral dispersal. while climatic conditions like temperature, humidity and wind could actually affect viral viability and spreading, their effect could be circumvented by a transmission mediated by "fast-moving" vectors like trucks, personnel and, potentially, wild species 35, 36 . more surprising could be the non-significant role of road density. however, it must be stressed that the available raster reported the overall density of roads, which could significantly differ from those preferentially used for live animal or their byproduct transportation, hindering the detection of an otherwise plausible risk factor. therefore, the mapping of the live animal transportation pathways could provide remarkable benefits in ibv (and other infectious diseases) epidemiology understanding and control. the present study demonstrates that ibv spreading potential is mainly affected by farm and poultry density overall, which can be reasonably claimed as a major risk factor. other environmental/climatic variables do not seem to affect ibv epidemiology, stressing the pivotal role of human action and thus highlighting the direct benefits that could derive from an improved management and organization of the poultry sector on a larger scale. actually, the integration of poultry production seems to provide a relevant constrain to ibv circulation, even though some differences were noted between the two considered companies. in fact, despite differences in management and applied control strategies likely playing a role, the presence in the same area of other minor poultry companies seems to represent a major issue, probably due to the less effective infection control ascribable to the sometimes lower organization capability and resources of small enterprises. the present study results emphasize the need of an active sharing of sequences and related molecular epidemiology data originating from all the actors in poultry production, allowing a proper depiction of the viral exchange dynamics, based on actual data rather www.nature.com/scientificreports www.nature.com/scientificreports/ than estimations. the obtained information would represent a fundamental substrate for the implementation of effective and shared efforts for the infection control on a broad regional scale. ibv strain sampling, diagnosis and sequencing. samples were collected for routine diagnostic purpose in the period 2012-2016 from poultry flocks belonging to the two main poultry companies (here named company a and company b) operating in italy, which account together for about 90% of italian poultry production. samples were obtained mainly from outbreaks of respiratory disease, following a standard protocol that enforced the collection of a pool of 10 tracheal swabs from randomly selected birds. for each sampling, collection date and farm localization were recorded. all considered samples had been performed in the context of routine diagnostic activity and no experimental treatments or additional assays were implemented during the study. therefore, no ethical approval was required to use specimens collected for diagnostic purpose. additionally, several samples from company a were already sequenced using the same protocol and published in franzo et al. 27 . when detailed information on sampling farm and time could be traced back, these samples were included in the study. the permission to use the collected samples for research purpose was obtained from each company. swab pools were resuspended in 2 ml of pbs and vortexed. thereafter, rna was extracted from 200 µl of the obtained eluate using the high pure viral rna kit (roche diagnostics, monza, italy) kit. diagnosis was performed by amplification and sanger sequencing of the hypervariable region of the s1 region using the primer pair described by cavanagh et al. 37 . obtained chromatograms quality was evaluated using finchtv (http://www. geospiza.com) and consensus sequences were generated using cromaspro (cromasproversion 1.5). sequence dataset preparation. all obtained sequences plus the reference dataset provided by valastro et al. (2016) were aligned using mafft 38 and a phylogenetic tree was reconstructed using iq-tree 39 selecting as the best substitution model the one with the lowest akaike's information criterion, calculated using jmodeltest 40 . the strains clustering with the gi-19 lineage (previously known as qx genotype) were selected and further evaluated for the presence of recombination in the considered region using rdp4 41 and gard 42 : to limit the computational burden the sequences were clustered using a 99% identity threshold using cd-hit 43 and a single representative sequence for each cluster was selected. these sequences plus the valastro et al. (2016) references were re-aligned and recombination analysis was performed. recombinant sequences, including the ones belonging to the same cluster, were removed from the dataset. finally, the dataset was re-expanded to the original size and sequences identical or closely related (p-distance <0.01) to the qx-based vaccines administered in italy were also excluded. to evaluate the distribution of italian gi-19 strains in the international scenario, an extensive dataset of s1 ibv sequences was downloaded from genbank and a phylogenetic tree was reconstructed as previously described. to reduce computational complexity and increase interpretation easiness (without losing information), only one sequence representative of all identical ones was selected using cd-hit and included in the analysis. the presence of an adequate phylogenetic signal was assessed by a likelihood mapping analysis performed with iq-tree. tempest was used to preliminarily evaluate the temporal signal of the italian qx phylogeny and therefore the applicability of molecular clock-based methods 20 . strain migration among integrated poultry companies. ibv qx strain migration among companies was evaluated using the structured coalescent-based approach implemented in the multitypetree extension of beast2 44 . according to this model, the considered population is divided in a series of demes, which can be imagined as different islands, featured by their own populations size and interconnected by a certain migration rate among them. in the particular italian qx scenario, the serially sampled (i.e. with known collection date) strains were used to infer the migration rate and history between the two integrated poultry companies (i.e. considered as different demes) over time. additionally, the bayesian approach implemented in beast allowed to contextually estimate other population parameters, including the time to most recent common ancestor (tmrca), evolutionary rate and population size. accounting for the presence of other farms and companies operating in the italian poultry sector, which could take part in or mediate the viral transmission among the investigated major companies, a third "ghost" deme (a deme for which no sequences were available) was added to the model 31 . the priori of the ghost deme size was set to one tenth of the other demes, according to the estimated poultry population distribution. however, broad priori distribution (i.e. relatively uninformative priori) was chosen to avoid constrains or biases in the parameter posterior estimation. for all analyses, the best substitution model (tn93 + g 4 ) was selected based on the bayesian information criterion, calculated using jmodeltest 40 , while the relaxed lognormal molecular clock model was selected based on marginal likelihood calculation and comparison using the path sampling and stepping stone method 45 . the final estimations were obtained performing a 200 million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer 1.5 and accepted only if mixing and convergence were adequate and the estimated sample size was greater than 200 for all parameters. parameter estimation was summarized in terms of mean and 95% highest posterior density (hpd) after the exclusion of a burn-in equal to 20% of the run length. maximum clade credibility (mcc) trees were constructed and annotated using treeannotator (beast package). results consistency was also evaluated performing a "traditional" serial coalescent analysis in beast 1.8.4 46 . the same substitution and clock model of the structured coalescent analysis were selected, while a nonparametric skyline population model was chosen to reconstruct the viral population dynamic over time 47 . independent (2020) 10:7289 | https://doi.org/10.1038/s41598-020-64477-4 www.nature.com/scientificreports www.nature.com/scientificreports/ analysis for each integrated company were also performed using the same approach but generating two new datasets including only the sequences collected from a specific company. however, sequences introduced from one company to the other were excluded from the company-specific analysis since they did not share a common evolution history. continuous phylogeography and determinants of ibv spreading. the history of qx dispersal was reconstructed over time using the continuous phyogeographic approach described by lemey et al., 48 using beast 1.8.4. substitution and clock models were selected as previously described. similarly, the gamma relaxed random walk was preferred over the other phylogeographic continuous diffusion models based on the marginal likelihood calculation and comparison using the path sampling and stepping stone method 45, 48 . the final estimations were obtained performing a 200 million generation markov chain monte carlo run, sampling parameters and trees every twenty thousand generations. results were visually inspected using tracer 1.5 and accepted only if mixing and convergence were adequate and the estimated sample size was greater than 200 for all parameters. the reconstruction of qx movements over time within italian borders was obtained using spread3, summarizing and visualizing the full posterior distribution of trees obtained in continuous phylogeographic analyses 49 . pattern and determinants of viral spreading were evaluated as described by (dellicour et al.) 19 , using the seraphim r library 50 . the history of lineage dispersal was recovered from the posterior trees generated using beast and annotated with ancestral longitude and latitude reconstruction. particularly, the distance, duration and velocity of spatial dispersal were recoded as vectors and used to generate different summary statistics of viral spreading, including dispersal velocity and maximal wave front distances (measured from the location of the tree root). several environmental/social variables were considered to determine if they were associated with the dispersal rate of ibv lineages. the environmental rasters describing the variables of are shown in supplementary fig. 2 . more in detail, the values in the raster (i.e. altitude, population density, poultry density, temperature, etc.) were used to associate a weight to the abovementioned vector. two models of spatial movements were considered: (1) "straight line (sl) path" model, assuming a straight movement between the starting and ending locations of each branch (i.e. the branch weight is computed as the sum of raster cells through which the straight line passes); (2) "least cost (lc) path" model, using a least cost algorithm (i.e. the branch weight is computed as the sum of the values of cells transition values between adjacent cells along the least-cost path). in this model, the analyzed environmental variable can be considered both as a conductance (i.e. enhancing viral dispersal through the cells with higher values) or resistance factor (i.e. allowing an easier dispersal through cells with lower values). both instances were evaluated for each considered factor. the obtained "environmental" weights were used to calculate a regression with the branch duration and the corresponding coefficient of determination (r 2 env ) was obtained. a null coefficient of determination (r 2 null ) was also calculated assuming the null raster (i.e. when only the spatial distance of each movement is assumed to affect branch duration). the statistic d = r 2 env -r 2 null was selected as final outcome, and describes how much the regression is strengthened when the spatial variation in the environmental variable is included. to account for the phylogenetic uncertainness, the d statistic was calculated for each tree of the posterior distribution. however, for computational constraints, the number of posterior trees was down-sampled to 1000 after discharging a 20% burn-in. only the environmental variables with more than 90% of d statistics > 0 were considered for further analysis. particularly, the significance of d statistic of those variables was assessed against a d null distribution obtained by randomizing 1000 times the phylogenetic nodes location under the constraint that branch length remained equal. a p-value was generated for each initial tree, therefore a percentage of the trees with p-value < 0.05 could be calculated, which can be interpreted as a posterior probability of observing a significant correlation between lineage movements and considered environmental variable. according to , a percentage of p-value < 0.05 greater than 50% was considered a strong evidence that the environmental variable is associated to viral movement speed 19 . molecular evolution and emergence of avian gammacoronaviruses s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification review of infectious bronchitis virus around the world effect of different vaccination strategies on ibv qx population dynamics and clinical outbreaks sjaak) & cook, j. k. a. factors influencing the outcome of infectious bronchitis vaccination and challenge experiments infectious bronchitis virus variants: a review of the history, current situation and control measures age-dependent immune responses and immune protection after avian coronavirus vaccination a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain porcine circovirus type 2 (pcv2) evolution before and after the vaccination introduction: a large scale epidemiological study genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza modelling the wind-borne spread of highly pathogenic avian influenza virus between farms avian influenza virus infections. ii. experimental epizootiology of influenza a-turkey-wisconsin-1966 virus in turkeys characterisation of influenza a viruses isolated from turkeys in england during avian influenza in caged laying chickens isolation of avian influenza virus in texas assessing the probability of introduction and spread of avian influenza (ai) virus in commercial australian poultry operations using an expert opinion elicitation transmission parameters of highly pathogenic avian influenza (h7n1) among industrial poultry farms in northern italy in 1999-2000 risk maps for the spread of highly pathogenic avian influenza in poultry explaining the geographic spread of emerging epidemics: a framework for comparing viral phylogenies and environmental landscape data exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) risk factors for the introduction of high pathogenicity avian influenza virus into poultry farms during the epidemic in the netherlands in 2003 risk factors for highly pathogenic h7n1 avian influenza virus infection in poultry during the 1999-2000 epidemic in italy a cross-sectional survey of australian chicken farms to identify risk factors associated with seropositivity to newcastle-disease virus wind-borne transmission of infectious laryngotracheitis between commercial poultry operations phylogenetic and epidemic modeling of rapidly evolving infectious diseases continued use of ibv 793b vaccine needs reassessment after its withdrawal led to the genotype's disappearance think globally, act locally: phylodynamic reconstruction of infectious bronchitis virus (ibv) qx genotype (gi-19 lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens control of avian influenza in poultry studies on australian infectious bronchitis virus. iv. apparent farm-to-farm airborne transmission of infectious bronchitis virus estimating population parameters using the structured serial coalescent with bayesian mcmc inference when some demes are hidden new routes to phylogeography: a bayesian structured coalescent approximation proroga e modifica dell' ordinanza 26 agosto 2005 e successive modificazioni, concernente: «misure di polizia veterinaria in materia di malattie infettive e diffusive dei volatili da cortile epidemiology and control of low pathogenicity avian influenza infections in rural poultry in italy coronaviruses in avian species -review with focus on epidemiology and diagnosis in wild birds coronaviruses in poultry and other birds longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions mafft multiple sequence alignment software version 7: improvements in performance and usability w-iq-tree: a fast online phylogenetic tool for maximum likelihood analysis jmodeltest 2: more models, new heuristics and parallel computing rdp4: detection and analysis of recombination patterns in virus genomes gard: a genetic algorithm for recombination detection cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences efficient bayesian inference under the structured coalescent improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty bayesian coalescent inference of past population dynamics from molecular sequences phylogeography takes a relaxed random walk in continuous space and time spread3: interactive visualization of spatiotemporal history and trait evolutionary processes seraphim: studying environmental rasters and phylogenetically informed movements spatial visualization with ggplot2 this research was partially founded by the grant (bird187958/18) from the department of animal medicine, production and health, university of padua. g.f., a.m. and m.c. planned the study, g.f., c.m.t., m.l., t.t., r.c., p.p. performed laboratory work and generated the sequences obtained in the present study, c.m.t. and m.l. curated the sequences dataset, g.f. analyzed the data, m.c., a.m., p.m., g.t., g.o., l.g. supervised the respective research groups, g.f. wrote the manuscript, c.m.t., m.l., m.c. revised and improved the manuscript. all authors reviewed and agreed on the current version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-64477-4.correspondence and requests for materials should be addressed to g.f.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-351564-nikcd44o authors: zhang, xiaozhan; deng, tongwei; lu, jianzhou; zhao, pandeng; chen, lulu; qian, mengwei; guo, yiwen; qiao, hongxing; xu, yaohui; wang, yan; li, xinzheng; zhang, guizhi; wang, zeng; bian, chuanzhou title: molecular characterization of variant infectious bronchitis virus in china, 2019: implications for control programmes date: 2020-01-24 journal: transbound emerg dis doi: 10.1111/tbed.13477 sha: doc_id: 351564 cord_uid: nikcd44o infectious bronchitis virus (ibv), an ongoing emergence enveloped virus with a single‐stranded positive‐sense rna genome, belongs to the gammacoronavirus genus in the coronaviridae family. ibv‐associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with ibv in china. here, we described two ib outbreaks with severe respiratory system or kidney injury in ibv‐vaccinated commercial poultry farms in central china. other possible causative viral pathogens, including avian influenza virus (aiv), newcastle disease virus (ndv) and kedah fatal kidney syndrome virus (kfksv), were excluded by reverse transcription‐polymerase chain reaction (rt‐pcr), and three virulent ibv strains, hen‐1/china/2019, hen‐2/china/2019 and hen‐101/china/2019, were identified. although the gross pathologic appearance of these two ib outbreaks was different, the newly identified ibv strains were all closely related to the ck/china/i0529/17 strain and grouped into gi‐19 genotype clade based on the sequencing and phylogenetic analysis of the complete s1 genes. moreover, there are still some evolutionary distance between the newly identified ibv strains, hen‐101/china/2019 in particular, and other gi‐19 strains, suggesting that chinese ibv strains constantly emerge and evolve towards different directions. in conclusion, this study provided an insight of the recently emerging ibv outbreaks in ibv‐vaccinated commercial poultry farms and identified the genetic characteristics of three virulent gi‐19 ibv strains, which shows the need to carry out proper preventive measures and control strategies. avian infectious bronchitis (ib), a common avian disease with high morbidity and mortality, has drawn great attention for causing devastating economic losses to poultry industry worldwide (jackwood & wit, 2013) . infectious bronchitis virus (ibv), the causative agent, is an ongoing emergence enveloped, single-stranded, positive-sense rna virus, with genome length about 27.6 kb. it is the prototype of the gammacoronavirus genus in the coronaviridae family (bande et al., 2017; masters & perlman, 2013) . ibv mainly targets the respiratory tract of its natural host-chicken, causing severe respiratory system disease, and spreads via respiratory and feco-oral routes. additionally, ibv also infects the uro-genital tract, digestive system and reproductive system, resulting in nephritis, salpingitis, proventriculitis and egg drop (bande et al., 2017; jackwood & wit, 2013; sjaak de wit, cook, & van der heijden, 2011) . a great number of ibv serotypes, genotypes and pathotypes have been identified worldwide since its first description in 1931 in america (bande et al., 2017; lin & chen, 2017) . to prevent ibv infection, researchers have developed various commercial inactivated and live attenuated vaccines over the past decades, which successfully prevented ibv infections (jordan, 2017) . as a highly mutable coronavirus, however, the continuous emergence of novel ibv strains greatly emasculate vaccines efficacy, and the low cross-protection rates of ibv vaccines inevitably hamper the prevention and control of the disease (fan et al., 2018; jordan, 2017) . since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with ibv in china, especially in the intensive poultry raising regions, such as henan, hebei and shandong provinces. herein, we reported two ib outbreaks in ibv-vaccinated commercial broiler farms in central china, and the complete s1 genes of the newly identified ibv strains were sequenced, and its genotype, phylogeny and variations were analysed further. this study systematically described the genotype and evolutionary characteristics of the emerging ibv strains and highlighted the importance of continuous extensive surveillance to help choose suitable vaccines and develop control programmes reasonably. case 1: in april 2019, an acute outbreak of fatal respiratory disease occurred in a commercial broiler farm in henan province, central china. according to the breeder's description, approximately onetenth of 20,000 25-day-old broilers, vaccinated with commercial live attenuated ibv vaccine h120 at 7-day-old, showed severe respiratory symptoms, such as gasping, nasal discharge, tracheal rales, sneezing, and some affected flocks also presented greenishyellow diarrhoea, watery eyes and lethargy. the outbreak started from 18 april 2019, and antibiotic-antimycotic combination therapy did not work. a total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to send to the laboratory for diagnosis. case 2: in september 2019, an outbreak of fatal nephritis disease occurred in another commercial layer farm in henan province. according to the breeder's description, the farm has approximate 9,000 layers with 21-day-old, which were vaccinated with commercial live attenuated ibv vaccine h120 at 7-day-old. the great majority of the flocks started to show moderate respiratory symptoms from 10 september 2019, and mitigated in a week with the antibiotic and traditional veterinary drugs combination therapies. since 20 september, about 200 chickens per day died acutely without obvious clinical symptom. six moribund layers were selected randomly to send to the laboratory for diagnosis. the sick chicken examined in this study was approved by the poultry farm owners. diagnosis and experimental protocols in this study were approved by the henan university of animal husbandry and economy animal care committee. the clinical samples used in this study were collected in strict accordance with the guidelines for animal ethics committees. all the chickens were euthanized by cervical dislocation and then examined the anatomical changes, samples of lungs, tracheas and kidneys were collected from sick chicken and stored at −80°c until used for diagnosis and virus isolation. the samples were homogenized and centrifuged to collect supernatants. total viral rna was extracted by using trizol (invitrogen, cat no: 15596026) according to the manufacturer's instructions, and further reversely transcribed to cdna as described in our previous report . ibv, aiv, ndv and kfksv (palya et al., 2019) , which can cause severe respiratory symptoms, nephritis and acute death, were analysed by rt-pcr using specific primers as previously described (nguyen et al., 2013) . moreover, the positive samples were homogenated and then isolated in 9-day-old specific pathogen-free (spf) eggs via the allantoic cavity, and the allantoic fluid was harvested sterilely and stored at −80°c. to identify the genotype of ibv strains identified in this outbreak, the complete s1 genes were amplified by using primers as follows: ibv-s1 forward: 5′-gaacaaaagaccgacttagt-3′ and ibv-s1 reverse: 5′-tatgtactcatctgtracagt-3′, which were designed based on the conserved regions flanked the s1 gene of ibv strains downloaded from the vipr database (http://www.viprb rc.org/brc/home.spg?decor ator=vipr). the correct size amplicons, with length about 2000 bp, were then cloned into pcloneez vector (clone smarter) and sequenced. the complete s1 genes of the newly identified strains were subjected to online blast program (https ://blast.ncbi.nlm.nih.gov/blast.cgi), and sequences sharing more than 95% nucleotide identify were downloaded for further genetic analysis. additionally, the s1 genes of representative ibv strains within different genotypes and lineages jiang et al., 2017; ma et al., 2019; valastro et al., 2016) were also downloaded for sequence alignment with clustal omega (https ://www.ebi.ac.uk/tools/ msa/clust alo/), and the phylogenetic tree was conducted by mega 7.0 with the neighbour-joining method using 1,000 bootstrap replicates, which were then visualized by itol v4 software (letunic & bork, 2019) . recombination events among ibv strains were further investigated using the rdp4 software, a widely used tool for analysing individual recombination events and overall recombination patterns, by seven different algorithms recombination detection program (rdp), bootscan, maxchi, geneconv, chimaera, siscan and 3seq (martin, murrell, khoosal, & muhire, 2017 ). previous studies have revealed that most of the variable amino acids in s1 region were distributed in the three hypervariable regions (hvrs), including hvr i (38 aa-67 aa), hvr ii (91 aa-141 aa) and hvr iii (274 aa-387 aa), which were associated with neutralizing epitopes and receptor-binding domain and usually employed to classify ibv genotypes (moore, jackwood, & hilt, 1997; valastro et al., 2016) . to further investigate the hvrs and mutations in the newly identified ibv strain, the 3d structure of hen-2/china/2019 s1 protein was constructed by homology modelling method. from the pdb database, the glycoprotein s1 structure of vaccine m41 strain (accession number: 6cv0) was downloaded and employed as the template for modelling s1 monomer (shang et al., 2018) . all the hvrs and mutations of hen-2/china/2019 and hen-101/china/2019 were located on the structure and visualized by pymol software (http://www.pymol.org/). among the organs collected from the morbid chicken among two ib outbreaks, the predominant histologic lesions were in the trachea and kidney, respectively. in case 1, a total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to examine the anatomical changes, and trachea, especially between bronchus and bronchioles, showed the gross pathologic appearance of typical tracheitis, with serous, catarrhal or caseous exudate in trachea (figure 1a-c) . moreover, some of them presented systemic colibacillosis or airsacculitis, and no obvious nephritis and proventriculitis were observed. in case 2, all the six moribund layers exhibited severe lesion in kidneys, which were characterized by pale, swollen and mottled (figure 1d,e) . some of them showed distended ureters filled with uric acid, but none of them presented tracheitis. to identify the causative agent of this outbreak, rt-pcr assays were used to detect the common potential viral pathogens, including ibv, ndv, aiv and kfksv. as shown in figure s1a to further characterize the biology and ecology of the newly identified ibv strains, we sequenced the complete s1 genes of phylogenetic relationship among ibv strains has been established on the analysis of complete s1 gene. as shown in figure 2 , the phylogenetic tree further revealed that the hen-1/china/2019, hen-2/china/2019 and hen-101/china/2019 strains were f i g u r e 2 phylogenetic analysis of the complete s1 genes of newly identified ibv in 2019. phylogenetic analysis based on the deducted amino acid sequences of complete s1 gene of hen-1/china/2019, hen-2/china/2019, hen-101/china/2019 and other ibv isolates available from genbank database. phylogenetic tree was constructed by mega 7.0 software using the neighbour-joining method with 1,000 bootstrap replicates. the seven different ibv genotype, with 35 distinct lineage ibv strains were illustrated by the colour scale with itol software. meanwhile, the available ibv vaccine strains in china were indicated by blue star, and the three ibv isolates in this study were indicated by red star clustered together with ck/china/i0529/17, qx and lx4 strains and grouped into the gi-19 genotype, which were distantly from the available commercial vaccines h120, 4/91 and ldt3-a. as a highly variable coronavirus, numerous ibv variants have been identified and nucleotide substitutions or recombination between field strains and vaccines occurred frequently. previous study revealed that the recombinant events have already occurred in european strains within the s1 gene (moreno et al., 2017) . we further investigated the recombinant events between gi-19 and other ibv genotypes using the rdp4 software. the results showed that several recombinant events occurred between gi-19 and other ibv genotypes with high score (p < .01, recombinant score >0.6), including strains of the newly identified genotype gi-28, gi-25 and unassigned strain ldt3 ( figure s2) . interestingly, the gi-19 genotype plays a two-faced role in these recombinant events, not only providing fragments for other strains, but also receiving fragments during infection. to further explore the characteristics of the newly identified ibv strains, amino acid polymorphism of the s1 glycoprotein was compared with other ibv strains of all 35 genotypes jiang et al., 2017; ma et al., 2019; valastro et al., 2016) . the result revealed that most of the amino acid mutations in s1 region of the newly identified ibv strains were located in the three hvr regions. (table 1) . compared with the available capsid structure of m41 strain in the pdb database (shang et al., 2018) , the p87l and q89k mutations are adjacent to hvr i and hvr ii region structurally (figure 3c ) and located in the major receptor-binding domain (19 aa-253 aa; promkuntod, van eijndhoven, de vrieze, grone, & verheije, 2014) ; the q97e, s117a, t120r, a130e and r131s are in hvr ii region; the t300i and h329y are in hvr iii region (figure 3d ), indicating that these mutations might change virus antigenicity. ib is an economically important avian disease that affects the poultry industries worldwide, particularly in the large poultry-producing countries, such as usa, china and brazil (bande et al., 2017; jackwood & wit, 2013) . the first case of ib in china dates back to early 1980s, and since then, the outbreaks were constantly emerging and numerous ibv strains were identified, of which strains in genotype gi-19 (refers to the qx-like or lx4) became predominant among chickens currently, although the qx strain was initially identified in 1996. additionally, other genotypes of ibv, such as gi-1 (mass), gi-7 (tw-i or tw-like), gi-13 (4/91-like), gi-16 (q1-like) and gi-22 (yn-like and saibk-like), were also frequently detected in china (han et al., 2018; li et al., 2012; lin & chen, 2017; mo et al., 2013) . importantly, several novel ibv genotypes, including gi-28, gi-29 and gvii-1, were identified recently jiang et al., 2017; ma et al., 2019) . all these indicated that the ibv strains circulating in china are genetically diverse, providing a potential platform for recombination and greatly challenging the current biological control measures. (bande et al., 2017; han et al., 2011; li et al., 2012) . gi-1 genotype vaccine h120 has been the most widely used vaccine in china until now, which shared a very low homology of identity with the emerging gi-19 strain hen-1/china/2019 (32.2%) and hen-2/china/2019 (32.2%) in glycoprotein s1 gene region, and even the other gi-19 strains identified recently. these results indicated the h120 vaccine could not provide effective protection against gi-19 genotype strains infection, which might explain the clinical phenomenon that a number of ibv outbreaks emerged in vaccinated flocks (lin & chen, 2017 characterization and localization of specific nonsynonymous mutations in capsid protein of the newly identified ibv strains in comparison with other ibv isolates. the multi-alignment of s1 glycoprotein of all ibv genotypes was conducted by clustal-omega, and schematic diagram based on the identified protein functional domains of mature s1 protein (without 1aa-19aa) was illustrated (a); 3d structure template of gi-1 genotype (m41 strain) was downloaded from pdb database, and the hvr regions of ibvs (b) and the location of mutation sites of hen-2/china/2019 (c and d) were visualized by pymol software global distributions and strain diversity of avian infectious bronchitis virus: a review identification and molecular characterization of a novel serotype infectious bronchitis virus (gi-28) in china immune protection conferred by three commonly used commercial live attenuated vaccines against the prevalent local strains of avian infectious bronchitis virus in southern china genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems a 15-year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china infectious bronchitis genome characterization, antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south china. infection vaccination against infectious bronchitis virus: a continuous challenge interactive tree of life (itol) v4: recent updates and new developments serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in guangxi infectious bronchitis virus variants: molecular analysis and pathogenicity investigation novel genotype of infectious bronchitis virus isolated in china detecting and analyzing genetic recombination using rdp4 corornaviridae molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain multiplex nested rt-pcr for detecting avian influenza virus, infectious bronchitis virus and newcastle disease virus novel orthobunyavirus causing severe kidney disease in broiler chickens mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins infectious bronchitis virus variants: a review of the history, current situation and control measures s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification. infection identification and genomic characterization of the emerging senecavirus a in southeast china the authors declared no potential conflict of interests with respect to the research, authorship and publication of this article. the data that support the findings of this study are available in genbank at ncbi, reference number [mn055627, mn055628 and mn635798] . these data were derived from the following resources available in the public domain ncbi. https://orcid.org/0000-0002-0280-4964zeng wang https://orcid.org/0000-0002-3274-4296 key: cord-353027-bc0un6kb authors: ali, ahmed; kilany, walid h; zain el-abideen, mohamed a; sayed, magdy el; elkady, magdy title: safety and efficacy of attenuated classic and variant 2 infectious bronchitis virus candidate vaccines date: 2018-08-06 journal: poult sci doi: 10.3382/ps/pey312 sha: doc_id: 353027 cord_uid: bc0un6kb vaccination programs against infectious bronchitis virus (ibv) in egypt depend on both classical and/or imported variant ibv strain vaccines. however, many ibv outbreaks associated with respiratory distress, nephropathy, and high mortalities were attributed to the circulation of both classical and new nephropathogenic ibv variant 2 strains. in the present study, we report the development of attenuated ibv candidate vaccines using the classic ibv strains (ibm41 and ib2) and a nephropathogenic strain (ibvar2). the wild-type (wt) viruses were attenuated through serial passages in embryonated specific pathogen free (spf) chicken eggs. virulence of the attenuated viruses was then tested via the ocular route inoculation and the in vivo back passage in day-old spf chickens. efficacy against homologous challenge was investigated also in day-old spf chickens. results showed that the viruses were successfully adapted to the embryo by the 100th (ibm41 and ib2) and 110th passages (ibvar2). the attenuated viruses were safe and showed no change of virulence in day-old spf chickens up to the 10th back passages. the efficacy experiment showed that the attenuated vaccines showed 90 to 100% protection against the homologous challenge based on ciliostasis score and protection percent. the att-ibm41 and att-ib2 vaccines were able to reduce the shedding of the challenge at 3 days post-infection (dpi) and no virus shedding was detected in both vaccinated groups by 5 dpi. in the att-ibvar2 vaccinated birds, only 20% of vaccinated birds shed the challenge virus with low titers (10(2.10±0.3) eid(50)/ml) at 3 dpi. in conclusion, the attenuated strains ibm41, ib2, and ibvar2 are efficient vaccine candidates against currently circulating classic and variant ib viruses, respectively. further studies to evaluate the field efficacy and combining these attenuated ibv strains to induce a wider protection against heterologous ibv challenge are suggested. avian infectious bronchitis (ib) is a highly contagious disease of chickens affecting the respiratory, renal, and reproductive systems. the ib was described as early as 1931 in north dakota, usa. since then, the ib virus (ibv) is circulating in different parts of the world with several ibv serotypes that led the ib control and prevention problem to persist (fabricant, 1998) . though the disease affects chickens of all ages, the clin-ical disease is more severe in young birds with high mortalities (cavanagh, 2005) . the secondary bacterial infections and/or other respiratory pathogens have been reported to exaggerate both clinical outcome and mortality during ibv infections (haghighat-jahromi et al., 2008; hassan et al., 2016 hassan et al., , 2017 . the ibv is a single-stranded enveloped rna virus and is a member of the genus gammacoronavirus belonging to the family coronaviridae (cavanagh, 2001) . the clinical signs of the ibv infection vary depending on the tissue tropism and the pathogenicity. clinical signs include coughing, sneezing, tracheal rales, and watery eyes. lesions in infected birds include degeneration of renal and ciliated respiratory epithelia (cavanagh, 2007) . the ibv epidemiology in egypt reported the circulation of the virus in poultry flocks with continuous evolution despite massive vaccination programs. different strains similar to massachusetts, 4238 d3128, d274, d08880, and 793b are frequently isolated from egyptian poultry (abdel-moneim et al., 2006; abdel-moneim et al., 2012; zanaty et al., 2016) . studies classified the egyptian ibv strains on the basis of their hvr3 sequences into egyptian variant 1 (egy/var-i), represented by the strain egypt/beni-suef/01 (abdel-moneim et al., 2006) and egyptian variant 2 (egy/var-ii), including recent egyptian ibv strains that resemble the israeli is/885/00 strain (abdel-moneim et al., 2012) . the continuous mutation and recombination events and the emergence of novel ibv variants urge researchers to establish a simple phylogeny-based classification system. thus, a recent study indicated that both egy/var-i and egy/var-ii egyptian ibv strains belong to a unique wild-type (wt) cluster confined to the middle east region (gi-23 lineage) (valastro et al., 2016) . vaccination remains the main control approach of ibv infection. however, the continuous genetic, antigenic, and tissue tropism changes of the circulating ibv caused continual vaccine failure events (cook et al., 2012) . ibv vaccines are based on live attenuated or killed vaccines derived from classical or variant serotypes. in egypt, mass-type and variant (e.g., 4/91 and cr88) vaccine strains were employed to provide broader protection in poultry. however, the uniqueness of the wt ibv lineage in the middle east (valastro et al., 2016) and the lack of cross protection between imported vaccine strains and field strains may explain the failure to establish an effective vaccination program against ibv (kim et al., 2013; toro et al., 2015) . several studies indicated the inability of the currently available vaccines to provide adequate protection against the egy/var-i and var-ii ibv strains (hassan et al., 2016; zanaty et al., 2016) . therefore, this study aimed to develop and evaluate live attenuated vaccines from ibv strains currently circulating in the middle east including an egy/var-ii strain belonging to the unique middle east lineage (gi-23). the candidate vaccine's safety was evaluated in both day-old and layer specific pathogen free (spf) chickens. finally, the protective efficacy of the attenuated strains was investigated against wt virus challenge. the ibv viruses used were obtained from the reference laboratory for veterinary quality control on poultry production (nlqp), animal health research institute, giza, egypt. the ibv strain ibv-eg/11539f/2011 (accession no.: jq839289) and ibv-eg/ m41-me01/2011 (accession no.: mg334195), classic mass-like strains belonging to the gi-1 lineage both viruses were isolated from 21-to 23-d-old commercial broilers suffering from respiratory problem. these chickens had a history of vaccination against ibv using h120 vaccine. the ibv strain eg/1212b/2012 (acces-sion no.: jq839287) was isolated from 14-d-old vaccinated chick suffering from respiratory symptoms and renal disease (zanaty et al., 2016) and identified as egy/var-ii. the viruses were designated ib2, ibm41, and ibvar2 for ibv-eg/11539f-2011, ibv-eg/m41-me01/2011, and eg/1212b/2012, respectively. the wt ibm41, ib2, and ibvar2 viruses were used for the homologous challenge at titers of 10 6.2 , 10 6.0 , and 10 5.0 eid 50 /0.2 ml, respectively. for attenuation purposes, the viruses were confirmed to be pure from extraneous viruses and bacteria at the reference laboratory for veterinary quality control on poultry production (nlqp), animal health research institute, giza, egypt in accordance with oie guidelines (eterradossi and britton, 2013 ) (data not shown). the viruses passaged for 100 times for ibm41 and ib2 viruses and for 110 times for ibvar2 via allantoic sac inoculation in 10-d-old spf eggs. inoculated eggs were incubated for 40 h at 37 • c. eggs that died within 24 h of inoculation were discarded. the allantoic fluid was harvested for subsequent passages. every 10 passages, viruses were confirmed by rt-pcr (huo et al., 2016) . the final embryo infective dose 50% (eid 50 ) of the attenuated viruses were determined in spf embryonated chicken eggs (reed and munech, 1938) . the attenuated viruses were designated att-ib2, att-ibm41, and att-ibvar2. in all experiments, white leghorn spf chickens were used to determine the safety and efficacy of attenuated viruses. the spf chickens were purchased from nile spf company (kom-osheim, fayoum, egypt). birds were kept in biosafety level iii chicken isolators and all experimental procedures were reviewed and approved by the ethical and animal welfare committee at me vac company. a total of 140-d-old spf white leghorn chicks were randomly divided into 7 groups of 20 chickens each. the chickens were maintained in biosafety level iii chicken isolators. the first group was kept as a pbs-inoculated negative control. for each virus strain, 2 chicken groups were inoculated via the ocular route with 10 4 eid 50 /bird of either the wt or the attenuated virus separately. all groups were observed for 21 days post-inoculation (dpi), and the ibv clinical signs were scored according to zhao et al. (2015) as follows: 0 = normal, 1 = mild depression, 2 = severe depressed, 3 = paralysis/prostration, and 4 = death. three chicks from each group were sacrificed at 3, 5, and 7 dpi for ciliostasis evaluation as well as kidney and tracheal gross lesions scoring (huang and wang, 2006) . forty-nine 25-wk-old white leghorn chicks were randomly divided into 7 groups (7 chickens/group). the chickens were maintained in isolators for adaptation and until they reached their normal egg production levels. the first group was kept as a pbs-inoculated negative control. two groups for each virus strain were inoculated via the ocular route with 10 4 eid 50 /bird of either the wt or the attenuated virus separately. all groups were observed for 14 dpi for clinical signs, daily egg production, and egg quality. to examine the in vivo reversion of virulence of the attenuated-ibm41, ib2, and ib-var2 viruses; 1-d-old spf chicks were divided into 4 groups of 3 chicks. the 3 inoculated groups received 10 4 eid 50 /bird of the attenuated viruses separately via the ocular route. the fourth group was kept as a pbsinoculated negative control. birds were observed daily for clinical signs for 5 d. at 5 dpi, the virus detection was confirmed by rt-pcr in kidneys and tracheal homogenates using a specific primer set for amplification of the hvr-3 of the ibv-s1 gene as previously described (selim et al., 2013) . the tissue homogenates were then used to inoculate a second set of birds. the back passage of the attenuated viruses was repeated for 10 times. at the 1st, 5th, and 10th passages of each virus, tracheal and kidney lesions were scored as described in the safety study. one hundred and twenty 1-d-old spf white leghorn chicks were randomly divided into 6 groups of 20 chickens each. the first 3 groups were vaccinated via the ocular route with 10 3.5 eid 50 /bird of att-ibm41 virus, att-ib2, and att-ibvar2 viruses, respectively. two groups served as unvaccinated challenge controls for wt ibm41, and ibvar2, respectively. the sixth group was kept as unvaccinated unchallenged control. at 21 days postvaccination, birds in each group were challenged with 10 5 eid 50 /bird of the homologous wt virus as shown in table 1 . all birds were observed daily for ib infection clinical signs for 15 dpi. at 3 and 5 dpi, 3 birds from each group were euthanized for gross lesions observation and ciliary activity evaluation. oropharyngeal swabs were collected from each group at 3, 5, and 7 dpi in 1 ml pbs to monitor virus shedding titers. the collected swab samples were processed on the same day of collection. swabs were vortexed and then centrifuged at 2000 rpm for 10 minutes at 4 • c to pellet the debris. the supernatants were then used for virus titration in 10-d-old embryonated spf chicken eggs and the eid 50 /ml was calculated (reed and munech, 1938) . ciliostasis evaluation in safety testing and postchallenge experiments, the tracheal ciliary activity was assessed as previously described (cook et al., 1999) . briefly, 3 sections of the upper, middle, and lower parts of the trachea were analyzed. the rings were placed in a petri dish containing minimum essential medium with 10% fetal bovine serum. the integrity and the ciliary movement of the tracheal epithelial cells were evaluated by inverted light microscope. ciliostasis scoring was 0 for the tracheal section showing complete movement, 1 for tracheal sections with 75 to 100% movement, 2 if the cilia in 50 to 75% of the trachea showed movement, 3 if the cilia in 25 to 50% of the trachea section showed movement, and 4 if the cilia in less than 25% of the trachea section showed movement or no movement at all. the average ciliostasis score for each group was calculated. the differences in ciliostasis scores and virus shedding titers at 3 dpi were estimated using one-way anova with tukey's post-test through graphpad prism version 5.00 for windows (graphpad software, san diego california usa, www.graphpad.com). the 3 viruses (ibv-eg/11539f-2011, ibv-eg/m41-me01/2011, and eg/1212b/2012) were successfully adapted to embryonated chicken eggs and showed the typical embryonic changes including dwarfing, stunting, and curling of embryos by the 10th or 14th passages for the classic (ib2 and ibm41) and the variant virus (ibvar2) (data not shown). by the 100th passage, the classic ib2 and ibm41 were efficiently growing to titers of 10 7.2 and 10 6.9 eid 50 /ml, respectively. however, the ibvar2 virus consistently reached the titer of 10 6.8 eid 50 /ml by the 110th passage. the wt viruses induced depression, ruffled feathers, and respiratory rales in day-old spf chicks; however, no mortality was recorded in any of the inoculated birds. upon necropsy, tracheal congestion was observed in the trachea in all wt viruses. the wt ib-var2 induced kidneys swelling and urate deposition. the attenuated viruses did not show any clinical signs in inoculated birds. no gross lesions in both trachea and kidneys were observed as compared to birds inoculated with the wt viruses. only the att-ibvar2 showed an average lesion score of 0.6 and 1.0 in the trachea and kidney, respectively. the wt viruses induced ciliostasis in the tracheal epithelium especially wt ibm41 and ibvar2 (3.6 ± 0. 14 and 3.9 ± 0.18, respectively) as compared to ib2 (3.1 ± 0.57). however, the attenuated strains had significantly (p < 0.001) lower tracheal ciliostasis (≤1.3) than those induced by wt viruses (figure 1) . the egg production dropped in the 3 wt viruses-inoculated chicken groups. egg quality deterioration including watery albumin, rough shell, and shelless eggs was also observed. chickens also showed mild respiratory signs with no mortality in the inoculated birds. neither clinical signs nor drop in egg production were observed in the attenuated viruses inoculated group with the exception of day 1 post inoculation where a slight drop was observed in all groups including the negative control group (figure 2) . the egg production declined to 28.6 to 42.8% between 3 and 7 dpi in wt viruses inoculated groups. meanwhile, in all attenuated viruses inoculated groups, the egg production was comparable to the negative control group (71.4 to 85.7%). the 3 attenuated viruses were consistently detected in tissue homogenates of inoculated birds at all passages. no clinical signs were observed in any of the inoculated birds after 5 passages. no significant differences were found between the tracheal and renal lesion scores of the 1st, 5th, and 10th passages of the attenuated ibm41, ib2, and ibvar2 viruses ( table 2) . challenge experiments were conducted separately for birds vaccinated with the classic viruses, att-ibm41 and att-ib2 and those vaccinated with att-ibvar2 using the homologous virus in each group. the att-ibm41 and att-ib2 vaccine candidates provided complete protection of the respiratory tract against homologous strain wt ibm41 challenge. the ciliary activity of the trachea of vaccinated birds was significantly higher than unvaccinated challenge control birds (p < 0.001) at 3 and 5 dpi (figure 3 ). calculation of protection score and percentage showed that the vaccine conferred 90 to 100% protection against homologous challenge ( table 3) . the att-ibm41 and att-ib2 vaccines were also able to reduce the shedding of the challenge at 3 dpi, since 40 and 50% of vaccinated birds, respectively, were active shedders with significantly low titers (p < 0.001). by 5 dpi, no virus shedding was detected in both vaccinated groups (table 4) . similarly, the att-ibvar2 protected birds from homologous wt ib-var2 virus challenge. none of the birds showed clinical signs, and the average ciliostasis score in the att-ibvar2 vaccinated birds was 1.3±0.7 that was significantly lower (p < 0.001) than the wt virus non-vaccinated challenge group (figure 3 ). only 20% of vaccinated birds shed the challenge virus with low titers (10 2.10±0.3 eid 50 /ml) (table 4 ). the widespread exposure to several serotypes and genotypes of ibv with limited cross-protection has led to the failure of currently available vaccines to protect chickens from the heterologous challenge (gelb et al., 1991) . though the ibv vaccination programs in egypt depend on both classical (mass 41 and h120) and/or imported variant (d278, cr88 or 4/91 strains) strain vaccines, many ibv outbreaks associated with respiratory distress, nephropathy, and high mortalities (>30%) were observed during last 10 yr (sid et al., 2015; hassan et al., 2016) . most of the outbreaks were mainly attributed to the nephropathogenic ibv variant 2 strains (hassan et al., 2016; zanaty et al., 2016; abozeid et al., 2017) . recent studies also indicated that the ibv strains circulating in egypt represent a distinct a day-old spf chicks received 10 4 eid 50 /bird of the attenuated viruses separately via the ocular route. birds were observed daily for clinical signs for 5 d. at 5 days post-infection, the virus detection was confirmed by rt-pcr in kidneys and tracheal homogenates. the tissue homogenates were then used to inoculate a second set of birds. at the 1st, 5th, and 10th passages; tracheal and kidney lesions were scored. wt, wild-type a,b protection score [1 -mean ciliostasis score for vaccinated challenged/mean ciliostatsis score for challenge group] × 100 and % of protected chicks showed an average score ≤20 out of all tested chicks (cook et al., 1999) . c na; not applicable. wt cluster confined to the middle east region (gi-23 lineage) (valastro et al., 2016) . the recently described protection studies with the chinese qx-like ibv (feng et al., 2015; huo et al., 2016; lim et al., 2015; zhao et al., 2015) and korean nephropathogenic ibv strain (lee et al., 2010) further confirmed the concept that the best protection against ibv challenge can be achieved by using homologous strain vaccines. in the present study, we report the development of an attenuated ibv candidate vaccines using the egyptian classic ibv strains (ibm41 andib2) and a nephropathogenicib-var2 strain belonging to the widely spread gi-23 lineage in the middle east. the wt viruses were attenuated through serial passage onto spf eggs. though the attenuation of ibv via egg passage is laborious and time-consuming, it is the most common technique of ibv vaccine development (jackwood et al., 2003) . by the 100th (att-ibm41 and ib2) and 110th passage (ib-var2), the viruses were successfully adapted to the embryo with acceptable growth characteristics. virulence of the attenuated virus was then tested via the ocular route in day-old spf chickens in comparison with the corresponding wt viruses. the effect of attenuated viruses on egg production in layer chickens and the in vivo back passage of the attenuated viruses was evaluated. the results confirmed that the viruses were efficiently attenuated compared to the wt parent viruses. the absence of adverse effect on egg production or change of virulence in day-old spf chickens up to the 10th passage further confirmed the safety of the attenuated viruses. it is worthy to note that the att-ibvar2 kidney lesion scores reported were mainly due to virus tropism to the kidney tissues which was reported with other nephropathogenic strains even after attenuation (hodgson et al., 2004; lim et al., 2015) . the mild ciliostasis observed in all attenuated strains seems to be consistent with other research studies that reported certain degrees of tracheal tissue damage which may be needed to induce ibv local immunity (jackwood et al., 2003; jackwood et al., 2015) . the levels of local tracheal immunity associated with variable tracheal tissue damage degrees were found to be positively correlating with the protection levels of ibv vaccines (awad et al., 2016) . efficacy of attenuated viruses against homologous challenge was investigated in day-old spf chickens. the heterologous challenge was not considered as both field data and broilers results confirmed that mass-type vaccines are not sufficient to protect birds against heterologous variant ibv strains (meir et al., 2004; bru et al., 2017) . recent studies highlighted the importance of depending on both quantification of ciliostasis and detection of viral shedding for the evaluation of protection levels afforded by the ibv vaccine candidates (bande et al., 2015) . 1.14 ± 1.38 * 2.14 ± 0.83 * 4.48 ± 0.59 2.10 ± 0.28 (10/10) (4/10) (5/10) (10/10) (2/10) 5 3 . 3 3± 0.77 nd c nd 3.05 ± 0.75 nd (7/10) (10/10) 7 1 . 5 7± 0.52 nd nd 2.17 ± 0.57 nd (6/10) (7/10) a dpi; days post-infection. b mean eid 50 /ml (positive/total tested). c nd; not detected. * indicate significant difference compared to the corresponding challenge virus group (p < 0.01). in the current study, both evaluation criteria were taken into consideration. the attenuated ibm41, ib2, and ib-var2 were capable to significantly reduce the clinical signs, lesions, and ciliostasis induced by the homologous challenge. moreover, a significant reduction of the virus shedding titers was observed in the vaccinated birds at 3 dpi and no virus was detected at 5 and 7 dpi. these findings indicate that the attenuated viruses are suitable vaccine candidates that can provide protection against the challenge with homologous strains. these findings provide evidence of the efficacy of the attenuated ibv strains ibm41, ib2, and ib-var2 as vaccine candidates against middle eastern classic and variant gi-23 ibv strains, respectively with protection % of ≥90%. however, further studies are required to evaluate the field safety and effectiveness of attenuated viruses, compared to other ibv vaccines. also, further research should be done to investigate the use of combinations of these attenuated ibv strains to induce a wider protection against heterologous ibv challenge considering the fact that several virulent ibv strains are co-circulating in the middle east. emergence of a novel genotype of avian infectious bronchitis virus in egypt s1 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view: 40 years of infectious bronchitis research breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes avian infectious bronchitis the early history of infectious bronchitis development and efficacy of a novel live-attenuated qx-like nephropathogenic infectious bronchitis virus vaccine in china attenuation of avian infectious bronchitis virus by cold-adaptation coinfection of avian influenza virus (h9n2 subtype) with infectious bronchitis live vaccine experimental co-infection of infectious bronchitis and low pathogenic avian influenza h9n2 viruses in commercial broiler chickens prevalence of avian respiratory viruses in broiler flocks in egypt recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m41 strain remains attenuated but induces protective immunity development of attenuated vaccines from taiwanese infectious bronchitis virus strains attenuation mechanism of virulent infectious bronchitis virus strain with qx genotype by continuous passage in chicken embryos attenuation, safety, and efficacy of an infectious bronchitis virus ga98 serotype vaccine evaluating protection against infectious bronchitis virus by clinical signs, ciliostasis, challenge virus detection, and histopathology cross-protective immune responses elicited by a korean variant of infectious bronchitis virus characterization of a novel live attenuated infectious bronchitis virus vaccine candidate derived from a korean nephropathogenic strain successful cross-protective efficacy induced by heat-adapted live attenuated nephropathogenic infectious bronchitis virus derived from a natural recombinant strain identification of a novel nephropathogenic infectious bronchitis virus in israel a simple method of estimating fifty per cent endpoints molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during 2012 co-infection with multiple respiratory pathogens contributes to increased mortality rates in algerian poultry flocks cross-protection by infectious bronchitis viruses under controlled experimental conditions s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification the sequence of the full spike s1 glycoprotein of infectious bronchitis virus circulating in egypt reveals evidence of intra-genotypic recombination safety and efficacy of an attenuated chinese qxlike infectious bronchitis virus strain as a candidate vaccine the authors would like to thank middle east for veterinary vaccines (me vac) co. research & development and laboratory animal team for their technical support during experimental safety and challenge studies. key: cord-323568-s0wmll4q authors: shang, jian; zheng, yuan; yang, yang; liu, chang; geng, qibin; luo, chuming; zhang, wei; li, fang title: cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins date: 2018-04-23 journal: plos pathog doi: 10.1371/journal.ppat.1007009 sha: doc_id: 323568 cord_uid: s0wmll4q as cell-invading molecular machinery, coronavirus spike proteins pose an evolutionary conundrum due to their high divergence. in this study, we determined the cryo-em structure of avian infectious bronchitis coronavirus (ibv) spike protein from the γ-genus. the trimeric ibv spike ectodomain contains three receptor-binding s1 heads and a trimeric membrane-fusion s2 stalk. while ibv s2 is structurally similar to those from the other genera, ibv s1 possesses structural features that are unique to different other genera, thereby bridging these diverse spikes into an evolutionary spectrum. specifically, among different genera, the two domains of s1, the n-terminal domain (s1-ntd) and c-terminal domain (s1-ctd), diverge from simpler tertiary structures and quaternary packing to more complex ones, leading to different functions of the spikes in receptor usage and membrane fusion. based on the above structural and functional comparisons, we propose that the evolutionary spectrum of coronavirus spikes follows the order of α-, δ-, γ-, and β-genus. this study has provided insight into the evolutionary relationships among coronavirus spikes and deepened our understanding of their structural and functional diversity. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as large enveloped rna viruses, coronaviruses are capable of adapting to new hosts with relative ease through mutations and recombinations [1] [2] [3] . as a result, coronaviruses infect a wide range of mammalian and avian species, and have genetically evolved into four major genera: α, β, γ, and δ [4] . coronaviruses from the four genera all contain envelope-anchored spike proteins that mediate viral entry into host cells [5, 6] . during viral entry, the spikes bind to host receptors through their s1 subunits and then fuse viral and host membranes through their s2 subunits. on the one hand, the spikes interact with host receptors and other host factors, hence needing to evolve for better adaptation to these host factors [7] [8] [9] [10] . on the other hand, they are exposed to the host immune system, thereby needing to evolve to evade the host immune surveillance [11] [12] [13] [14] . consequently, the spikes are the most divergent among all coronavirus proteins [6] . the s1 subunits are particularly divergent, with little or low sequence similarities across different genera [15] . how coronavirus spikes have evolved to their current diverse structures imposes a major evolutionary conundrum. traces of protein evolution can often be found more reliably in their tertiary structures and related functions than in their primary structures, because proteins generally need to evolve within certain structural and functional constraints [16, 17] . to decipher the evolutionary puzzles surrounding coronavirus spikes, extensive structural studies have been carried out using both x-ray crystallography and cryo-electron microscopy (cryo-em) [12] [13] [14] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . these studies have resulted in structure determinations of s1 from the α-and β-genera and spike ectodomains from the α-, β-, and δ-genera. coronavirus spikes exist in two distinct conformations: the pre-fusion structures are present on mature virions and have a clove-like shape with three s1 heads sitting on top of a trimeric s2 stalk [12] [13] [14] [18] [19] [20] ; the post-fusion structures are the membrane-fusion state and have a dumbbell-like shape with three s2 subunits forming a six-helix bundle structure [29] [30] [31] [32] [33] [34] [35] . whereas the structures of s2 from different genera are similar to each other in both the pre-and post-fusion states, the s1 subunits from different genera diverge structurally and they also recognize a variety of host receptors [6, 7] . s1 contains two domains, n-terminal domain (s1-ntd) and c-terminal domain (s1-ctd), either or both of which can function as the receptor-binding domain. the s1 domains from different genera contain structural features that are unique to their genus. s1-ctds are particularly diverse, with low or no structural similarity across different genera [15] . overall, these previous studies have provided structural snapshots of coronavirus spikes from α-, β-, and δ-genera. however, because the structures of γ-coronavirus spikes were still missing, we lacked a clear picture of the evolutionary relationships among coronavirus spikes from different genera. in the current study, we determined the cryo-em structure of avian infectious bronchitis coronavirus (ibv) spike, the first such structure from the γ genus. the ibv spike possesses structural features that are unique to different other genera, suggesting that it falls in the middle of an evolutionary spectrum of coronavirus spikes. we also discuss how the structural evolution of coronavirus spikes has affected their functions as cell-invading molecular machinery. overall, our study has filled in a critical gap in the structural, functional and evolutionary studies of coronavirus spikes, and deepened our understanding of viral evolutions in general. expression and purification of ibv spike ectodomain ibv spike gene (virus strain m41; genbank number abi26423.1) was synthesized (genscript) with codons optimized for insect cell expression. its ectodomain (residues 20-1084) was cloned into pfastbac vector (life technologies inc.) with a n-terminal honeybee melittin signal peptide and c-terminal gcn4 and his 6 tags. it was expressed in sf9 insect cells using the bac-to-bac system (life technologies inc.) and purified as previously described [21] . briefly, the protein was harvested from cell culture medium, and purified sequentially on ni-nta column and superdex200 gel filtration column (ge healthcare). ibv s1-ctd (residues 248-495) was expressed and purified in the same way as the ibv spike ectodomain, although it only contains a c-terminal his 6 tag and does not contain the gcn4 tag. for sample preparation, aliquots of ibv spike ectodomain (3 μl, 0.35 mg/ml, in buffer containing 2 mm tris ph7.2 and 20 mm nacl) were applied to glow-discharged cf-2/1-4c c-flat grids (protochips). the grids were then plunge-frozen in liquid ethane using a fei markiii vitrobot system (fei company). for data collection, images were recorded using a gatan k2 summit direct electron detector in the direct electron counting mode (gatan), attached to a fei titan-krios tem, at arizona state university. the automated software serialem [36] was used to collect~2,000 total movies at 37,700x magnification and at a defocus range between 1 and 3 μm. each movie had a total accumulated exposure of 53.66 e/å 2 fractionated in 50 frames of 200 ms exposure. data collection statistics are summarized in s1 table. for data processing, whole frames in each movie were corrected for beam-induced motion and dose compensation using motioncor2 [37] and~1,400 best images were manually selected (we manually discarded micrographs with only carbon field of view or thick ice after motion correction as well as micrographs with defocus parameter higher than 4.5 μm after ctf estimation). the final image was bin-averaged to lead to a pixel size of 1.02 å. the parameters of the microscope contrast transfer function were estimated for each micrograph using gctf [38] . particles were automatically picked and extracted using relion [39] with a box size of 320 pixels. initially,~802,000 particles were subjected to 2d alignment and clustering using relion, and the best classes were selected for an additional 2d alignment.~5,000 best particles were applied for creating the initial 3d model using relion.~170,000 particles selected from 2d alignment were then subjected to 3d classification and the best class with 100,000 particles were subjected to 3d refinement to generate the final density map. the final density map was sharpened with modulation transfer function of k2 operated at 300kv using relion post-processing. reported resolutions were based on the gold-standard fourier shell correlation (fsc) = 0.143 criterion, and fourier shell correction curves were corrected for the effects of soft masking by high-resolution noise substitution [40] . data processing statistics are summarized in s1 table. the initial model of ibv spike ectodomain was obtained by fitting the seven parts (s1-ntd, s1-ctd, two parts of sd1, two parts of sd2, and s2) of the porcine delta coronavirus spike structure (pdb id: 6b7n) individually into the cryo-em density map of ibv spike using ucsf chimera [41] and coot [42] . manual model rebuilding was carried out using coot based on the well-defined continuous density of the main chain; the side chain assignments were guided by the densities of n-linked glycans and bulky amino acid residues. the structural model of the ibv spike in the pre-fusion state was refined using phenix [43] with geometry restrains and three-fold noncrystallographic symmetry constraints. refinement and model rebuilding in coot were carried out iteratively until there were no further improvements in geometry parameters and model-map correlation coefficient. the quality of the final model was analyzed with molprobity [44] and emringer [45] . the validation statistics of the structural models are summarized in s1 table. ibv pseudovirus entry assay ibv pseudovirus entry assay was carried out as previously described [46] . briefly, full-length ibv spike gene was inserted into pcdna3.1 (+) plasmid. retroviruses pseudotyped with ibv spike and expressing a luciferase reporter gene were prepared through co-transfecting hek293t cells (source: american type culture collection) with a plasmid carrying env-defective, luciferaseexpressing hiv-1 genome (pnl4-3.luc.re) and the plasmid encoding ibv spike. the produced ibv pseudoviruses were harvested 72 hours post transfection, and then used to enter df-1 cells (source: american type culture collection) and hek293t cells. after incubation for 5 hours at 37˚c, the medium was changed and cells were incubated for an additional 60 hours. cells were then washed with pbs and lysed. aliquots of cell lysates were transferred to optiplate-96 (perkinelmer), followed by addition of luciferase substrate. relative light units (rlus) were measured using enspire plate reader (perkinelmer). all the measurements were carried out in quadruplicates. recombinant ibv s1-ctd was assayed for its cell-binding capability using flow cytometry as previously described [13] . briefly, hek293t and df-1 cells were incubated with recombinant ibv s1-ctd containing a c-terminal his 6 tag (40 μg/ml) at room temperature for 30 minutes, followed by incubation with phycoerythrin (pe)-labeled anti-his 6 antibody for 30 minutes. the cells were then analyzed for the binding of ibv s1-ctd using flow cytometry. the total surface area and buried surface area of coronavirus s1-ctds were calculated using the pisa server at the european bioinformatics institute (http://www.ebi.ac.uk/pdbe/prot_ int/pistart.html) [47] . specifically, for each trimeric spike protein, a pdb file containing all of the six s1 domains (including three copies of s1-ctds and three copies of s1-ntds) was submitted to the pisa server, and the total surface area and buried surface area for each s1-ctd were calculated. for the spike proteins used for the above analysis, all their s1-ctds were in the "lysing down" state. the structures of mers-cov and hku1 spikes were not included in the above analysis because the former contain at least one s1-ctd in the "standing up" state and the latter contains long stretches of missing residues in its s1 domains, both of which would interfere with the above analysis. we constructed the ibv spike ectodomain (from ibv strain m41) in the pre-fusion state by replacing its transmembrane anchor and intracellular domain with a c-terminal gcn4 trimerization tag, followed by a his 6 tag ( fig 1a) . we expressed the protein in insect cells and purified the protein to homogeneity. we collected cryo-em data on ibv spike ectodomain, calculated a density map at 3.93å resolution (fig 1b; s1 fig) , built an atomic model of the structure and refined it (fig 1c and 1d) . the final structural model contains all of the residues from 21 to 1022 (except residues 702-710) as well as glycans n-linked to 20 sites. data collection and model statistics are shown in s1 table. the overall structure of ibv spike ectodomain resembles the pre-fusion structures of coronavirus spikes from the α-, β-, and δ-genera [12] [13] [14] [18] [19] [20] . it has a clove-like shape, with three s1 heads forming a crown-like structure and sitting on top of a trimeric s2 stalk. each overall structure of ibv spike ectodomain in the pre-fusion conformation. (a) schematic drawing of ibv spike ectodomain. s1: receptor-binding subunit. s2: membrane-fusion subunit. gcn4-his 6 : gcn4 trimerization tag followed by his 6 tag. s1-ntd: n-terminal domain of s1. s1-ctd: c-terminal domain of s1. ch: central helix. fp: fusion peptide. hr1 and hr2: heptad repeats 1 and 2. residues in shaded regions (n-terminus, hr2, gcn4 tag, and his 6 tag) were not included in the structural model. question mark indicates that the exact location of fp is uncertain; the range of fp used for making figures is consistent with a previous structural study on β-genus mouse hepatitis coronavirus spike [18] . monomeric subunit of s1 contains two major domains, s1-ntd and s1-ctd, and two subdomains, sd1 and sd2 (fig 2a and 2b ). the s1-ctds from three different subunits sit on the top and center of the spike trimer, whereas the three s1-ntds are located on the lower and sd1' and sd1": two parts of sd1. sd2' and sd2": two parts of sd2. (b) structure of monomeric s1. s1-ntd is colored in cyan. s1-ctd is colored in green. sd1 is colored in magenta. sd2 is colored in orange. ã indicates putative sugar-binding site. partial ceiling on top of the s1-ntd core is labeled. putative receptor-binding motif loops (rbms) in s1-ctd are also labeled. (c) structure of trimeric s1. three s1 subunits are colored differently. (d) structure of monomeric s2. the structural elements are colored in the same way as in fig 1a. (e) structure of trimeric s2. dotted line indicates residues 702-710 that are missing in the structural model. the structural elements of subunit a are colored in the same way as in fig 1d. subunits b and c are colored in light purple and light pink, respectively. all structures are viewed from the side. https://doi.org/10.1371/journal.ppat.1007009.g002 structure, function, and evolution of ibv spike protein outer side to s1-ctds (fig 2c) . sd1 and sd2 connect s1 to s2. the interface of trimeric s2 contains three central helices; each subunit of s2 contains one (fig 2d and 2e ). each subunit of s2 also contains two heptad repeat regions, hr1 and hr2, and a fusion peptide (fp) ( fig 2d and 2e ). in the post-fusion structure of trimeric s2, three copies of hr1 and three copies of hr2 would refold into a six-helix bundle structure, and fp would insert into the target membrane [29] [30] [31] [32] [33] [34] [35] . as in the structures of other coronavirus spikes, the hr2 region (residues 1022-1076) in the pre-fusion ibv spike is disordered (figs 1a and 2d) . the exact residue range of coronavirus fp remains unknown, although biochemical studies have identified a region in coronavirus s2 that associates with membranes and likely corresponds to fp ( fig 2d) [48, 49] . in the following sections of this paper, we will compare the structures and functions of ibv spike to those of the spikes from the other three genera, and discuss the evolution of coronavirus spikes. ibv s1-ntd takes the same galectin fold as the s1-ntds from the other three coronavirus genera, but it contains unique structural features (fig 3a-3d ). its core structure is a twelvestranded β-sandwich, which consists of two six-stranded antiparallel β-sheet layers stacked together through hydrophobic interactions (figs 2b and 3c). the topology of the β-sandwich core is identical to that of human galectins (s2 fig). underneath the core structure is another β-sheet and an α-helix, which are also present in the s1-ntds from the other three coronavirus genera. above the core structure are some loops that form a partial ceiling-like structure. this structure is not present in human galectins or s1-ntds from α-or δ-genus, but becomes a more extensive ceiling-like structure in β-coronavirus s1-ntds (fig 3a, 3b and 3d ). based on the structure and function of β-coronavirus s1-ntds, we previously predicted that s1-ntds from all of the genera have a galectin fold, and further correlated the galectin fold to their functions as viral lectins [15] . recent structural studies, including the current one, have confirmed our previous structural predictions (s2 fig) . these studies also have unexpectedly revealed that the presence and extent of the ceiling-like structure on top of the core structure are unique structural features for s1-ntds from different genera. it has been known that ibv spike binds sugar [50] . a recent study further confirmed that the sugar-binding domain in ibv spike is its s1-ntd [51] . to date no structural information is available for the complexes of coronavirus s1-ntds and their sugar ligand. mutagenesis study showed that in the s1-ntd from β-genus bovine coronavirus (bcov), the sugar-binding site is located in the pocket formed between the core structure and the ceiling [24] . in the structure of host galectins, despite no ceiling, the sugar-binding site is located in the same place [52] . based on the structural similarity between the s1-ntds from different coronavirus genera, the sugar-binding site in ibv s1-ntd might also be located in the pocket formed between the core structure and the partial ceiling (figs 2b and 3c) . the structure determination of ibv s1-ntd provides insight into the structural and functional evolution of coronavirus s1-ntds. we hypothesized that coronavirus s1-ntds originated from host galectins based on the structural similarities between coronavirus s1-ntds and host galectins [23, 24] . as host proteins, galectins are not recognized by the host immune system. in comparison, coronavirus s1-ntds are under the host immune pressure to evolve. the gradual structural evolution of the ceiling on top of the core structure may have led to three functional outcomes. first, the ceiling could provide better protection to the sugar-binding site from host immune surveillance, which appears to be a common feature of viral lectins [53] . this hypothesis on protected sugar-binding sites in viral lectins is also consistent with the "canyon hypothesis" which states that receptor-binding sites on viral surfaces are hidden from the host immune surveillance [54] . second, the ceiling is also involved in the quaternary packing of s1, which will be discussed later in this paper. third, in the structure of s1-ntd from βgenus mouse hepatitis coronavirus (mhv), the outer surface of the ceiling has further evolved the capability to bind a protein receptor ceacam1, while the presumed sugar-binding pocket has lost its capability to bind sugar [23] . hence, the structural development of the ceiling is a possible indicator for the evolution of s1-ntds in the direction of α-and δ-genera, then the γgenus, and finally the β-genus. furthermore, we performed quantitative structural comparisons of s1-ntds from different genera by calculating the z-score and r.m.s.d. between each pair of the proteins (fig 3e) . the result confirmed that s1-ntds are relatively conserved among different genera, as reflected by the generally high z-scores and low r.m.s.d. in terms of structural distances to α-coronavirus s1-ntds, δ-coronavirus s1-ntds are the closest, β. although each subunit of nl63 s1 contains two copies of s1-ntds (i.e., s1-ntd1 and s1-ntd2), s1-ntd2 was used in structural comparisons with the s1-ntds from the other genera because it occupies the same location as the s1-ntds from the other genera in quaternary structures of the spikes (see fig 5a) . (b) structure of s1-ntd from δ-genus porcine delta coronavirus (pdcov) (pdb id: 6b7n). (c) structure of s1-ntd from γ-genus ibv. (d) structure of s1-ntd from β-genus sars coronavirus (pdb id: 5x58). ã indicates sugar-binding site or putative sugar-binding site in sugarbinding s1-ntds from each genus. core structure, partial ceiling, and extensive ceiling are labeled. arrows from panels (a) to (d) indicate evolutionary direction. (e) quantitative structural comparisons among s1-ntds from different genera using software dali [58] . both z-score and r.m.s.d. were calculated for each pair of the proteins. pdb ids for nl63, pdcov and sars s1-ntds are the same as in panels (a)-(d). pdb ids for mouse hepatitis coronavirus (mhv) and mers coronavirus are 3jcl and 5x5f, respectively. ceacam1b (pdb id: 5vst), whose β-sandwich fold is topologically different from that of coronavirus s1-ntds [59] , was used as a negative control. n.d.: no detectable structural similarity. https://doi.org/10.1371/journal.ppat.1007009.g003 coronavirus s1-ntds are the farthest, and γ-coronavirus s1-ntds fall in the middle. moreover, the structural similarity between α-and δ-coronavirus s1-ntds is slightly higher than that between two β-coronavirus s1-ntds, suggesting that s1-ntds within β-genus have diverged slightly more than those between the α-and δ-genera. taken together, s1-ntds from the four genera form an evolutionary spectrum in the order of α-, δ-, γ-, and β-genus, with α-coronavirus s1-ntds probably being the most ancestral (fig 3a-3d ). the structure of ibv s1-ctd is significantly different from the structures of s1-ctds from the other genera (fig 4a-4d; s3 fig) . its core structure is a β-sandwich containing two β-sheet layers: one is five-stranded and antiparallel, and the other is two-stranded and parallel (figs 2b and 4c; s3 fig) . the interactions between the two β-sheet layers are present but minimal. in contrast, the core structures of α-coronavirus and δ-coronavirus s1-ntds are both standard β-sandwich folds with extensive interactions between the two β-sheet layers: one is threestranded and antiparallel, and the other is three-stranded and mixed (fig 4a and 4b; s3 fig) . even more drastically different are the β-coronavirus s1-ctds, which contain only one fivestranded antiparallel β-sheet layer with the other layer turning into an α-helix and a coil ( fig 4d; s3 fig) . despite these dramatic structural differences, the s1-ctds from all genera share the same structural topology (i.e., connectivity of secondary structural elements) (s3 fig) . moreover, the additional structural motifs on the edge of the core structure are also diverse among different genera (s3 fig). in the ibv s1-ctd, two extended loops on the edge of the core structure function as putative receptor-binding motifs (rbms) by potentially binding to an unknown receptor (see below) (figs 2b and 4c) . in both the α-and δ-coronavirus s1-ctds, three short discontinuous loops are located in the same spatial region; they function as the rbms in α-coronavirus s1-ctds and putative rbms in δ-coronavirus s1-ctds (fig 4a and 4b ). in β-coronavirus s1-ctds, a long continuous subdomain is located in this spatial region and functions as the lone rbm (fig 4d) . structural variations of the rbms in the s1-ctds within each of the genera further lead to different receptor specificities [7] . in sum, ibv s1-ctd contains a weakened β-sandwich core structure and two extended rbm loops; the former structural feature falls between the β-sandwich cores of α-and δ-genera and the βsheet core of β-genus, whereas the latter structural feature falls between the three short discontinuous rbm loops of α-and δ-genera and a single long continuous rbm subdomain of βgenus. to investigate the function of ibv s1-ctd, we performed two assays to detect possible interactions between ibv s1-ctd and its potential receptor on the host cell surface. first, we carried out an ibv-spike-mediated pseudovirus entry assay in the presence of recombinant ibv s1-ctd (s4a fig). to this end, retroviruses pseudotyped with ibv spike (i.e., ibv pseudoviruses) were used to enter host cells. in the absence of recombinant ibv s1-ctd, ibv pseudoviruses entered df-1 cells (chicken fibroblast) efficiently, which was consistent with a previous report showing that df-1 cells are permissive to live ibv (strain m41) infections [55] . as a negative control, their entry into hek293t cells (human kidney) was inefficient. recombinant ibv s1-ctd reduced the efficiency of ibv pseudovirus entry into df-1 cells in a dose-dependent manner, likely because it competed with ibv pseudoviruses for an unknown receptor on the host cell surface. second, we examined the binding of recombinant ibv s1-ctd to the host cell surface using a flow cytometry assay (s4b fig). to this end, recombinant ibv s1-ctd was incubated with df-1 cells, and subsequently cell-bound s1-ctd was detected using flow cytometry. recombinant ibv s1-ctd bound to the surface of df-1 cells efficiently, but not the surface of hek293t cells. taken together, ibv s1-ctd binds to a yetto-be-identified receptor on the surface of chicken cells and hence functions as a receptorbinding domain (rbd). thus, the s1-ntd and s1-ctd of ibv spike may both function as rbds. because coronavirus s1-ctds from the α-and β-genera all use the additional structural features on the edge of their core structure as their rbms, it is likely that the two extended loops in the same spatial region in ibv s1-ctd function as the rbms. coronavirus s1-ctds represent remarkable examples of divergent evolution of viral proteins. the core structures and the rbm regions of s1-ctds are both divergent among different genera (fig 4a-4d; s3 fig) . the core structures are β-sandwiches for α-and δ-coronavirus s1-ctds, weakened β-sandwiches for γ-coronavirus s1-ctds, and single β-sheet layer for βcoronavirus s1-ctds. the rbms are three short discontinuous loops for α-and δ-coronavirus s1-ctds, two reinforced loops for γ-coronavirus s1-ctds, and a single continuous subdomain for β-coronavirus s1-ctds. hence the s1-ctds form an evolutionary spectrum, with αand δ-coronavirus s1-ctds on one end, β-coronavirus s1-ctds on the other end, and γcoronavirus s1-ctds in between. we performed quantitative structural comparisons of s1-ctds from all four genera (fig 4e) . the result confirmed that s1-ctds are relatively poorly conserved among different genera, as reflected by the generally low z-scores and high r.m.s.d. in terms of structural distances to α-coronavirus s1-ctds, δ-coronavirus s1-ctds are the closest, β-coronavirus s1-ctds are the farthest, and γ-coronavirus s1-ctds fall in the middle. the functional outcomes of the core structure evolution are not clear, but the evolution of the rbms may have led to the following two functional outcomes. first, the diversity of the rbms from three short loops to two extended loops and then to a long subdomain may allow coronaviruses to explore a wider variety of receptors. second, the reinforced rbm regions in both β-and γ-coronavirus s1-ntds facilitate quaternary packing of s1, which will be discussed later in this paper. taken together, the s1-ctds from different genera form an evolutionary spectrum in the order of α-, δ-, γ-, and β-genus, although the evolutionary direction could go either way (fig 4a-4d ). curiously, coronavirus s1 from different genera take two types of quaternary packing modes (fig 5a-5d ) [12] [13] [14] [18] [19] [20] . ibv s1 takes a cross-subunit quaternary packing mode where the s1-ntd and s1-ctd from different subunits pack together (fig 5c) . specifically, in the trimeric ibv spike, one s1-ctd packs against two s1-ctds from the other subunits as well as one s1-ntd from another subunit. the putative rbms of ibv s1-ctd and the partial ceiling structure, function, and evolution of ibv spike protein of ibv s1-ntd are both involved in the cross-subunit packing. as a result, the putative rbms of ibv s1-ctd are partially concealed, disallowing their full access to the host receptor. hence ibv s1-ctd in the current structure was captured in a "lying down" state, and would need to "stand up" on the spike trimer for efficient receptor binding. this potential conformational change of ibv s1 can minimize the exposure of the putative rbms in its s1-ctd to the immune system, thereby functioning as a possible strategy for viral immune evasion. β-coronavirus s1 also takes the cross-subunit packing mode, with the rbm of its s1-ctd and the ceiling of its s1-ntd both involved in the cross-subunit packing (fig 5d) [18] [19] [20] . in contrast, α-and δ-coronavirus s1 both take an intra-subunit packing mode where the s1-ntd and s1-ctd from the same subunit pack together (fig 5a and 5b ) [12] [13] [14] . the rbms of α-and δ-coronavirus s1-ctds are involved in the intra-subunit packing. whether s1 packs in the intra-subunit or cross-subunit mode, the rbms of s1-ctds are concealed or partially concealed in their "lying down" state, and would need to switch to the "standing up" state for receptor binding. overall, β-and γ-coronavirus s1 both take the cross-subunit quaternary packing mode, whereas α-and δ-coronavirus s1 both take the intra-subunit quaternary packing mode. we examined whether the quaternary structures of coronavirus s1 can lead to functional differences of coronavirus spikes. first, in both β-and γ-coronavirus spikes, the rbms of their s1-ctds and the ceiling of their s1-ntds have evolved to facilitate the cross-subunit packing. these additional structural features further evolved to gain other functions: the rbms of s1-ctds recognize diverse protein receptors, whereas the ceiling of the s1-ntds either protects the sugar-binding site or recognizes a new protein receptor [7] . second, to investigate the structural restrain on s1-ctds that may hinder their potential conformational change, we calculated the total and buried surface areas of the s1-ctd on the spikes from different genera. the result did not reveal systematic difference between intra-subunit s1 packing and crosssubunit s1 packing in the buried surface area of s1-ctds. however, it is worth noting that the s1-ctd from β-genus sars-cov has the smallest buried surface area (in both absolute value and percentage) (s2 table) . the relative small buried surface area of sars-cov s1-ctd indicates less structural restraint on the s1-ctd from other parts of the spike s1, possibly allowing the s1-ctd to switch to the "standing up" and receptor-accessible conformation more easily. the "standing up" conformation of sars-cov s1-ctd may also weaken the structural restraint of s1 on s2 (discussed in more detail later), potentially allowing membrane fusion to proceed more easily [56] . indeed, frequent "standing up" of sars-cov s1-ctd has been observed [19] . overall, compared to the intra-subunit quaternary packing of α-and δ-coronavirus s1, the cross-subunit quaternary packing of β-and γ-coronavirus s1 may have allowed their s1 to evolve additional functions in receptor recognition; moreover, the s1-ctd from βgenus sars-cov spike has a relatively small buried surface area, which may be responsible for its dynamic receptor-binding conformation. the structure and function of ibv s2 are highly similar to those of s2 from the other coronavirus genera. in the pre-fusion structure of ibv s2, hr2 is disordered, whereas hr1 and fp each consist of several α-helices and connecting loops (the exact residue range of fp is not clear) (fig 2d) . in the post-fusion structure, hr1 would refold into a long α-helix, hr2 would refold into a mixture of α-helices and coils, three copies of hr1 and hr2 would pack into a six-helix bundle structure, and fp would also refold and insert into the target membrane (s5a fig) [6, 29] . ibv s2 is locked in the pre-fusion state because of the structural restraint from s1. specifically, because of the cross-subunit quaternary packing of trimeric ibv s1, hr1 and fp of ibv s2 are structurally restrained by two s1-ctds from the other subunits and sd1 from another subunit, respectively (s5b fig). the structural restraints from s1 on s2 can be weakened by the standing up of s1-ctds (which allows receptor binding) and can be lifted completely upon proteolysis removal of s1. the packing between s1 and s2 in ibv spike is the same as those in β-coronavirus spikes [18] [19] [20] . however, in α-and δ-coronavirus spikes, the packing between s1 and s2 becomes different due to the intra-subunit quaternary packing of their trimeric s1: hr1 and fp are restrained by one s1-ctd and one sd1 from another subunit, respectively (s5c fig) [12 -14] . other than the differences in s1/s2 packing, the structural and functional similarities of coronavirus s2 from different genera suggest evolutionary conservation of coronavirus s2. the fast evolutionary rates of viruses, particularly rna viruses, make it difficult to trace their evolutionary history [1] [2] [3] . envelope-anchored coronavirus spike proteins guide viral entry into cells; they are the fastest evolving coronavirus proteins due to viral needs to engage diverse host receptors, maximize membrane-fusion efficiency, and evade host immune surveillance [7] [8] [9] [10] [11] [12] [13] [14] . coronavirus spikes from four different genera are divergent, and their evolutionary relationships pose a major puzzle in the virology field [6] . because viral proteins need to function under certain structural and functional constraints, evolutionary information of viral proteins can be more reliably found in their tertiary structures and related functions than in their primary structures [16, 17] . although extensive structural studies including both x-ray crystallography and cryo-em have been done on coronavirus spikes, a critical piece that was still missing is the structure of γ-coronavirus spikes, preventing a clear understanding of the evolutionary relationships among coronavirus spikes [12] [13] [14] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . in this study, we determined the cryo-em structure of ibv spike ectodomain, the first such structure from the γ-genus, which bridges the divergent structures of coronavirus spikes into an evolutionary spectrum and provides insight into the evolutionary relationships among coronavirus spikes. our study compares the structures and functions of coronavirus spikes from the four genera, and illustrates the structural and functional evolution of these proteins. first, coronavirus s1-ntds from all genera share the same structural fold and possibly evolutionary origins with host galectins. from α-and δ-genera to γ genus and then to β genus, the s1-ntds have evolved from simple galectin-fold core structure with an exposed sugar-binding site, to having a partial ceiling on top of the core structure, and to having an extensive ceiling to protect the sugar-binding site from host immune surveillance (the outer surface of the ceiling in one βcoronavirus can even bind to a novel protein receptor). the partial ceiling in γ-coronavirus s1-ntds and the extensive ceiling in β-coronavirus s1-ntds are also involved in the quaternary packing of s1. second, coronavirus s1-ctds from different genera are very diverse, but still form an evolutionary spectrum with α-and β-coronavirus s1-ctds at two ends and δand γ-coronavirus s1-ctds in the middle. the core structures of s1-ctds have diverged from β-sandwich to weakened β-sandwich and then to β-sheet, whereas the rbms have diverged from short loops to extended loops and then to a long subdomain. the functional significance of the core structure evolution is not clear, but the rbm evolution may allow the viruses to expand receptor recognition and also contributes to the quaternary packing of s1. third, from α-and δ-genera to β-and γ-genera, the quaternary packing of s1 has diverged from simple intra-subunit packing to more complex cross-subunit packing. the cross-subunit quaternary packing of β-and γ-coronavirus s1 may have allowed their s1 to evolve additional functions in receptor recognition. moreover, the relatively small buried surface area of the s1-ctd from β-genus sars-cov may allow the s1-ctd to be more dynamic for receptor binding. finally, the s2 from all four genera are structurally and functionally conserved, although there are some differences in their s1/s2 packing. quantitative structural comparisons also demonstrate that in terms of structural distances to α-coronavirus s1, δ-coronavirus s1 is the closest, β-coronavirus s1 is the farthest, and γ-coronavirus s1 is the intermediate. we also calculated the phylogenetic tree using the amino acid sequences of 29 coronavirus spikes from different genera, and the result showed that in terms of amino acid sequence distances to αcoronavirus spikes, δ-coronavirus spike is the closest, β-coronavirus spike is the farthest, and γcoronavirus spike is the intermediate (s6 fig). taken together, coronavirus spikes from different genera form an evolutionary spectrum, with α-coronavirus spikes on one end, followed by δ-coronavirus spikes and γ-coronavirus spikes, and β-coronavirus spikes on the other end. because of their fast evolutionary rates, viruses are perfect model systems for studying evolution. our study has demonstrated that despite structural divergence among coronavirus spikes, particularly in their s1 where low or little structural similarities can be detected, we can still trace the evolutionary relationships among these viral proteins through detailed analyses of their structures and functions. our study also reveals that coronavirus spikes have evolved to remarkable diversity to expand their receptor recognition, facilitate membrane fusion, and evade host immune surveillance, while conserving basic membrane-fusion mechanisms. the evolutionary approaches that coronaviruses take and the evolutionary edges that they gain are good examples of viral evolution and deepen our understanding of evolution in general. supporting information s1 table. buried surface area of coronavirus spike s1-ctds. (b) structural topology of the core structures of α-, γ-, and δ-coronavirus s1-ntds. (c) structural topology of the core structures of β-coronavirus s1-ntd. pdb ids of coronavirus s1-ntds are the same as in fig 3. β-strands are shown as arrows. the two layers of the core structures are colored in green and magenta, respectively. n ã and c ã indicate n-and c-terminus, respectively. numbering of the secondary structures only counts secondary structural elements in the core region. (tif) s3 fig. structural topology of coronavirus s1-ctds. (a) structural topology of the core structures of α-and δ-coronavirus s1-ctds. (b) structural topology of the core structure of γcoronavirus s1-ctd. (c) structural topology of the core structure of β-coronavirus s1-ctd. pdb ids of coronavirus s1-ctds are the same as in fig 4. β-strands are shown as arrows. αhelices are shown as cylinders. coil is shown as a curled line. the two layers of the core structures are colored in green and magenta, respectively. receptor-binding motifs (rbms) are colored in red and the relative lengths of the rbms are labeled in parentheses. in both γ-and δ-coronavirus s1-ctds, the rbms have not been experimentally identified and thus their functions are putative. n ã and c ã indicate n-and c-terminus, respectively. numbering of the secondary structures only counts secondary structural elements in the core region. (tif) s4 fig. function of ibv s1-ctd. (a) ibv pseudovirus entry into cells in the presence of recombinant ibv s1-ctd. entry efficiency was characterized by luciferase activity accompanying entry. rlu: relative light units. mock: no ibv pseudoviruses were added. entry: ibv pseudovirus entry in the absence of any recombinant ibv s1-ctd. (b) flow cytometry assay for the binding of recombinant ibv s1-ctd to the surface of cells. cell-bound ibv s1-ctd was detected using antibodies recognizing its c-terminal his 6 tag. cells only or cells plus antibody without ibv s1-ctd were used as negative controls. statistic analyses were performed using two-tailed t-test. arrow in the pre-fusion structure indicates the direction in which hr1 would need to extend to reach the post-fusion conformation. (b) packing between s1 and s2 in ibv spike. trimeric s1 and one monomeric s2 are shown. structural elements in monomeric s2 are colored in the same way as in panel (a). three s1 subunits are colored differently. (c) packing between s1 and s2 in porcine delta coronavirus spike (pdb id: 6b7n). trimeric s1 and one monomeric s2 are shown. s1 and s2 are colored in the same way as in panel (b). all structures are viewed from the side. the phylogenetic tree was constructed using the neighbor-joining method as previously described [57] . horizontal scale bars represent average numbers of substitutions per amino acid position. the genbank accession numbers of the selected spikes are marked before each virus name. 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interferon response by infectious bronchitis coronavirus immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus touring protein fold space with dali/fssp structural and molecular evidence suggesting coronavirus-driven evolution of mouse receptor initial cryo-em images were collected using fei tecnai tems maintained at the characterization facility of the university of minnesota. final cryo-em data were collected at the john m. cowley center for high resolution electron microscopy of arizona state university. we thank dr. dewight williams for helping us prepare grids and collect data. initial image analysis and computation work were performed using the workstations at the basic sciences computing laboratory of the university of minnesota supercomputing institute. key: cord-352511-gkm7i62s authors: yamada, yoshiyuki; liu, xiao bo; fang, shou guo; tay, felicia p. l.; liu, ding xiang title: acquisition of cell–cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells date: 2009-07-02 journal: plos one doi: 10.1371/journal.pone.0006130 sha: doc_id: 352511 cord_uid: gkm7i62s coronavirus host and cell specificities are determined by specific interactions between the viral spike (s) protein and host cell receptor(s). avian coronavirus infectious bronchitis (ibv) has been adapted to embryonated chicken eggs, primary chicken kidney (ck) cells, monkey kidney cell line vero, and other human and animal cells. here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the s protein determines the infectivity of ibv in cultured cells. expression of s protein derived from veroand ck-adapted strains showed efficient induction of membrane fusion. however, expression of s protein cloned from the third passage of ibv in chicken embryo (ep3) did not show apparent syncytia formation. by construction of chimeric s constructs and site-directed mutagenesis, a point mutation (l857-f) at amino acid position 857 in the heptad repeat 1 region of s protein was shown to be responsible for its acquisition of the cell–cell fusion activity. furthermore, a g405-d point mutation in the s1 domain, which was acquired during further propagation of vero-adapted ibv in vero cells, could enhance the cell–cell fusion activity of the protein. re-introduction of l857 back to the s gene of vero-adapted ibv allowed recovery of variants that contain the introduced l857. however, compensatory mutations in s1 and some distant regions of s2 were required for restoration of the cell–cell fusion activity of s protein carrying l857 and for the infectivity of the recovered variants in cultured cells. this study demonstrates that acquisition of the cell–cell fusion activity in s protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins. interspecies adaptation, replication and transmission in cells are essential steps for an animal virus to emerge successfully in a human population. virus-cell and cell-cell membrane fusion, mediated by fusion proteins associated with viral envelope, is crucial for the entry of enveloped viruses into cells and for rapid spread of infection to the neighboring cells. this membrane fusion process may, therefore, be a limiting point for efficient adaptation and infection of an animal virus in cells from a different host species. in this study, we report that acquisition of the cell-cell fusion activity by point mutations in the spike (s) protein of avian coronavirus infectious bronchitis virus (ibv) plays a critical role in adaptation and/or selection of a variant that infects cultured cells. coronavirus is a large family of enveloped, positive-stranded rna viruses that cause respiratory and intestinal infections in avian and mammalian species [1] . ibv, the prototype member of coronavirus, causes highly contagious diseases in chicken and is a constant threat to the poultry industry. coronavirus was traditionally considered to have narrow host specificities [2] . however, the outbreaks of severe acute respiratory syndrome (sars), a serious zoonotic transmission event caused by a novel coronavirus, demonstrate that a certain coronvirus species may exhibit wider host specificities and suggests the possibility of crossspecies transmission of animal coronaviruses to human [3, 4] . cross-species transmission was also observed in coronavirus transmissible gastroenteritis virus (tgev) and human coronavirus oc43 [5] [6] [7] . these events highlight the importance of understanding the mechanisms of interspecies adaptation and transmission of coronavirus. the beaudette strain of ibv was previously adapted to embryonated chicken eggs. this embryo-adapted ibv strain was subsequently adapted to cultured cells originated from chicken and monkey. for example, the virus was adapted by serial passages to primary chicken kidney (ck) cells [8, 9] and the african green monkey kidney cell line vero cell [8] [9] [10] [11] [12] [13] . furthermore, the veroadapted ibv is able to infect cultured human and animal cell lines [14, 15] . in a previous report, a total of 49 amino acid substitutions was found during adaptation of ibv from chicken embryo (ep3) to vero cells (p65) [10] [11] [12] . among them, 26 amino acid substitutions are in the s protein [10] . in this study, expression of s protein cloned from ibv strains ep3, ck, passage 7 (p7) and p65 of vero-adapted ibv showed induction of cell-cell fusion by s(ck), s(p7) and s(p65) constructs. however, no formation of syncytial cells was observed in cells expressing s(ep3). construction of chimeric s constructs and site-directed mutagenesis studies identified a leucine to phenylalanine substitution at the amino acid position 857 in the heptad repeat 1 region (l857-f) that confers the non-fusogenic s protein to fusogenic. re-introduction of the f857-l mutation back to the genome of vero-adapted ibv (p65) showed rescue of virus containing the f857-l mutation. however, compensatory mutations occurred in the s1 region that could rescue the cell-cell fusion activity of s constructs carrying the f857-l mutation. cells were maintained in dmem supplemented with 10% newborn calf serum. the vero-adapted ibv and recombinant vaccinia/t7 virus was propagated and titrated on vero cells. virus stocks were kept at 280uc until use. cell monolayers grown on 4 well slide chambers were infected with vaccinia/t7 virus for 1 hour followed by transfection of indicated plasmids using the effectene transfection kit (qiagen). at 12 hours post-transfection, cells were washed with phosphate buffered saline (pbs) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in pbs for 15 minutes, and permeabilized with 0.2% triton x-100. immunofluorescent staining was performed by incubation of cells with rabbit anti-ibv s polyclonal antibodies and subsequently with fitcconjugated anti-rabbit igg. cells were examined by fluorescent microscopy and digital images were collected. protein samples were prepared from cells harvested at 12 hours post-transfection, separated by sds-page and transferred to pvdf membranes. the membranes were incubated with rabbit anti-ibv s polyclonal antibodies or mouse anti-b-tubulin monoclonal antibody (sigma aldrich), and subsequently with hrp-conjugated anti-rabbit or -mouse igg (dako). polypeptides were detected using the enhanced chemiluminescence (ecl) detection reagents (amersham). vero cells were transfected as described above, and harvested at 12 hours post transfection. cells were washed once with pbs, resuspended in blocking buffer containing 20% fbs and 1% bsa in pbs, and incubated on ice for 30 minutes. subsequently, cells were incubated with 0.1% saponin in facs washing buffer containing 2.5% fbs and 0.05% sodium azide in pbs for 10 minutes at room temperature when required. immunofluorescent staining was carried out with 1:100 diluted rabbit anti-ibv s polyclonal antibodies, and 1:20 diluted fitc-conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. viral rna was extracted from the culture supernatants or infected cells using the rneasy mini kit (qiagen) according to the manufacturer's instructions. rt-pcr was performed using the expand reverse transcription and high fidelity pcr kits (roche). the pcr products were cloned into pcrh-xl-topoh vector (invitrogen) and sequenced by automated sequencing. construction of the full-length ibv cdna clones from p65 of vero-adapted ibv was previously reported [16, 17] . the f857-l point mutation was introduced into the corresponding fragment using quickchange site-directed mutagenesis kit (stratagene), and subsequently ligated into the full-length cdna clone. the full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion) according to the manufacturer's instructions with certain modifications. briefly, 30 ml of transcription reaction with a 1:1 ratio of gtp to cap analog were sequentially incubated at 40.5uc for 25 minutes, 37.5uc for 50 minutes; 40.5uc for 25 minutes, and 37.5uc for 20 minutes. the transcripts were extracted with phenol/chloroform. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and one electrical pulse at 450 v, 50 mf was given using a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 mm dish or a six-well plate and further cultured in mem without fbs. the s genes from different passages of the vero-adapted ibv strain and ck-adapted ibv were amplified and cloned into pkt0 vector [18] . chimeric s constructs were made by overlapping pcr [19] . point mutations were introduced by site-directed mutagenesis using the quikchange tm kit (stratagene). all constructs were confirmed by automated nucleotide sequencing. cell-cell fusion activity of s proteins cloned from ep3 and ck-adapted beaudette strain of ibv acquisition of the cell-cell fusion activity is essential for selection and adaptation of coronavirus ibv from chicken embryo to cultured cells [10] . sequence comparison of two s protein constructs, s(ep3) and s(ck), cloned from ep3 and ck-adapted ibv strains, respectively, showed amino acid substitutions at 31 positions (fig. 1a) . the cell-cell fusion activity of these two s constructs was analyzed by transfection into vero cells using the vaccinia/t7 recombinant virus system. western blot analysis showed the presence of major forms of s protein, including the 180-kda glycosylated (s*) and 130-kda unglycosylated full-length s (s) and the cleaved s1 and s2 species (s1/s2) in cells expressing the two constructs (fig. 2a, lanes 2 and 3) . it was noted that the expression level of s(ck) was higher than s(ep3) (fig. 2a) . as a negative control, cells transfected with ibv n protein were included, and the expressed n protein was detected by western blot with anti-n antibodies (fig. 2a, lane 1) . immunofluorescent staining of vero cells expressing s(ck) clearly showed syncytia formation at 12 hours post-transfection (fig. 2b , panel s(ck)). however, in vero cells expressing s(ep3), no obvious syncytia was observed (fig. 2b , panel s(ep3)). in the negative control cells, no fusion of the transfected cells was detected (fig. 2b, panel n) . to investigate the possibility that intrinsic differences in cell surface translocation of the two s constructs may affect their cellcell fusion activity, cell surface expression of the two proteins was analysed by flow cytometry after immunofluorescent staining with anti-s antiserum. as shown in fig. 2c acquisition of the cell-cell fusion activity by mutation of a conserved leucine residue to phenylalanine (l857-f) in the heptad repeat 1 region of s protein to map the amino acid mutation(s) responsible for acquisition of the cell-cell fusion activity of s(ck), three chimeric constructs were first made. construct ep3-ck(1) was made by replacing the c-terminal 412 amino acid region of s(ep3) with the corresponding region from s(ck), ep3-ck(2) was made by replacing the cterminal 280 amino acid region of s(ep3) with the corresponding region from s(ck), and ck-ep3 was made by replacing the nterminal 882 amino acid region of s(ep3) with the corresponding region from s(ck) (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the glycosylated and unglycosylated forms of the full-length s protein (fig. 3b , lanes [3] [4] [5] . immunofluorescent staining showed cell-cell fusion and syncytia formation in cells expressing both ep3-ck(1) and ck-ep3 (fig. 3c , panels ep3-ck(1) and ck-ep3), but not ep3-ck(2) (fig. 3c , panel ep3-ck (2)). the relative cell-cell fusion activities of these s constructs were semiquantitatively defined by comparing the average size of syncytia induced by different s constructs with the average size (considered as 1) of cells expressing s(ep3), and are listed in the order from high to low as follows: ck-ep3.ck = ep3-ck(1)&ep3 = ep3-ck(2) (.indicates the relative activity is within 1 fold higher, and &indicates more than 1 fold higher). these results demonstrate that the region between amino acids 750 and 882 may determine the fusogenic difference between s(ep3) and s(ck). examination of this region showed two amino acid substitutions from s(ep3) to s(ck), i.e. n826 to s and l857 to f (fig. 1b) . to determine which amino acid substitution dictates the fusogenic change, three mutant constructs were made. constructs ck3(s826-n) and ck(f857-l) were made by mutation of the s826 and f857 residues in s(ck) to n and l, respectively (fig. 3a) . construct ep3(l857-f) was made by mutation of the l857 residue in s(ep3) to f (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the fulllength forms (fig. 3b , lanes 6-8). immunofluorescent staining showed formation of syncytia in cells expressing ck(s826-n) (fig. 3c , panel ck(s826-n), suggesting that mutation of s836 to n did not affect the cell-cell activity of s(ck). cell-cell fusion and syncytia formation were also observed in cells expressing ep3(l857-f) but not ck(f857-l) (fig. 3c , panels ep3(l857-f) and ck(f857-l)), demonstrating that the l857-f mutation introduced into s(ep3) renders the protein fusogenic in cultured cells. on the other hand, mutation of the f857 residue to l totally abolishes the fusion activity of s(ck) (fig. 3c , panel ck(f867-l). the relative cell-cell fusion activities of these s constructs are ck(s826-n).ep3(l857-f)&ep3 = ck(f857-l). these results confirm that s(ck) gains the cell-cell fusion activity by l857-f mutation in the heptad repeat 1 region of the protein. the f857-l substitution was then introduced into the s constructs cloned from vero-adapted ibv (p7) and (p65), respectively, generating s(p7) and s(p65) (fig. 3a) . expression of these constructs showed cell-cell fusion and syncytia formation in cell expressing wild type s(p7) and s(p65), but not the mutant s proteins (fig. 3c , panel p7, p7(f857-l), p65 and p65(f857-l)). the expression levels of both f857-l mutants were lower than the wild type constructs, but no significant difference in the s1/s2 cleavage was observed (fig. 3b, lane 9-12 ). the relative cell-cell fusion activities of these s constructs are p65.p7&ep3 = p7(f857-l) = p65(f857-l). these data indicate that s protein acquires its cell-cell fusion activity by the l857-f mutation during adaptation to both ck and vero cells. further mutations of the l857 residue to other amino acids based on s(ep3) were made. as shown in fig. 3a , the l857 was mutated to y, s, e, i and k, respectively. expression of these mutant constructs showed that mutations of l857 to y and s exhibited similar effect on cell-cell fusion as the l857-f mutant. cell-cell fusion and syncytia formation were observed in cells expressing these two mutants (fig. 3c) . however, much less cell-cell fusion and smallersized syncytia were observed in cells expressing l857-e, l857-k and l857-i mutant constructs (fig. 3c) . the relative cell-cell fusion activities of these s constructs are ep3(l857-y).ep3(l857-s).ep3(l857-e).ep3(l857-i) = ep3(l857-k).ep3. introduction of the f857-l substitution back to the genome of vero-adapted ibv and analysis of its effect on viral infectivity in cultured cells the f857-l mutation was then introduced back to the genome of vero-adapted ibv by using an infectious clone system based on p65 [16, 17] to test its influence on viral recovery and infectivity. in vitro synthesized full-length transcripts derived from wild type (ribv) and mutant (fl) clones were introduced into vero cells by electroporation. at 3 days post-electroporation, syncytia formation the recombinant wild type and mutant viruses (p0) were recovered from the culture media at 3 and 6 days post-electroporation, respectively, and further propagated on vero cells for 5 passages. total rna was extracted from the culture media of cells infected with each passage of the mutant virus and rt-pcr was carried out to amplify the s gene. the rt-pcr products were cloned, 10 bacterial clones were randomly chosen from p0, and the complete nucleotide sequence of the s gene was determined to confirm if the recovered virus maintains the f857-l substitution. as shown in table 1, l857 was found in all 10 clones. however, only five clones had an identical sequence with the original mutant s gene (type fl), and additional mutations at other positions were found in the other five clones (table 1) . among them, two clones contain a t773-s substitution (flv1), one contains an i769-v substitution (flv2), and two contain q523-l and i769-v substitutions (flv3) ( table 1 ). these results demonstrate that the recovered fl mutant virus from p0 contains a mixed population of quasispecies. to investigate which quasispecies would become dominant in the subsequent passages, sequencing analysis of bacterial clones containing the pcr fragments from p1, p3 and p5 was performed. in the four clones chosen from p1, a homogenous s gene with both q523-l and i769-v (flv3) mutations was found (table 1) . subsequent sequencing of clones derived from p3 and p5 each showed that six out of 10 clones from p3 and two out of 10 clones from p5 are flv3 (table 1 ). the dominant clones contain an additional proline to serine substitution at amino acid position 327 (flv4) ( table 1) . the recovered viruses were then plaque-purified. compared to wild type ibv, ribv showed similar growth kinetics in vero cells (fig. 4b) , but formed slighlty smaller plaques (fig. 4a) with lower expression level of s protein (fig. 4c) . a total of 20 mutant viruses was plaque-purified from passages 3 and 5, and the s gene of all purified viruses was shown to share the same sequence as flv4 ( table 1 ). the flv4 mutant virus formed similar-sized plaques as ribv (fig. 4a) with slightly lower expression of s protein (fig. 4c) . interestingly, the mutant virus produced up to 10-fold higher titers of virus, compared to ribv (fig. 4b) . restoration of the cell-cell fusion activity of s(p65) protein carrying the f875-l mutation by compensatory mutations in the s1 region the cell-cell fusion activity of s proteins cloned from the mutant ibv construct fl and the four variants (flv1, flv2, flv3 and flv4) was analyzed by expression in vero cells. once again, expression of these constructs led to the detection of s1 and s2 species as well as the full-length forms (fig. 5a) . higher levels of s protein were detected in cells expressing s(flv3) and s(flv4), comparing to cells expressing the other two s constructs (fig. 5a) . immunofluorescent staining showed the formation of giant syncytia in cells expressing s(flv3) and s(flv4) (fig. 5b , panels s(flv3) and s(flv4)), but much smaller syncytia were observed in cells expressing s(flv2) (fig. 5b, panel s(flv2) ). no obvious cell-cell fusion was observed in cells expressing s(fl) and s(flv1) (fig. 5b , panels s(fl) and s(flv1)). the relative cell-cell fusion activities of these s constructs are flv4.flv3.flv2&ep3 = fl = flv1. these results confirm that acquisition of the cell-cell fusion activity is an important step for adaptation of ibv in cultured cells. since amino acid difference between s(flv2) and s(flv3) was only at the 523 th residue, the s(fl(q523-l)) construct was also created and expressed. the results showed that it displayed a similar cell-cell fusion activity as flv2 ( = fl(i769-v)) ( fig. 5a further enhancement of the cell-cell fusion activity of s protein and adaptation of ibv to cell culture by g405-d substitution vero-adapted ibv gradually increased its infectivity in vero cells by serial passages and a significant difference between p7 and p65 was observed [10] . comparison of amino acid sequences between s(p7) and s(p65) revealed a single mutation at the amino acid position 405 (g405-d) in s(p65) (fig. 1a) . to analyze the possibility that the enhanced infectivity of p65 virus is due to the enhanced cell-cell fusion activity of the corresponding s protein, s(p7) and s(p65) constructs were created and expressed in vero cells. efficient induction of cell-cell fusion was observed in cells transfected with both constructs (fig. 6a, panels s(p7) and s(p65) and 6b, lanes 3 and 4) . comparatively, significantly larger syncytia was observed in cells expressing s(p65) construct than in cells expressing s(p7) (fig. 6a) , demonstrating that the additional g405-d mutation in s(p65) may enhance its cell-cell fusion activity. the g405-d mutation was then introduced into s(ep3) and s(ck) and expressed (fig. 6a) , showing that introduction of g405-d mutation into s(ck) drastically enhanced its cell-cell fusion activity (fig. 6b) . interestingly, introduction of the mutation into s(ep3) and expression of the construct in vero cells showed formation of small syncytial cells (fig. 6b) . the relative cell-cell fusion activities of these s constructs are ck(g405-d).p65.ck&p7.ep3(g405-d).ep3. avian coronaviruses have been isolated from chicken, turkey and pheasant and may exist in many other avian species [20] . ibv is usually associated with respiratory disease in domestic fowl, and was believed to have a limited host range. chicken is the only natural host. similarly, coronaviruses originated from human and other animal species were considered to have narrow host specificities until the identification of sars-cov as the causative agent of sars outbreaks in 2003. the current model of animal origin of sars-cov highlights the importance of cross-species adaptation and transmission of animal coronaviruses to human. cross-species transmission of virus infection has long been recognized as a way for the emergence of many zoonotic diseases. the molecular basis for this phenomenon would lie on the rapid adaptation of certain viruses to a changing environment through selection of minor variants from quasispecies, accumulation of mutations, recombination between minor variants, and reassortment of their genomes. a better understanding of the underlying mechanisms that control these events would be essential for providing safeguards to limit the impact of these devastating diseases. in this study, we show that acquisition and enhancement of the cell-cell fusion activity by amino acid substitutions in the s protein are critical for interspecies adaptation and infectivity of ibv to cultured cells. data present clearly show that the l857-f mutation in the heptad repeat 1 region of s proteins derived from cell-culture-adapted ibv is important for adaptation of the virus to cell culture systems, and an additional mutation in the s1 region (g405-d) could enhance this process. as s protein carrying the l857-f mutation is able to induce cell-cell fusion, but losses the activity when f857 was mutated back to l, it suggests that induction of cell-cell fusion is an essential step in adaptation/ selection of ibv to cultured mammalian cells. coronavirus s protein is the major determinant for viral entry and host specificity. it is a class i fusion protein and mediates viral entry by specific binding of the s1 domain to a host cell receptor [21] [22] [23] [24] [25] . the cellular receptors for several coronaviruses have been identified, including members of the cacinoembryonic antigen family of cell adhesion molecules as the receptor for mhv, angiotensin converting enzyme ii for sars-cov and human coronavirus nl63, and aminopeptidase n for human coronavirus 229e and tgev [26] [27] [28] [29] [30] . to date, the receptor(s) for ibv has not been identified in its native or adapted host cells. it is assumed that a mammalian counterpart on vero cells could be used as a receptor for ibv at low affinity, and might have structural and functional similarities to the native receptor on chicken cells. at the initial stages of the adaptation process, a certain proportion of ep3 would weakly bind to this molecule and gains entry into the cells by endocytosis. in addition, binding of ibv to sialic acid was reported to be important for adaptation of the virus to human cells [31, 32] . the beaudette strain of ibv was also reported to have an additional binding activity to heparin-like structures [33] . these additional binding activities may help to initiate infection and thus allow the virus to adapt to the new host receptor by mutation. to uncoat the engulfed virion and to establish subsequent infection cycles as well as to spread infection to neighboring cells, acquisition of virus-cell/cell-cell membrane fusion and enhancement of the cell-cell fusion activity would be an essential step for successful selection/adaptation of virus to the new host cells. membrane fusion mediated by coronavirus, similar to other viruses, is a multistep process. it includes binding of the s protein to one or more receptors, conformational changes of the protein to a fusionactive form and the actual fusion process. the membrane-fusion activity of coronavirus s protein is mainly associated with domains in the s2 region of the protein [34] [35] [36] [37] [38] . in this study, we demonstrate that l857-f substitution in the s2 region of the ibv s protein confers the s protein from non-fusogenic to fusogenic. this mutation may affect one of these fusion steps and thus modify the fusion activity of s protein and syncytia formation. at the same time, the virus was successfully adapted to the cultured cells with enhanced infectivity, confirming that acquisition of membrane fusion is an important step in selection/adaptation of ibv to cell culture and may also play a crucial role in cross-species adaptation and transmission of ibv in cultured cells. further enhancement of the cell-cell fusion activity of ibv s protein was achieved by a single amino acid substitution (g405-d) in p65 virus. this mutation, meanwhile, enhances the infectivity of the virus in cultured cells. the enhancement effect by mutations in the s1 region and its significance on viral infectivity was further demonstrated by cloning and expression of s gene derived from the ibv variants rescued from the full-length transcripts containing the f857-l mutation. in variants flv3 and flv4, additional amino acid substitutions (q523-l and i769-v) greatly enhanced the cell-cell fusion activity of the l857-containig s protein and the infectivity of the recovered virus. the involvement of residues in the s1 region in the cell-cell fusion activity of s protein was also demonstrated in other coronaviruses [25] . these results illustrate the complexity of the fusion process and the involvement of multiple domains in the induction of membrane fusion. it is worth mentioning that the cell-cell fusion activity of various s constructs was approximated by the degree of cell-cell fusion induced in cells overexpressing individual constructs. in a previous report, we showed nice correlation between the cell-cell fusion activity of two s constructs and their expression levels in the cells [39] . this correlation was also observed in this study with more wild type and mutant s constructs. based on data generated from adaptation [10] and cell-cell fusion studies presented here, a model of two-step adaptation process is proposed (fig. 7) . in this model, the adaptation was divided into primary and secondary adaptation (fig. 7) . early passages of vero-adapted ibv, including p7, p12, p14 and p20, belong to the primarily adapted group (fig. 7) . other cell cultureadapted strains, including the ck-adapted and beaudette-us strains cac39114 and cac39300 [8, 9] , and the vero-adapted strain aav98206 described by youn et al. [13] , also belong to this group (fig. 7) . late passages of vero-adapted ibv, including p36, p50 and p65, contain the additional g405-d amino acid substitution and belong to the secondarily adapted group (fig. 7) . except in the cell culture-adapted ibv strains, the l857 residue was found to be absolutely conserved in all coronaviruses sequenced so far. as structural information for this ibv s protein is currently lacking [40, 41] , we are unclear the overall role of this residue on the formation and stability of the six-bundle structure of the protein. further structural and functional studies are required to delineate the precise roles of this mutation in the fusion process. mutation of this residue to either a ser or a tyr showed similar effect on the cell-cell fusion activity of the s protein as a phe. on the other hand, when the residue was mutated to ile, glu or lys, a much lower cell-cell fusion activity of the s protein was observed. interestingly, mutations in the s1 and some distant s2 regions could compensate the effect of f857-l mutation. this may explain why s protein from several other coronaviruses, such as mhv and human coronavirus, could induce efficient virus-cell/cell-cell fusion although a conserved leu residue was found at the equivalent position [42, 43] . it is worth mentioning that the cell-cell fusion activities of different s constructs were qualitatively and semi-quantitatively determined in cells overexpressing individual s constructs using the vaccinia/t7 expression system. attempts to obtain more rigorous quantitative data were unsuccessful. as shown in this figure 7 . diagram showing a two-step adaptation process of chicken embryo-adapted ibv to vero cells. also shown are the numbers of amino acid changes during each adaptation process. a the accession no. for s genes from ep3 is dq001338, p7 is dq001337, p65 is dq001339. b the accession no. for this vero-adapted strain is aav98206. c the accession no. for these two strains are cac39114 and cac39300. doi:10.1371/journal.pone.0006130.g007 study, s protein is inefficiently translocated to the cell surface, probably due to the presence of an er retention signal [44] . since cell surface expression of s protein is a prerequisite for the induction of cell-cell fusion, disruption of the er-retention signal may facilitate cell surface expression as well as quantitative analysis of the cell-cell fusion activity of the protein. as virus-cell/cell-cell fusion is essential for efficient propagation of viral infection, attempts to interfere this process with fusion inhibitors, such as peptides or small molecules, are being made for several viruses, including hiv, sars-cov and influenza virus. the involvement of multiple domains in the induction of cell-cell fusion demonstrated here would complicate the design of such inhibitors. furthermore, mutations in regions beyond the target sequence, in the case of coronaviruses the s1 and some distant s2 regions, may lead to the emergence of drug-resistant strains. understanding of the fusion mechanisms in more detail would, therefore, help design more efficient inhibitors. conceived and designed the experiments: dxl. performed the experiments: yy xbl sf fpt. analyzed the data: yy xbl sf dxl. wrote the paper: xbl dxl. the molecular biology of coronaviruses the biology and pathogenesis of coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immune-mediated genetic evolution and tropism of transmissible gastroenteritis coronaviruses circulation of genetically distinct contemporary human coronavirus oc43 strains replication and morphogenesis of avian coronavirus in vero cells and their inhibition by momensin coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated 3b gene: functional characterization of the 3b gene in pathogenesis and replication single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication induction of p53-independent cell cycle arrest at s-and g 2 /m-phase in cells infected with the coronavirus infectious bronchitis virus promotes viral replication identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the replication and infectivity of coronavirus in cultured cells further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i a 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins human aminopeptidase n is a receptor for human coronavirus 229e sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of hela cells by avian infectious bronchitis virus is dependent on cell status heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam1 is responsible for fusogenic activation and conformational changes of the spike protein fusogenic properties of uncleaved spike protein of murine coronavirus jhmv the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam1 receptor specificity of the virus strain conformational changes in the spike glycoprotein of murine coronavirus are induced at 37uc either by soluble murine ceacam1 receptors or by ph 8 coronavirus spike protein inhibits host cell translation by interaction with eif3f structural basis for coronavirusmediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus mhv-a59 acquired fusion activity of a murine coronavirus mhv-2 variant with mutations in the proteolytic cleavage site and the signal sequence of the s protein infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent key: cord-345630-bam3pa70 authors: lee, han-jung; shieh, chien-kou; gorbalenya, alexander e.; koonin, eugene v.; la monica, nicola; tuler, jeremy; bagdzhadzhyan, anush; lai, michael m.c. title: the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase date: 1991-02-28 journal: virology doi: 10.1016/0042-6822(91)90071-i sha: doc_id: 345630 cord_uid: bam3pa70 abstract the 5′-most gene, gene 1, of the genome of murine coronavirus, mouse hepatitis virus (mhv), is presumed to encode the viral rna-dependent rna polymerase. we have determined the complete sequence of this gene of the jhm strain by cdna cloning and sequencing. the total length of this gene is 21,798 nucleotides long, which includes two overlapping, large open reading frames. the first open reading frame, orf 1 a, is 4488 amino acids long. the second open reading frame, orf 1 b, overlaps orf 1 a for 75 nucleotides, and is 2731 amino acids long. the overlapping region may fold into a pseudoknot rna structure, similar to the corresponding region of the rna of avian coronavirus, infectious bronchitis virus (ibv). the in vitro transcription and translation studies of this region indicated that these two orfs were most likely translated into one polyprotein by a ribosomal frameshifting mechanism. thus, the predicted molecular weight of the gene 1 product is more than 800,000 da. the sequence of orf 1 b is very similar to the corresponding orf of ibv. in contrast, the orf 1 a of these two viruses differ in size and have a high degree of divergence. the amino acid sequence analysis suggested that orf 1 a contains several functional domains, including two hydrophobic, membrane-anchoring domains, and three cysteine-rich domains. it also contains a picornaviral 3c-like protease domain and two papain-like protease domains. the presence of these protease domains suggests that the polyprotein is most likely processed into multiple protein products. in contrast, the orf 1b contains polymerase, helicase, and zinc-finger motifs. these sequence studies suggested that the mhv gene 1 product is involved in rna synthesis, and that this product is processed autoproteolytically after translation. this study completes the sequence of the mhv genome, which is 31 kb long, and constitutes the largest viral rna known. mouse hepatitis virus (mhv), a murine coronavirus, contains a single-stranded, positive-sense rna genome (lai and stohlman, 1978; wege eta/., 1978) . the genomic organization is well understood lai, 1990) . it contains 8 genes, each of which is expressed from the 5'-end of a polycrstronic mrna species. these mrnas have a 3'-coterminal, nestedset structure (lai et al., 1981) . starting from the 5'-end of the genome, the genes are named 1, 2a, 2b, 3, and so on until gene 7 (cavanagh eta/., 1990) . genes 2b, 3, 6, and 7 encode the four known viral structural proteins, i.e., he (hemagglutinin-esterase), s (spike), m (membrane), and n (nucleocapsid) proteins, respectively. the remaining genes presumably encode nonstructural proteins, most of which are yet to be identified in the virus-infected cells. the nucleotide sequences of genes 2 to 7 have been determined for two strains, a59 and jhm, of mhv (armstrong eta/., 1983 (armstrong eta/., , 1984 skinner and siddell, 1983, sequence data from this article have been deposlted with the embugenbank under accession no. m55148 . ' to whom correspondence should be addressed. 1985; schmidt et a/., 1987; luytjes et a/., 1987 luytjes et a/., , 1988 shieh et al., 1989) . altogether these seven genes account for roughly 9.5 kb. the remaining gene, gene 1, which is the 5'-most gene, has been estimated to be longer than the size of all of the other genes combined (pachuk eta/., 1989; baker et al., 1990) . only the 5'-terminal 5.3 kb in jhm strain and the 3'-terminal 8.4 kb of this gene in a59 strain have so far been sequenced baker et al., 1989; pachuk et al,, 1989; bredenbeek et al., 1990) . the corresponding gene of an avian coronavirus, infectious bronchitis virus (ibv), has been completely sequenced and shown to be 20 kb long . this ibv gene consists of two open reading frames (orfs), which can be translated into a polyprotein via a ribosomal frameshifting mechanism (brierley et a/., 1987 (brierley et a/., , 1989 . again, the gene products have yet to be detected in the virus-infected cells. the size of mhv gene 1 has not been determined. from the approximate sizes of the cdna clones, it has been estimated to be roughly 22-23 kb (pachuk et al,, 1989; baker et al,, 1990) . comparison of the published partial sequences of gene 1 showed that ibv and mhv share sequence similarity in the 3'-terminus of the gene ( , 1990) , and yet their 5'-ends are diverged (soe eta/., 1987; baker et al., 1989) . thus, the evolutionary relationship of these two viruses in gene 1 is not clear. several pieces of evidence suggest that gene 1 may encode proteins which are directly involved in viral rna synthesis: first, since mhv does not contain rna polymerase (brayton et al., 1982) , this enzyme has to be synthesized from the incoming virion genomic rna. this translation is only possible if the gene is located at the 5'-end of the genome. second, rna recombination studies using temperature-sensitive (ts) mutants indicated that the ts lesions affecting rna synthesis are localized within the gene 1 region (keck et al., 1987) . this conclusion has been confirmed by rna recombination mapping studies (baric et a/., 1990) . third, the 3'-half of the gene 1 sequences of ibv and mhv-a59 contains the sequence motifs for rna polymerase and helicase, which are the activities expected to be involved in rna synthesis gorbalenya et a/., 198913; bredenbeek et a/., 1990) . however, these postulated functions have not been directly demonstrated. at least one enzymatic activity, i.e., an autoprotease , has been associated with the gene product. the presence of the protease activity suggests that the gene 1 product is likely to be processed into multiple proteins. the properties of the rna polymerase of coronavirus are of considerable interest since the coronavirus rna synthesis utilizes an unusual mechanism of discontinuous transcription, probably involving a free leader rna species (lai, 1988) . the understanding of the rna polymerase should shed further light on the mechanism of rna synthesis. to this end, we have obtained the complete sequence of gene i of the jhm strain of mhv. this gene is nearly 22,000 nucleotides long and contains two overlapping orfs, similar to the corresponding ibv gene. sequence analysis shows that the mhv gene may have undergone extensive divergence from the ibv gene, particularly at its 5'-half. several functional domains were identified, which may be important for the processing and the enzymatic activities of its gene product. virus and cells. the plaque-cloned jhm strain of mhv (makino et a/., 1984) was used throughout this study. the virus was propagated on dbt cells (hirano et a/., 1974) at m.o.i. of 1. virus was harvested and purified from the medium, and viral rna was prepared as previously described (makino et a/., 1984) . cdna cloning. the cdna c\ones encompassing , i i i i , i i i / , i i i i i 1 2000 2500 3000 3500 3 orf la/lb 0 orf lb , i i i i , i i i i , i i i i i ' ' 1000 1500 2000 2500 fig. 2. hydropathy profiles of the predicted amino acid sequences of orf 1 a and orf 1 b. values above the lrne are hydrophobic and values below the line are hydrophilic. the hydropathicrty was calculated using a moving window of 40 amino acids, with a value plotted every 16 residues (kyte and doolittle, 1982) . gene 1 were obtained by using specific synthetic oligo-gonucleotides were derived from rna sequence analynucleotides as primers and purified virion genomic sis of the rnase tl-resistant oligonucleotides which rna as template. initially, the sequences of these olihad been mapped to either gene 1 or 2 (shieh et al, , 13, 600 13,650 nucleotide number 13,700 fig. 3. dragram of the codon preference in the region between orf 1 a and orf 1 b. the codon usage patterns for the three reading frames of the predicted amino acid sequences at the junction between the orf 1 a and orf 1 b are shown. the two stop codons at 13600 (tag) and 13679 (taa) are marked. the codon usage table was generated for genes 3, 6, and 7, which encode the viral structural proteins, of mhv-ihm (schmidt er a/., 1987; skinner and siddell, 1983) and used for comparison with orfs 1 a and 1 b. the parameters used are a window length of 25 and a maximum scale of 1.1 (gribskov et al., 1984) . l bv-m42 5' 12337 gauaagaauuauuuaaacggguacgggguagcagug----aggcucggcugauaccccuugcuagugg 3' ii i lllll 11111111111 lllllli iiii i ii ii 11-11111 iii illll mhv-jhm 5' 13643 gacacgaauuuuuuaaacggguucgggguacaaguguaaaugcccgucuuguacccugugccagugg 3' mhv-a59 5' 284 llllllll 11111111111 iiiiiiiiiiiiiiiltriiiiiiiiiiiiiiiiiiiiiiiiiiii gacacgaacuuuuuaaacggauucgggguacaaguguaaaugcccgucuuguacccugugccagugg 3 comparison of the rna sequences and the proposed secondary structure of the mhv-jhm, mhv-a59 and ibv rnas at the junction between orf 1 a and orf 1 b. (a) alignment of nucleotide sequences. the first nucleotides are numbered according to boursnell era/. (1987) for ibv, and bredenbeek ef al. (1990) for mhva59, and termrnation codons are underlrned. (b) tertiary rna structure at the region of ribosomal frameshifting. the potential signal for ribosomal frameshifting is boxed, and the stop codon is underlined. arrows indicate the differences in the rna sequence of mhv-jhm in comparison with that of ibv (boldfaced) and mhv-a59 (outlined). soe et al., 1987) . cdna synthesis was were trimmed with t4 dna polymerase and ligated to performed by the general method of gubler and hoff-ptz18u (united states biochemical corp.) either by man (1983) . the double-stranded cdna molecules blunt-end ligation or ecorl linker ligation. the recombi4, and 7) . generating a 0.5.kb rna. translation was performed in a rabbit reticulocyte lysate system using [35s]methionine. translation products were analyzed directly (lanes l-3) or after immunoprecipitation using the orf 1 a-specific antiserum (lanes 4-6) or rabbrt preimmune serum (lanes 7-9). m indicates molecular weight markers in kilodaltons; lanes 3. 6, and 9, translation of ptz(orfaug). nant dnas were transformed into escherichia co/i strain mvl 190 competent cells (dagert and ehrlich, 1979) . homopolymer dc tailing to the 3'-end of the cdnas using terminal transferase were also used to anneal to pstl-linearized pbr322 with oligo(dg) tails and transformed into e. co/i strain mc1 061. specific cdna clones were identified using 5'-end-labeled oligonucleotides as probes and confirmed by subsequent hybridization to viral mrna . once the sequences of the cdna clones were obtained, oligonucleotides complementary to the 5'-ends of these clones were synthesized to serve as primers for addi-tional cdna cloning to obtain overlapping cdna clones. dna sequencing. sequencing was performed as previously described . both chemical modification (maxam and gilbert, 1980) and dideoxynucleotide chain termination @anger et a/., 1977) methods were used directly on plasmid dna (chen and seeburg, 1985) . construction of recombinant plasmids for the frameshifting analysis. subcloning and mutagenesis of cdna clone t-l 2 was accomplished using synthetic oligonucleotides and polymerase chain reaction (pcr). briefly, oligomer #166 (5'-gatcgaattcctttacat-ggtgaaggggtg-3') which extends from nucleotide 13,147 to 13,167 of gene 1 and contains mismatches at both nucleotides 13,154 and 13,156, and oligomer #199 (5'-catatgacacaggatcctttatgcc-3') which is complementary to nucleotides 13,529 to 13,553 and includes the bamhl site at nucleotide 13,537, were used for dna amplification by pcr according to the standard procedures (saiki et al., 1988) . the resulting pcr dna product encompasses sequences from nucleotide 13,147 to 13,537 with a specific mutation (t to a) at nucleotide 13,154 and another (t to g) at nucleotide 13,156, resulting in the introduction of an atg codon. the dnawas then digested with ii i iill iii iiii lllll ir iii1 iiiii iii qts~gvcvvcnspti the overall alignment was generated by combining segments aligned by programs optal (gorbalenya et al., 1989a) and multalin (corpet, 1988) . it consists of four distinct pieces separated by regions that could not be aligned with certainty. for the latter regions, only the total numbers of amino acid residues are indicated. the amino acid numbers of the first and the last residues of each aligned segment are indicated in parentheses. two dots, identical residues; single dots, similar residues. conserved cys residues are highlighted by boldface. asterisks, putative catalytic residues of proteases; arrows, putative cleavage sites for bc-like proteases. box, the putative cleavage site for 3clpr0 in ibv substituted by a kr dipeptide in mhv-jhm. the ibv sequence was from boursnell et al. (1987) . mhva: orf la of mhv. ibvfl: orf la of ibv. previously described . the resulting in vitro transcription and translation. recombinant rna was translated in the mrna-dependent rabbit replasmids ptz(orfaug) and ptz (frsh) shin and morrison (1989) and analyzed by electrophoresis on 7.5 to 15% polyacrylamide gel. computer analysis of nucleotide and amino acid sequences. sequence data were analyzed on a vax 1852 using the gcg sequence analysis software package developed by genetics computer group of university of wisconsin. detailed comparative analyses of coronavirus protein sequences were done by programs multalin (corpet, 1988 ) optal (gorbalenya et a/., 1989a), dothelix (leontovich et al., 1990) , and site (koonin et a/., 1990) . the programs dothelix and site are parts of the genbee program package for biopolymer sequence analysis. to clone the gene i region, which represents more than two thirds of the mhv genome, a synthetic oligonucleotide (oligo 30; 5'-ctgaatftgggg-gttggg-3') was initially used as a primer for cdna synthesis . the sequence of this oligonucleotide was based on the sequence analysis of the rnase tl-resistant oligonucleotide no. 30, which had previously been mapped to gene 2 (makino et al., 1984) . the resulting cdna clones contained inserts ranging from 0.5 to 3 kb in size. these cdna clones detected only the genomic rna on northern blots of intracellular rnafrom mhv-infected cells (data not shown). based on the nested-set structure of mhv 9 . a schematic presentation of the relationship between the orf 1 a of mhv-jhm and ibv. the two orf 1 a are shown to scale. the designation of regions, for which specific functional predictions could be made, and of regions of similarity between the two viruses are shown in the bottom of the figure. high similarity, statistical significance over 10 sd (standard deviation), when aligned by the program optal (gorbalenya eta/., 1989a,b) ; moderate similarity, significance of 3 to 10 sd. the alignments in the regions, with predicted functions, were significant at the level of at least 5 sd. regrons of similarity between the two viruses are joined. vertical arrows, putative cleavage sites for 3clpr0'). horizontal arrows, putative papain-like proteases (two copies in mhv-jhm, and one copy in ibv). mrnas (lai et a/., 1981) , this result indicated that these cdna clones represent part of gene 1. the 5'ends of these dnas were sequenced, and synthetic oligonucleotides complementary to these sequences were generated to prime further cdna synthesis for walking toward the 5'-end of gene 1. in this way, overlapping dna clones which encompass about 11 kb at the 3'-end of gene 1 were obtained (fig. 1) . cdna clones representing the 5'-terminal 6.2 kb of gene 1 were derived as described . the cdna clones spanning the gap between the two cdna groups were obtained by using specific primers representing both the sequences downstream and upstream of the gap as primers for first-strand and second-strand cdna synthesis, respectively. the overlap of these cdna clones was determined by southern blotting and confirmed by dna sequencing. the complete cloning of jhm gene 1 indicated that the size of gene 1 is approximately 22 kb in length (fig. l) , longer than that of ibv , and agrees with the previous estimate for the gene 1 of the a59 strain of mhv (pachuk et al., 1989) . analysis of the nucleotide sequence and the predicted amino acid sequence. the complete mhv-jhm gene 1 sequence was obtained from the cdna clones as indicated in fig. 1 . this sequence has been deposited with genbank (accession no. m55148), and will not be duplicated in this publication. the complete sequence of gene 1 contains 21,798 nucleotides preceding the ucuauac, which is the transcriptional initiation site for gene 2 . analysis of the sequence revealed two large, overlapping open reading frames (orfs), orf 1 a and orf 1 b (fig. 1 a) . orf 1 a is 4488 amino acids long and has a predicted molecular weight of 499,319, which includes the coding region for p28 protein at its n-terminus . the hydropathy plot (kyte and doolittle, 1982) shows that orf 1 a has several long stretches of hydrophobic regions at the carboxy-terminal region, which indicate potential membrane-spanning domains (fig. 2) . orf 1 b, which overlaps orf 1 a for 75 nucleotides but is located at a different reading frame, is 2731 amino acids long with a predicted molecular weight of 308,483. the orf 1 b sequence is very similar to that of mhv-a59 in both nucleotide and predicted amino acid sequences (bredenbeek et al., 1990) . only minor substitutions were noted between the two strains (data not shown). the orf 1 b starts with cug instead of aug. the first potential initiator codon aug is located 399 nucleotides downstream of the first amino acid the numbers of amino acid residues to the known or postulated termini of the respective viral 3clpro and between the aligned segments are indicated. for mhv 3clpro, the postulated n-terminus is at amino acid residue 3350 ( fig. 8 gorbalenya et a/. (1989b) , except bwyv (veidt et a/., 1988) and sbmv (wu et al., 1987) . riore. in the aa position columns, the amino acid positions of the respective q residues are indicated. the arrows show the predicted cleavage sites, abbreviations: mpl , mp2, putative membrane proteins flanking the 3clpr0 at the n-and c-sides, respectively. pol: polymerase motif. hel: helicase motif. gfl: growth factor-like domain. the data on ibv was obtained from gorbalenya ef al. (1989b) . the sequence analysis was performed using the computer program as described under materials and methods. codon in orf 1 b. nevertheless, the codon preference plot suggests that the 399 nucleotides upstream of the first aug are most likely translated together with the downstream sequences using the same reading frame (fig. 3) . in light of the corresponding sequences of ibv and mhv-a59 bredenbeek et a/., 1990) , this result suggests that this region could be translated via a ribosomal frameshifting mechanism (brierley et al,, 1989) . comparison of tertiary structure of rna in the frameshift regions. it has been proposed that the nucleotide sequences in the overlapping regions between orf 1 a and orf 1 b in ibv and mhv-a59 rnas are able to fold into a pseudoknot tertiary structure, which is essential for efficient frameshifting and, thus, expression of the downstream orf 1 b (brierley et a/., 1989; bredenbeek et al., 1990) . comparison of the primary sequence revealed that the corresponding region of mhv-jhm contains a "slippery" sequence, uuuaaac, similar to that of ibv (fig. 4a) . the possible folding of rna in this region into a pseudoknot tertiary structure is similar among ibv, mhv-a59, and mhv-jhm (fig. 4b) . it is interesting to note that the nucleotide changes between mhv-jhm and ibv in either the stem or loop regions are compensated by mutations at the complementary positions (fig. 48) . this suggests the significance of the putative tertiary structure in ribosomal frameshifting. only two nucleotides differ between mhv-jhm and mhv-a59 in this region; they are located at the regions immediately upstream and downstream of the uuuaaac sequence. ribosomal frameshifting in vitro. to confirm that the orf 1 a and 1 b of mhv-jhm could be translated into one polypeptide by ribosomal frameshifting, we cloned the region spanning from nucleotide 13,147 to 14,164 of gene 1 into an expression vector under the control of the t7 promoter for in vitro translation studies. because of the lack of a translational initiation codon, an atg codon was introduced by pcr-mediated mutagenesis at nucleotide 13,154-l 3,156. if the translation of this transcript terminates at the uaa stop codon in orf 1 a, a 19-kda protein will be produced. however, if the -1 translational frameshift occurs, a 37-kda protein will be synthesized. as shown in fig. 5 , the in vitro translation of this rna yielded both proteins (lane 2). the 37-kda protein was heterogeneous; the smaller proteins may represent aberrant translational initiation or specific processing of the translation products. the addition of protease inhibitors in the rabbit reticulocyte lysates did not alter this translation pattern (data not shown). the antiserum prepared against the amino acid sequence just upstream of the frameshift (unpublished) precipitated both proteins (lane 5). surprisingly, the major products precipitated by this antiserum migrated faster than the respective primary translation products, suggesting that protein processing had occurred. none of the proteins was immunoprecipitated by the preimmune serum. as controls, the transcripts containing either the 5'-or the 3'-halves [ptz(orf""g)] of the orf did not yield the 37-kd protein. as predicted, only the products of the 5'-half were precipitated by this antibody (fig. 5b, lane 4) . these results are in agreement with the results obtained with ibv (brierly et a/., 1987) and mhv-a59 (bredenbeek et a/., 1990) . analysis of sequence homology among mhv-jhm, mhv-a59, and ibv. the comparison of nucleotide and predicted amino acid sequences between mhv-jhm and ibv revealed considerable similarity between the two. the dot matrix comparison of the amino acid sequences shows that orf 1 b is very similar between mhv and ibv (fig. 6) . overall, there are 47.7% similarity at nucleotide level and 52.8% at amino acid level. similar to the orf 1 b of ibv, the mhv orf contains the polymerase and helicase motifs at the corresponding positions (gorbalenya et al., 1989b ) (data not shown). the putative zinc-binding domain is also largely conserved between the two viruses. on the other hand, two of the residues implicated in metal binding for ibv (gorbalenya et al., 198913) are replaced in mhv, suggesting that the specific structures of the putative "fingers" may differ (fig. 7) . the orf 1 b of mhv-jhm and mhv-a59 are also very similar (95.9% at nucleotide level, and 94.9% at amino acid level) (data not shown). in contrast, the orf 1 a is more diverged (fig. 8) . the mhv orf 1 a is longer than the corresponding ibv orf by 537 amino acids. the c-terminal half of the orf 1 a is relatively conserved between mhv-jhm and ibv, while the n-terminal half is very diverged (fig. 6) . the alignment of amino acids in orf la of mhv-jhm and ibv showed that there are four possible stretches of moderate homology which are separated by highly diverged sequences (fig. 8) . analysis of the functional domains of orf la. although orf la is highly diverged between mhv-jhm and ibv, common functional domains could be identified in this orf of both viruses by detailed amino acid sequence analysis (see materials and methods) (fig. 9 ). two hydrophobic, potentially membrane-anchoring regions are present in the c-terminal half. there are three cysteine-rich domains, one of which contains a segment distantly resembling growth factors and their receptors (gorbalenya et al., 1989b) . in both coronaviruses, homologous domains of about 300 residues each have been identified to be related to the putative 3clike proteases (3clpro) of picorna-, coma-, nepo-, poty-, sobemo-and luteoviruses (gorbalenya et al., 1989b) . the sequences of the putative coronavirus 3c-like proteases possess certain unusual features distinct from that of other viral 3clike proteases (fig. 10 , and see discussion). the search for sequences resembling the cleavage sites for the 3c-like proteases revealed six conserved putative target sites for the mhv and ibv 3c-like proteases (table 1 ) (see discussion). these potential cleavage sites are localized in the orf 1 b and the c-terminal half of the orf la. interestingly, the n-terminal one of these cleavage sites marks the n-end of the putative 3clike protease itself. finally, there is a region of moderate conservation between mhv and ibv, which contains short segments resembling those around the catalytic cys and his residues of papain-like proteases (fig. 11) . this region is duplicated in the mhv genome, but not in ibv, at an upstream site in the orf la. this upstream papain-like cysteine protease has been identified as the one responsible for the cleavage of p28 from the n-terminus of the gene 1 protein . a domain of considerable conservation between mhv and ibv (x domain in fig. 9 ) has been found next to the putative coronavirus papain-like proteases. interestingly, a homologous conservative domain also flanks the putative thiol proteases of alpha-and rubiviruses (a. e. gorbalenya, unpublished observations) . the complete sequence of gene 1 of mhv presented in this paper shows that this gene is probably the largest known viral gene among rna viruses. evidence was presented suggesting that the two orfs in this gene may be translated into a large polyprotein. this interpretation is consistent with the lack of the transcriptional initiation signal (ucuaaac) in the entire gene 1 sequence except at the extreme 5'-end. although the putative "slippery" sequence (uuuaaac) between the orf 1 a and 1 b (brierly et a/., 1989) is similar to the transcriptional initiation signal, no major subgenomic mrnas have been detected within this gene. thus, this gene most likely encodes a single polyprotein of at least 800 kda. the total size of the rna genome of mhv is approximately 3 1 kb, which is considerably larger than any of the other known viral rna. the evolution of the coronavirus rna genome into such a large rna may have reflected the unusual mechanism of coronavirus rna synthesis. the complexity of the discontinuous mode of coronavirus rna synthesis (lai, 1988) suggests that the coronavirus rna polymerase needs a variety of different enzymatic activities. the amino acid sequence of gene 1 of mhv shows considerable similarity to that of ibv. the orf 1 b is particularly conserved. its degree of conservation between mhv and ibv is higher than that for any of the other genes in the coronavirus genomes. the orf 1 b contains the polymerase, helicase, and metal-binding motifs (gorbalenya el a/., 1989b) , suggesting that this region may be directly involved in rna synthesis. these structural features are conserved between these viruses. the proposed pseudoknot structure which is important for the ribosomal frameshifting for cotranslation of orf 1 a and orf 1 b (brierley et a/., 1989) is also highly conserved. this fact has previously been recognized in the partial sequence of gene 1 of mhv-a59 (bredenbeek eta/., 1990) . the sequence differences between mhv-a59 and mhv-jhm within this junction region are located at the nucleotides which do not affect the putative pseudoknot structure. in contrast, orf la is much more diverged. it is nearly 2 kb longer than the orf la of ibv, and contains several stretches of sequence which are not present in the ibv genome. these nonhomologous stretches of sequence are interspersed between the conserved regions. furthermore, a papain-like protease domain, which is present once in the ibv genome, is duplicated in the 5'-half of the orf la of mhv. the n-terminal sequence including ~28, which is cleaved by the papain-like protease of mhv , is also highly diverged between mhv and ibv. thus, it appears that the 5'-end of orf 1 a has undergone considerable sequence rearrangement and possibly recombination, while the remaining sequences in gene 1 are almost colinear between mhv and ibv. in contrast to the orf 1 b which contains sequence motifs related to the synthesis of rna, the orf la contains several domains suggestive of other functions. first of all, there are two long stretches of hydrophobic domains, which are conserved between ibv and mhv. the presence of these domains suggests that the gene 1 products may be anchored to the membrane. this possibility is consistent with the finding that mhv rna synthesis occurs on the membrane fractions in the infected cells (brayton et al., 1982) . second, there are three cysteine-rich regions, which are also homologous between mhv and ibv. the function of the cys-rich domains is still not clear. however, it has been noted with ibv that the c-terminal cys-rich domain is related to that of the growth factors and their receptors (gorbalenya et al., 1989b) . third, there is a 3c-like protease domain (3clpro) in the 3'-half of orf 1 a, which is also conserved in ibv. the putative catalytic his and cys residues previously predicted in ibv have also been observed in mhv (fig. 10) . however, the putative coronavirus proteases remain unique in that they do not contain a conserved asp(glu) residue that could serve as the third catalytic residue as suggested for the other 3c-like proteases (gorbalenya et al., 1989b) . furthermore, the unusual substitution of tyr for gly in the putative substrate-binding region, described previously in ibv, is also observed in the putative mhv 3clp" (fig. 10) . the potential cleavage sites for this 3c-like protease have been identified to be mainly in orf 1 b and the c-terminus of orf 1 a (gorbalenya et al., 1989b) . these sites (qs) are either conserved or converted to qa in mhv (table 1 ). the potential cleavage at q/s and q/a sites by picornavirus 3clp" has been demonstrated previously (parks and palmenberg, 1987) . two qg dipeptides proposed to be cleaved in ibv were substituted in mhv by qc in one case, and by kr dipeptide in another (table 1) . substitution of a c (unlike several other residues) for g in a cleavage site for encephalomyocarditis virus protease did not abolish processing in an in vitro system (parks et a/., 1989) . dibasic dipeptides are cleaved in the polyproteins of flaviviruses (strauss and strauss, 1988) . thus, these postulated cleavage sites are potentially cleavable by mhv 3clpro despite the divergence. these cleavages could separate different functional domains of the gene 1 polyprotein into distinct protein products. whether these sites are indeed cleaved in mhv-infected cells remains to be studied. fourthly, the n-terminal portion, which is the most diverged region, contains a papain-like protease domain as pointed out previously for ibv (gorbalenya et al., 1989b) . the papain protease domain is duplicated in the mhv orf 1 a (fig. 1 1) and is homologous with the known proteases (fig. 12) . this protease is probably involved in the cleavage of the n-terminus of the gene 1 polyprotein (baker eta/., 1989) , which has been demonstrated in mhv-infected cells (denison and perlman, 1987) . site-specific mutagenesis studies demonstrated that this protease has cys and his at its active site (unpublished observation). the possible presence of the protease domains suggests that the gene 1 polyprotein is processed into many proteins. it has been shown that there are at least five to six complementation groups involving mhv rna synthesis, five of which have been mapped within gene 1 (leibowitz eta/., 1982; baric eta/., 1990) . these proteins conceivably participate in various aspects of mhv rna synthesis. none of the proteins have been detected so far. sequence of the nucleocapsid gene from murine coronavirus mhv-a59 sequence and topology of a model intracellular membrane protein, el glycoprotein, from a coronavirus murine coronavirus gene 1 polyprotein contains an autoproteolytic activity identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein. 1. viroi establishing a genetic recombination map for murine coronavirus strain a59 complementation groups completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus the primary structure and expresslon of the second open reading frame of the polymerase gene of the coronavirus mhv-a59; a highly conserved polymerase is expressed by an efficient rlbosomal frameshifting mechanism an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas and genes of coronavirus supercoil sequencing: a fast and simple method for sequencing plasmid dna multiple sequence alignment with hierarchical clustering prolonged incubation in calcium chloride improves the competence of escherychia co/i cells identification of putative polymerase gene product in cells infected with murine coronavirus a59 a comprehensive set of sequence analysis programs for the vax purification and amino acid sequence of chicken liver cathepsin l an ntp-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rna viral replication coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis the codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression a simple and very efficient method for generating cdna libraries replication and plaque formation of mouse hepatitis virus (mhv-2) in mouse cell line dbt culture multiple recombination sites at the 5'-end of murine coronavirus rna a method for localization of motifs in amino acid sequences. biopolim. kletka a simple method for displaying the pathic character of a protein replication of coronavirus rna coronavirus: organization, replication and expression of genome mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv 3 the rnaof mouse hepatitis virus genetic analysis of murine hepatitis virus strain jhm. 1. viral a method for generation of complete local similarity maps between two amino acid sequences. dothelix program of the genbee package primary structure of the glycoprotein e2 of coronavirus mhv-a59 and identification of the trypsin cleavage site sequence of mouse hepatitisvirus a59 mrna 2: indications for rna recombination between coronavirus and influenza c virus analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus sequencing end-labeled dna with base-specific chemical cleavages. ln evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein cloning and characterization of a mouse cysteine proteinase molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus strain a59 proteolytic cleavage of encephalomyocarditis viral capsid region substrates by precursors to the 3c enzyme site-specific mutations at a picornavirus vpb/vpl cleavage site disrupt in vitro processing and assembly of capsid precursors primer-directed enzymatic amplification of dna with a thermostable dna polymerase dna sequencing with chain-terminating inhibitors nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome the 5'.end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderpnmed transcription production and properties of chimeric antibody molecules coronavirus mhv-jhm mrna 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame. 1. gem viral coronavirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein coding sequence of coronavirus mhv-jhm mrna 4 sequence and translation of the murine coronavirus y-end genomic rna reveals the n-terminal structure of the putative rna polymerase. 1. viral coronaviruses: structure and genome expression. 1. gem viral replication of the rnas of alphaviruses and flaviviruses nucleotide sequence of beet western yellows rna genomic rna of the murine coronavirus jhm. 1 sequence and organization of southern bean mosaic virus rna we thank dr. susan baker for advice throughout the course of the study. we also thank lisa banner and daphne shimoda for editorial assistance. a.e.g. and e.v.k. are most grateful to professor v. i, ago1 for constant support, to dr. l. i. brodsky for supply of the gen-bee program package, and to dr. key: cord-335310-61wibso4 authors: chen, hui-wen; huang, chen-yu; lin, shu-yi; fang, zih-syun; hsu, chen-hsuan; lin, jung-chen; chen, yuan-i; yao, bing-yu; hu, che-ming j. title: synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection date: 2016-08-15 journal: biomaterials doi: 10.1016/j.biomaterials.2016.08.018 sha: doc_id: 335310 cord_uid: 61wibso4 the ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. a major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. herein, a facile approach to formulate synthetic virus-like particles (svlps) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. using an avian coronavirus spike protein as a model antigen, svlps were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. as compared to inoculation with free proteins, vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the svlps. the study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture. vaccine is historically the most effective countermeasure against infectious threats, as agents resembling pathogens are administered to mount an immune response against specific targets. amidst continuing and emerging viral threats, vaccine technology continues to advance with the aim of effectively promoting antiviral immune responses, and a major development effort lies in retaining or integrating virus-like features in vaccine formulations for improved immune processing. several morphological and antigenic characteristics of viral particles have been demonstrated to promote immune potentiation. for example, particles at the nanoscale have been shown to have better lymphatic transport as compared to smaller subunit antigens [1, 2] . in addition, the display of multiple antigens on a single particle facilitates more effective antigen presentation to immune cells [1] . as compared to traditional vaccine formulations, vaccines preserving virus-like features have shown superior capability in eliciting immune responses [3e5] . these results and observations have also prompted material scientists to apply synthetic nanomaterials towards mimicking viral features for vaccine development [6e9] . given their high radii of curvature, synthetic nanoparticles frequently possess high surface energies that induce adsorption of biomolecules in a phenomenon known as protein corona formation. in protein-rich media, strong nanoparticle/protein association occurs spontaneously as a means to passivate surface energies, and the resulting particles are encased in a protein layer that dictates the particles' interactions with the environment [10, 11] . while protein corona formation is gaining increasing scientific interest owing to its implications in biomedical applications [10, 12, 13] , we herein demonstrate harnessing this phenomenon can be beneficial towards mimicking viral features for vaccine applications. we show that synthetic virus-like particles (svlps) with close semblance to native virions in physicochemical properties and antigen display can be facilely prepared through spontaneous antigen-particle association in optimized incubation conditions. using 100 nm gold nanoparticles (aunp), a biologically inert material commonly used for biomedical research [14e16] , and a spike glycoprotein derived from an avian infectious bronchitis virus (ibv), a single-stranded positive-sense rna virus that belongs to the family coronaviridae [17] , we controlled the incubation condition to prepare spike glycoprotein-laden svlps (fig. 1) . the morphological features and antigen display by the svlps were compared to native ibv viral particles using nanoparticle tracking analysis and immunogold staining. in addition, vaccination potency between the svlps and free spike glycoproteins was compared in an avian model of coronavirus infection. a commercial whole inactivated virus (wiv) formulation that is the current standard vaccine for ibv management was examined in parallel. coronaviruses are a major viral family of which the most publicized examples include the pathogens behind severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [18] . in animals, ibv is a prime example of coronavirus that infects the respiratory and urogenital tracts of chickens, posing a serious economic threat as one of the most important pathogens in the poultry industry. the ibv spike glycoprotein, which forms the large, pental-shaped spikes on the surface of the virion, is chosen as the antigen candidate as it is implicated as a determinant of virus pathogenicity. among coronaviruses, spike glycoproteins possess a variety of biological functions, including triggering cell attachment, inducing cell-cell fusion, and binding to cellular receptors [19, 20] . as spike glycoproteins are the primary targets in ongoing vaccine development efforts for coronavirus vaccinations, the present study has broad implications across both human and animal disease management [21, 22] . s. frugiperda sf9 (atcc crl-1711) insect cells were cultured in grace's insect cell medium (invitrogen, carlsbad, ca) and supplemented with 10% fbs (thermo fisher, rockford, il) and 1% p/ s/a antibiotics (biological industries, beit-haemek, israel) at 27 c. 100 nm gold nanoparticle (aunp) solution was purchased from sigma-aldrich (st. louis, mo). avian coronavirus ibv strain 2575/98 was propagated in 10-dayold specific-pathogen-free (spf) chicken embryos via the allantoic route as previously described [23] . the virus titers of ibvs were determined with the method of reed and muench [24] in spf chicken embryos and expressed as 50% embryo infectious dose (eid 50 ) [25] . the virus-containing allantoic fluid was concentrated and purified using sucrose gradient solution as previously described to derive the native virions [23] . full spike (s) protein of avian coronavirus ibv was cloned and expressed using the bac-to-bac baculovirus expression system (invitrogen). briefly, a recombinant plasmid was constructed by inserting full spike protein gene of ibv strain 2575/98 (accession no. dq646405) [26] into the pfastbac-1 vector using the following primer set: ibv-s-bamhi-f: 5 0 -ttggg atccg atgtt ggtga agtca c-3 0 ; ibv-s-sali-f: 5 0 -cttgt cgaca ttaaa cagac ttttt aggt-3 0 . the recombinant pfastbac-1 shuttle vector was then transposed to the bacmid in e. coli strain dh10bac, and recombinant bacmid was purified using the hipure plasmid midiprep kit (invitrogen). sf9 cells were used for transfection with the recombinant bacmid, and recombinant baculoviruses were then harvested in the supernatant and designated rbac-2575s. recombinant spike proteins (r2575s) were harvested from sf9 cells infected with rbac-2575s (multiplicity of infection ¼ 1). sf9 cells were washed and lysed with the i-per insect cell protein extraction reagent (thermo fisher). recombinant proteins were purified using the glycoprotein isolation kit, cona (thermo fisher) according to the manufacturer's instructions. after purification, r2575s protein was stored in 10% sucrose at à20 c. citrate-buffered 100 nm gold nanoparticles were washed repeatedly in water to remove the citrate stabilizer, and the resulting pellet was resuspended in 10% sucrose. protein solutions ranging in concentrations between 100 mg/ml to 3 mg/ml of purified spike proteins were then mixed with 1 â 10 11 /ml of gold nanoparticles (determined by nanoparticle tracking analysis) in 10% sucrose. the mixtures were bath sonicated for 1 min followed by incubation in an ice bath for 30 min. the nanoparticles were then removed from unbound spike proteins via centrifugation at 1500g for 3 min. following 3 centrifugal washes with 10% sucrose, pelleted nanoparticles were mixed with 1x pbs and sonicated in a bath sonicator for 30 s. dispersible, stabilized svlps were retrieved and their protein content was quantified using a bca protein assay (thermo fisher) with 25 ml of 1 â 10 11 particles/ml following the manufacturer's protocol. visualization of unstable nanoparticles and colloidally stable svlps was performed using a 200 kv high resolution transmission electron microscope (fei tecnai tf20). particle stability was assessed by monitoring the size of svlps for 7 days. particle size, polydispersity index (pdi), and concentrations were measured by nanoparticle tracking analysis using nanosight ns-500 (malvern, uk) at a concentration of 1 â 10 8 particles/ml based on the manufacturer's instructions. particle size and zeta potential were also measured by dynamic light scattering using zetasizer nano zs at a concentration of 1 â 10 10 particles/ml (malvern, uk) based on the manufacturer's instructions. antigen display was examined using freshly prepared svlps. antigen retention was examined by mixing svlps in protein-poor (pbs) or in protein-rich (10% bsa) conditions for varying periods of time. at 0, 3, 10, and 24 h marks, svlps were pelleted from their respective solutions. the particles were then processed using a previously published protocol with sds-page loading buffer for protein removal and quantification [27] . ibv spike proteins eluted from the svlp were analyzed in 6% discontinuous sds-page under non-reducing condition. protein gel was then transferred onto a 0.45 mm nitrocellulose membrane (bio-rad). after transfer, the membrane was soaked in blocking buffer (5% skim milk in pbs) at room temperature for 1 h and probed with anti-s monoclonal antibody (mab) for another 1 h. after three washes, the membrane was incubated with peroxidase-conjugated goat anti-mouse igg (h þ l) (jackson immunoresearch laboratories, west grove, pa) in blocking buffer at room temperature for 1 h. after three washes, the protein blots were detected with either tmb membrane peroxidase substrate (kpl) or enhanced chemiluminescence (ecl) substrate (pierce). band intensities were analyzed via imaging analysis using imagej. presence of ibv spike proteins on the svlps was further verified by immunogold staining, and purified ibv 2575/98 virions were used as a control. briefly, 3 ml of svlp or virion samples were deposited onto a glow-discharged carbon-coated grid for 2 min. the virion sample was fixed with 4% paraformaldehyde for 5 min. after 3 washes with pbs, the samples were blocked with 1% bsa for 15 min. the samples were then incubated with anti-s mab for 1 h. after pbs washes, the samples were incubated with 6 nm goldconjugated goat anti-mouse igg (jackson immunoresearch laboratories) for another 1 h. after pbs washes, native virions were further stained with 1% uranyl acetate for 15 s. all experiments were performed at room temperate. particles were visualized under a 200 kv high resolution transmission electron microscope (fei tecnai tf20). the care and use of animals were approved by the institute animal care and use committee, national taiwan university (approval no. ntu-102-el-89). all animal experiments were carried out in accordance with the approved guidelines. 8-week old balb/c mice were injected with 50 ml of pbs, free protein formulation, or svlps containing 2 mg of viral antigens via the intrafootpad route. after 24 h, the mice were sacrificed and the popliteal lymph nodes were harvested (n ¼ 6). cryosections (6 mm) were made and fixed for 10 min in acetone, followed by 8 min in 1% paraformaldehyde. sections were blocked by 5% normal goat serum (invitrogen) in pbs for 10 min and stained with anti-s mab for 4 h at room temperature. after washes, sections were further incubated with fitc-conjugated anti-mouse igg (jackson immu-noresearch laboratories) for 1 h at room temperature. nuclei were counterstained with dapi (invitrogen). fluorescence signal was observed under a fluorescence microscope (leica dmi8), and quantified via imaging analysis using imagej. 8-week old balb/c mice were injected intramuscularly in the thigh with 100 ml of formulations containing pbs, free protein, or svlps (10 mg of viral antigens) mixed with the complete freund's adjuvant. mice blood was collected on day 14 and 21 for antibody titer quantification (n ¼ 4e5 per group). three-week-old spf chickens were obtained from jd-spf biotech (miaoli, taiwan). chickens were randomly divided into four different experimental groups (n ¼ 4e6 per group) receiving pbs, free protein (r2575s), whole inactivated virus (wiv) vaccine (merial laboratories, lyon, france), or svlps. briefly, free protein or svlps (10 mg of viral antigen in 100 ml) were emulsified with the complete freund's adjuvant and administered via an intramuscular route. the commercially available wiv vaccine (oily-adjuvanted) was administered to chickens according to the manufacturer's recommendation (0.3 ml per chick). chicken sera and tears were collected on day 0 (before immunization), 14, and 21 post-immunization. all chickens were intranasally challenged with ibv 2575/98 live virus (10 6 eid 50 ) on day 21, and were observed for disease signs for 7 days. chickens were sacrificed on day 28. for serum iga and igg virus-specific elisa, 100 ng of purified ibv 2575/98 virions was diluted with coating buffer (15 mm na 2 co 3 and 35 mm nahco 3 , ph 9.6) and coated onto flatbottomed microtiter plates (nunc) at room temperature overnight. the wells were washed with pbst (0.1% tween 80 in pbs) three times and blocked with blocking reagent (5% skim milk in pbst) at 37 c for 1 h. after washes, 100 ml of chicken serum was added and incubated at room temperature for 1 h. following three washes, 100 ml of peroxidase-conjugated goat anti-chicken igy (h þ l) or iga (jackson immunoresearch) in blocking buffer was added into each well and incubated at room temperature for 1 h. after three washes, 100 ml of sureblue reserve tmb microwell peroxidase substrate (kpl) was added to each well and incubated in the dark at room temperature for 10 min. the reaction was stopped by adding 100 ml of tmb stop solution (kpl). the od was measured at 450 nm using an automated plate reader (thermo fisher). for total tear iga quantification, elisa was performed with chicken iga elisa kit (ab157691, abcam) according to the manufacturer's protocol. on day 28 post immunization, chicken spleens were minced and passed through a 70-mm cell strainer (corning) to obtain single-cell suspensions. red blood cells (rbcs) were lysed using an rbc lysis buffer (ebiosciences), and cells were resuspended in rpmi 1640 medium (gibco, grand island, ny) containing 10% fbs. viable cells were determined by trypan blue staining. 10 6 splenocytes were plated in 96-well u-bottom plates (corning), and were stimulated with 1 mg of purified ibv 2575/98 virions in the presence of brefeldin a (golgiplug, bd biosciences) for 6 h at 37 c. for the quantification of cytokine expression, the stimulated splenocytes were lysed, and total rna was isolated by trizol (invitrogen) according to the manufacturer's manual. real-time rt-pcr was performed using iscript (bio-rad) and iq sybr green supermix kit (bio-rad) with previously described primers for chicken ifn-g and gapdh [28] . melting curve analysis following real-time pcr was conducted to verify the specificity for each primer set. all obtained ct values were normalized to gapdh. the relative expression of chicken ifn-g (fold change of naive control) was determined by a 2 àddct method [29] . disease signs of chickens were recorded on a daily basis after virus challenge. the clinical score index of ibv infection was interpreted according to a previously described method [30] . the clinical signs were evaluated as: 0 ¼ no clinical signs; 1 ¼ lacrimation, slight shaking, watering feces or tracheal rales; 2 ¼ lacrimation, presence of nasal exudate, depression, water feces, apparent sneezing or cough; 3 ¼ high degree of lacrimation, nasal exudate, and severe watery feces; 4 ¼ death. after necropsy, gross lesions at the tracheas and kidneys were recorded. chicken kidneys were further harvested and homogenized in tryptose phosphate broth (bd biosciences). viral load in kidneys was assessed by quantitative rt-pcr described below. 2.11. viral rna quantification rna in chicken kidneys was extracted using trizol (invitrogen) according to the manufacturer's manual. for viral load assessment, quantitative rt-pcr was performed with iscript (bio-rad) and iq sybr green supermix kit (bio-rad) using previously described primer sets that target the s protein gene of ibv (rc2u and rc3l) [31] and chicken 28s rrna [32] . quantitative rt-pcr experiments were performed in duplicates. data was expressed as arbitrary units. data was analyzed by anova followed by dunnett's multiple comparison tests using graphpad prism (graphpad software, san diego, ca). p values smaller than 0.05 were considered significant. following aunp incubation in solutions of different protein concentrations, the resulting nanoparticles were pelleted from free proteins and re-dispersed through sonication in pbs. consistent with previous studies on nanoparticle/protein interactions [33] , it was observed that higher protein concentrations yielded particles with increased colloidal stability as evidenced by the disappearance of a discernable pellet and a purple solution characteristic of aunp suspensions ( fig. 2a) . svlps prepared from the 3 mg/ml protein suspension were readily dispersible and manifest as distinct, nonclustered nanoparticles under transmission electron microscopy (fig. 2b) , indicating passivation of the high particle surface energy upon sufficient protein coating. in contrast, particle preparations with lower protein content (1000 mg/ml) yielded clustered aunps. to further characterize svlps, we assessed aunps, svlps, and native ibv virions (fig. 2c) using nanoparticle tracking analysis, which examines particle samples on a particle-by-particle basis via tracking of scattered laser light from individual particles [34] . between aunps and svlps, we observed an overall reduction in the light scattering intensity. given that aunps are known to scatter light at an extraordinary efficiency, the intensity reduction in svlps can be attributed to successful protein coating, which restricts light passage to the aunp surfaces. likewise, native virions have the lowest light scattering under the analysis as they are comprised entirely of organic materials. the result demonstrates the feasibility of studying the evolution of nanoparticle protein corona formation using nanoparticle tracking analysis, which reveals changes in light scattering and size simultaneously. upon examining the size distributions of the different particles, svlps showed a broader distribution as compared to the sharply distributed 100 nm aunps. protein corona formation increased the nanoparticle size from 100.6 nm (pdi ¼ 0.012) to 139.2 nm (pdi ¼ 0.073) and increased the zeta potential from à23.2 mv to à16.7 mv (fig. 2c,d) . in comparison to native ibv virions, which have an average diameter of 147.3 nm (pdi ¼ 0.081) and a zeta potential of à16.6 mv, the svlps are similar in overall physicochemical properties. examination of particle stability showed that the svlps remained stable in pbs over a 7-day period with its size ranging from 136.7 nm (pdi ¼ 0.071) to 140.2 nm (pdi ¼ 0.091) (fig. 2e) . analysis of antigen display with freshly prepared svlps showed that 1 â 10 11 aunps retained 23.5 ± 2.2 mg of spike proteins, corresponding to approximately 900 ibv spike proteins per particle. western blotting using analysis revealed a sharp protein band of approximately 160 kda (fig. 2f) , which is characteristic of the viral antigen [17] . transmission electron microscopy and immunogold staining further highlight the similarity between svlps and native ibv virions. it was observed that immunogold clustered around the svlps, mirroring the staining pattern on the native virions (fig. 2g) . these observations demonstrate the close semblance between the svlps and native virions regarding their physicochemical properties and antigen display. examination of antigen retention in protein-poor (1x pbs) and protein-rich (10% bsa in 1x pbs) conditions also shed light on the characteristics of the protein corona around the svlps. in pbs, particle-bound antigen level remained steady over a span of 24 h, yielding similar ibv spike protein band intensities across the different incubation samples (fig. 2h) . a rapid drop-off in particlebound spike protein was observed upon incubation in 10% bsa. immediate retrieval of svlps from the bsa solution resulted in~65% reduction in the spike protein level, and at the 24 h mark,~25% of the initial antigen remained on the svlps. this observation suggests the formation of two distinctive corona layers distinguishable by their interaction dynamics with surrounding biomolecules, reflecting the presence of both a reversible "soft corona" and an irreversible "hard corona" that have been frequently observed in prior nanoparticle studies [35e37]. the results indicate that approximately 200e250 ibv spike proteins are stably bound to each svlps. these proteins are expected to remain in the particulate form in complex biological environments upon in vivo administration. to examine antigen delivery and lymphatic transport by the svlps as compared to free spike proteins, svlp formulation was administered to mice through a footpad injection. popliteal lymph nodes, which are the draining lymph nodes by the footpads, were subsequently collected and sectioned for immunofluorescence assay. ibv spike protein-specific immunofluorescence staining showed a significantly enhanced antigen delivery by the svlps as compared to the free protein formulation, resulting in an increased number of fluorescent punctates (green) in the lymph node sections (fig. 3a) . imaging analysis on multiple lymph node sections showed that the svlp formulation increased lymphatic delivery by approximately 6 fold (fig. 3b) . the observation of increased delivery attests to the strong protein/particle binding in the "hard corona" layer as the particle carrier is capable of facilitating antigen transport in vivo. the enhanced lymph node localization of the svlps is consistent with prior observations on nanoparticles and virus-like particles [2] . owing to their nanoscale morphology and physicochemical properties, these nanoparticles are known to facilitate free lymphatic drainage via convective transport [38, 39] as well as cell-mediated lymphatic delivery via increased cellular uptake [2] . immunogenicity of the svlps was also examined following intramuscular inoculation in mice. anti-ibv igg serum titers were compared between mice vaccinated with svlps and with free ibv spike proteins (fig. 3c) , and it was observed that the svlps elicited significantly higher igg levels, demonstrating improved vaccination potency over the free protein formulation. the improved immunogenicity can be explained in part by the enhanced antigen delivery to the lymph node, where a high number of antigen presenting cells reside. in addition, the particulate nature of the svlps likely also favors other immune activation mechanisms, such as improved cellular uptake, enhanced complement activation [38] and presentation by follicular dendritic cells [40] . these nanoparticle-specific immunological features make the svlps a promising vaccine candidate for disease management. to evaluate the svlps' effectiveness against viral infections, we vaccinated spf chickens with free ibv spike proteins or svlps (10 mg of total viral antigens) via the intramuscular route. as an additional reference, a commercial wiv vaccine for ibv was administered based on the manufacturer's suggested dosage. following vaccination, blood and tear were collected for analysis and a live ibv challenge was performed (fig. 4a) . elisa analysis showed that the svlps were superior in generating both igg and iga titers as compared to the free protein formulation and the wiv vaccine (fig. 4b,c) . the total iga in the tears of the vaccinated chickens were also quantified. despite that intramuscular vaccination is generally known to be non-ideal for promoting mucosal immunity [41] , elevation of tear iga level was observed for all three vaccine formulations (fig. 4d) . it is expected that mucosal vaccination in future studies may further increase tear iga levels and better highlight the differences among the formulations in eliciting mucosal immunity. besides humoral immunity, cellular immunity, a major component of effective antiviral immune responses [42] , was analyzed using splenocytes extracted on day 28. the svlp sample showed a significant increase in the ifn-g mrna level as compared to the control, free protein, and the wiv vaccine samples (fig. 4e) , demonstrating superior promotion of antigen-specific cellular immunity. we further examined the effect of the different vaccinations in protecting against a viral challenge. clinical scores evaluated based on stamina, posture, and voice show that the svlp group had the lowest overall symptoms, on par with animals vaccinated with the wiv formulation (fig. 5a,b) . in comparison, vaccination with the free protein formulation was less effective and highly variable in moderating the disease symptoms. on day 28, necropsies were performed to examine the tracheas and kidneys, which are characteristic sites for infections by ibv [43] . as indicated in the gross lesion photos, the best antiviral protection was observed in the svlp-immunized group, whereas organs from the free protein group and the wiv vaccine group showed observable mucus secretion and petechiae in tracheas (fig. 5d, upper panel, arrowed) and swollen lesions and hemorrhages in kidneys (fig. 5d, lower panel, arrowed) . the prophylactic effect of the svlp vaccination was further demonstrated by examining the viral load in kidneys. analysis by quantitative rt-pcr showed that immunization with svlps more consistently reduced the viral content, resulting in the lowest relative viral mrna expression across the animal samples (fig. 5c) . the results further corroborate the protective effect by the svlp vaccination, which enhanced both humoral and cellular immunity for increased protection against the viral challenge. coronavirus spike proteins are the primary antigenic signatures on coronaviruses as they contribute to the characteristic crown-like morphology underlining this virus family. as these proteins comprise the outermost layer of coronaviruses, the spike proteins have a pivotal role in viral pathogenesis and are recognized as the primary target for vaccine preparations [44] . present vaccination strategies for coronaviruses include recombinant viruses and viruslike particles, and there is a continuing effort in developing new strategies for improving vaccine potency and safety [22] . to the best of our knowledge, incorporating coronavirus spike protein with synthetic nanoparticles has not been previously explored. by exploiting the high surface energies of synthetic nanoparticles, spontaneous assembly of svlps covered with ibv spike proteins were demonstrated. the strong particle/antigen association resulted in virus-sized particulates displaying ibv spike proteins, and the svlps elicited strong immune protection against a live ibv challenge. the enhanced immunopotentiation by the particle carrier is consistent with previous studies and echoes the curious observation that gold nanoparticles not only promote humoral but also cellular immune responses upon association with antigens [14, 15] . as the increased cellular immune response suggests that the nanoparticles may play a role beyond a passive antigen carrier, future studies examining the impact of nanomaterials and nanoparticle surface energies on immunological interactions are warranted. it should be noted that the phenomenon of protein corona formation is an evolving field of study in which scientists continue to examine nanomaterials in biological medium with increasing complexity [45e47]. subtle changes on the environment and on nanoparticle properties can have dramatic and unpredictable impact on the overall corona identity with significant biological implications. to demonstrate a practical utility for the protein corona phenomenon, the present study adopts a reductionist approach in examining protein-particle interactions. aunps are incubated in a highly controlled condition with proteins of a singular species to form svlps with virus-mimetic features, and the dynamics of such association are expected to vary with different biomolecules and nanomaterials [48] . in general, inorganic nanoparticles promote stronger protein adsorption as compared to organic nanoparticles as inorganic nanoparticles tend to have higher surface energies. decreasing particle size also tends to increase biomolecule interactions as it increases radii of curvature of nanoparticle surfaces. other forces, such as electrostatic interactions, van der waals forces and covalent interactions all play intertwining roles in governing the nano-bio interface, and factors including nanoparticle functionalizations, buffer conditions, and biomolecule species have significant impact on the corona formation [48] . nonetheless, in a controlled and optimized condition, the phenomenon may be exploited to facilely prepare formulations with defined characteristics and favorable biological performance. the present work takes advantage of this spontaneous interaction between nanomaterials and biomolecules towards improving vaccine development. this strategy may find practical applications in disease management against coronaviruses as well as other infectious threats. in summary, we demonstrate by incubating viral antigens with synthetic nanoparticles in optimized conditions, spontaneous formation of protein corona induces the assembly of virus-like nanostructures with viral antigens encasing the particulate core. results from the present study validate the successful preparation of svlps via nanoparticles' innate tendency to induce protein coating. in comparison to typical virus-like particle preparations, the present strategy offers practical advantages owing to its simple and facile process. amidst the growing health threats of coronavirus infections as well as the ongoing economic impact of ibv infections, virus-like particles are garnering increasing scientific interest as vaccine candidates owing to their improved efficacy in comparison to subunit antigens [49, 50] . in the present study, vaccination with the svlps resulted in enhanced humoral and cellular immune responses, improving protection against an avian model of coronavirus infection as compared to free protein antigens and a commercial wiv vaccine. strong immunity against the viral challenge following svlp vaccination was evidenced by multiple criteria, including improved physical symptoms, reduced organ lesions, and decreased overall viral load. the enhanced immunopotentiation by the svlps is attributable at least in part to increased lymphatic delivery 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s proteins the study was supported by the ministry of science and technology (103-2321-b-002-066, 104-2321-b-002-023, 105-2321 -b-001-055) and national taiwan university (104r7320). key: cord-354547-eomm1sl5 authors: wang, jibin; fang, shouguo; xiao, han; chen, bo; tam, james p.; liu, ding xiang title: interaction of the coronavirus infectious bronchitis virus membrane protein with β-actin and its implication in virion assembly and budding date: 2009-03-16 journal: plos one doi: 10.1371/journal.pone.0004908 sha: doc_id: 354547 cord_uid: eomm1sl5 coronavirus m protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. the m protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. in this study, we showed co-purification of the m protein from coronavirus infectious bronchitis virus (ibv) with actin. to understand the cellular factors that may be involved in virion assembly, budding and maturation processes, ibv m was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. this interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids a159 and k160 on the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system blocks the infectivity of the clone, although viral rna replication and subgenomic mrna transcription were actively detected. disruption of actin filaments with cell-permeable agent cytochalasin d at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. however, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. this study reveals an essential function of actin in the replication cycle of coronavirus. enveloped viruses acquire their envelope by budding from the host cell. in this process, viral envelope proteins gather at a special membranous structure and cooperate with other viral components to induce budding [1] . for example, some viruses including human immunodeficiency virus bud from the plasma membrane and release the virion from host cells by pinching-off. others are budding at intracellular membranes [2, 3] . in this way, virions are wrapped within intracellular membrane-bound compartments, such as the endoplasmic reticulum (er) and golgi apparatus, and the newly budded viruses exit the cell by using the cellular secretory pathway [2] . however, the detailed mechanisms of viral assembly and budding, especially the host factors that are involved in these processes, are yet to be revealed for many viruses. in this study, we report that interaction between coronavirus membrane protein (m) and actin with functional implication in facilitating virion assembly and budding. coronavirus is an enveloped virus with a large, positivestranded rna genome of about 27 to 31 kilobases in length. the avian coronavirus infectious bronchitis virus (ibv) belongs to the third group of coronaviruses genus. like many other coronavriuses, ibv virion is built from four structural proteins, including the nucleocapsid (n) protein with which the genomic rna is packed, the spike (s) protein that forms the prominent coronavirus spikes, the m protein which is the most abundant component of coronavirus, and the envelope (e) protein which is a minor but yet critical component in virion assembly [4] . some group ii coronaviruses also encode an additional structural protein, the hemagglutinin-esterase (he). coronaviruses are known to assemble and bud at membranes of the intermediate compartment (ic) , locating between the er and golgi complex [5] . the m protein is a type iii membrane protein and a key player in coronavirus assembly. it spans the membrane bilayer three times, leaving a short amino-terminal domain on the virion exterior surface (or exposed luminally in intracellular membranes) and a large carboxy-terminal tail in the virion interior (or in the plasma) [6] . lateral interactions between m proteins are thought to mediate the formation of the virion envelope [7] . when expressed alone, m protein accumulates in the golgi complex in the form of homomultimeric complexes [8, 9] . however, in combination with the e protein, m is retained in the budding compartment and incorporated into virus-like particles (vlps) with similarity in size and shape to authentic virions, demonstrating that the m and e proteins are the minimal requirements for envelope formation for most coronaviruses, [10] . the m protein appears to interact with s and he proteins, and the s-m-he protein complexes can be detected in cells infected with the bovine coronavirus [6] . the m protein was also shown to interact with the mouse hepatitis virus (mhv) nucleocapsid consisting of the genomic-size mrna 1 and n protein in a pre-golgi compartment, probably at the er membrane. it may interact directly with the genomic rna through the packaging signal, initiating the m-nucleocapsid interaction [11] . there is also a detectably direct interaction between m and n proteins in the nucleocapsid, which may further stabilize the m-genomic rna interaction [11] . actin is the most abundant protein in a typical eukaryotic cell, accounting for about 15% in some cell types [12] . the protein is highly conserved, differing by no more than 5% between species as diverse as algae and humans. it polymerizes in a helical fashion to form actin filaments (or microfilaments) that form the cytoskeleton, a three-dimensional network inside a eukaryotic cell. actin filaments provide mechanical support for the cell, determine the cell shape, enable cell movements (through lamellipodia, filopodia, or pseudopodia), and participate in certain cell junctions, in cytoplasmic streaming and in cell contraction during cytokinesis [13] . in the present study, actin was shown to be co-purified with the ibv particles and was identified as a potential interacting protein of the ibv m protein. the interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescent staining. mutation of amino acids a159 and k160 in the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system showed no infectious virus could be recovered, although viral rna replication and subgenomic mrna transcription were detected. furthermore, treatment of the infected cells with cell-permeable agent cytochalasin d at early, but not late, stages of the replication cycle showed no release of the virion to the cultured media, suggesting that disruption of actin filaments might block virion assembly and budding. yeast two-hybrid screening yeast two-hybrid screening was performed with the pretransformed matchmaker human bone marrow cdna library (clontech) according to the instructions of the manufacturer. in brief, the bait plasmid was transformed into yeast strain ah109, and transformants containing the bait plasmid were mated with the pretransformed cdna library. candidates were initially selected on sd-ade/-his/-leu/-trp plates, and plasmid dna was isolated from positive clones and sequenced according to the instructions of the manufacturer. h1299 and vero cells were cultured in rpmi-1640 and complete dulbecco's modified eagle's medium (invitrogen), respectively, supplemented with 10% new born calf serum (sterile) and 1% penicillin/streptomycin (invitrogen) and maintained at 37uc in humidified 5% co 2. constructs containing plasmid dna under the control of a t7 promoter were transiently expressed in mammalian cells using a vaccinia virus-t7 system. briefly, 90% monolayers of h1299 cells were infected with 10 plaque forming units (pfu)/cell of the recombinant vaccinia virus (vtf-3), which express the t7 rna polymerase gene, for 2 hours at 37uc prior to transfection. the plasmid dna was transfected into vtf-3 infected cells using lipofectamine 2000 reagent according to the instructions of the manufacturer (invitrogen). hela cells transfected with appropriate plasmids were lysed with tntg lysis buffer (30 mm tris-hcl ph 7.4, 150 mm nacl, 1% np40, and 10% glycerol) in the presence of 16protease inhibitor mixture (sigma) at 24 hours post-transfection. total cell lysates were immunoprecipitated with appropriate antibodies for 2 hours at 4uc and further incubated for 2 hours at 4uc after adding buffer-balanced protein a agarose beads. the beads were washed three times and subjected to electrophoresis on sds-12% polyacrylamide gel. ibv m protein was transiently expressed in h1299 cells grown on 4-well chamber slides (iwaki). after rinsing with phosphatebuffered saline (pbs), cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% triton x-100, followed by incubating with specific antibodies diluted in fluorescence dilution buffer (pbs with 5% newborn calf serum) at room temperature for 2 hours. cells were then washed with pbs, incubated with fitc-conjugated anti-rabbit secondary antibodies (dako) in fluorescence dilution buffer at 4uc for 1 hour and with alexa fluor 488 phalloidin (molecular probe) at rt for 20 minutes before mounting. confocal microscopy was performed on a zeiss microscope. samples were lysed with 26sds loading buffer and subjected to 10% sds-page. proteins were transferred to pvdf membrane (bio-rad) by using a semi-dry transfer cell (bio-rad, trans-blot sd), and blocked overnight at 4uc with 10% nonfat milk in pbs-t. the membranes were probed with specific primary antibodies followed by anti-mouse or anti-rabbit secondary antibodies conjugated with harderadish peroxidase (sigma). membranebound antibodies were detected with the enhanced chemiluminescence (ecl) detection reagents (amersham, uk). the virus was layered onto 20% tne-buffered sucrose solution (tne buffer: 50 mm tris-hcl, ph 7.4, 100 mm nacl, 1 mm edta) and centrifuged at 175,0006g for 3 hours at 4uc (cushion). the resulting virus pellets were resuspended in tne buffer, layered onto 10-50% linear sucrose gradient prepared with tne buffer, and centrifuged at 175,0006g for 18 hours at 4uc. aliquots of fractions starting from the top of the gradient were analyzed by sds-page. in vitro assembly and transcription of full-length cdna clones were carried as previously described [14] . briefly, plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d and e). bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). all the fragments were ligated with t4 dna ligase at 16uc overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.8% agarose gels. full-length transcripts and n transcripts (using a linearized pkt0-ibvn containing ibv n gene and the 39-utr region as templates) were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tex) according to the manufacturer's instructions. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs (14) . rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and electroporated with one pulse at 450 v, 50 mf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbscontaining dmem in a 60 mm dish and further cultured in dmem without fbs. reverse transcription, rt-pcr, and real-time pcr viral rna was reverse-transcribed to cdna using superscript iii (invitrogen) with modification to the protocol as follows: random hexamers (300 ng) and total rna (5 mg) were incubated for 10 minutes at 70uc. the remaining reagents were added according to the manufacturer's recommendation and the reaction was incubated at 55uc for 1 hour followed by 20 minutes at 70uc to inactivate the rt. for rt-pcr, a forward primer in the leader sequence and a reverse primer were used to generate a product by pcr. quantitative real time rt-pcr was conducted using smart cycler ii (cepheid) with sybr green (cepheid) to detect subgenomic cdna with primers (7.5 pm) optimized to detect a product spanning the leader sequence to the 59 end of m gene or genomic cdna with primers (7.5 pm). the cdna from the rt reaction of each virus was diluted 1:10, and 1 ml was used for each pcr, with a total reaction volume of 25 ml. m gene was cloned by digesting pibvm with ncoi and ecori, and inserted into ecori/ncoi digested pgbkt7 to generate pgbkt7-m. the pcr fragment for actin was amplified by using primers 59-cgcggatccatggatgatgatatcgccgcg-39 and 59-ccgctcgaggaagcatttgcggyggacgat-39. the pcr fragment was digested with bamhi and xhoi and ligated into bamhi and xhoi digested pxj-myc to generate pxj-myc-actin. the deletion constructs md1, md2, md3, md4 and md5 were made by two rounds of pcr as described previously [15] . the primer used for md1 is 59-aactgcagttaagc-aagccactgaccctc-39. the primers used for md2 are 59-tcttttgtaggttataagtgtgaaccagac-39 and 59-gtctggttcacactta taacctacaaaaga-39. the primer for md3 is 59-aactgcagccgcttt ggtcaccag-39. the primers for md4 are 59-tgtgagggtccagac-cacttg-39 and 59-caagtggtctggaccctcaca-3. the primers for md5 are 59-ggtcagtggc tttgtgaa-ccagac-39 and 59-gtctggttcacaaagccactgacc-3. all constructs were confirmed by automated nucleotide sequencing. in a previous study, it was observed that a cellular protein with migration properties on sds-page similar to actin was consistently co-purified with highly purified ibv particles [16] . however, as no suitable antibodies against actin were readily available at the time, the identity of this protein was not established. this issue was revisited in this study. confluent monolayers of vero cells were infected with ibv at a multiplicity of infection of approximately 2 pfu/cell. the supernatants were collected at 18 hours post-infection and centrifuged at 4,000 rpm for 20 minutes to remove cell debris. the virus particles were spun down by ultracentrifugation through a 2 ml sucrose cushion (20%), and purified using a 10-50% sucrose gradient. following ultracentrifugation, 11 fractions were collected from top to bottom, and the presence of viral proteins was checked by western blot with anti-n antibodies, showing that the ibv n protein was detected in fractions 7-11 with majority of the protein located in fractions 8-10 ( fig. 1, top panel) . analysis of the same fractions by western blot with anti-actin antibodies showed that actin appeared in the same fractions as n protein, with the majority of the protein detected in fractions 8-10 ( fig. 1 , middle panel). fractionation of total lysates from cells without ibv infection under the same conditions showed that actin was mainly detected in fractions 3-4 ( fig. 1, bottom panel) . these results demonstrate that actin could indeed be co-purified with the virus particles. however, it is currently uncertain if actin could be incorporated into the virions. in an attempt to search for ibv proteins that may interact with b-actin, ibv structural and nonstructural proteins were used as baits to screen a human cdna library in yeast two-hybrid screening. among all the ibv proteins used, only the c-terminal cytoplamsic portion of the ibv m protein was able to interact with b-actin. to confirm the interaction by co-immunoprecipitation, the fulllength cdna for b-actin was amplified by rt-pcr from hela cells, cloned into an expression vector with a c-myc tag at its nterminus (myc-actin), and co-transfected into hela cells with the ibv m. analysis of cells expressing the myc-tagged actin either on its own or together with the m protein by western blot with anti-myc monoclonal antibody showed the detection of the myctagged actin (fig. 2, lanes 1 and 3) . similarly, analysis of cells expressing the m protein either on its own or together with the myc-tagged actin by western blot with anti-m polyclonal antibodies showed the detection of the full-length glycosylated (two upper bands) and unglycosylated (25 kda) forms of the m protein (fig. 2, lanes 5 and 6) . the same cell lysates were then subjected to immunoprecipitation with anti-myc antibody. western blot analysis of the precipitates with the same anti-myc antibody showed the detection of the myc-tagged actin expressed either on its own or together with the m protein (fig. 2, lanes 7 and 9) . western blot analysis of the same precipitates with anti-m antibodies showed the detection of the m protein only in cells coexpressing the two proteins (fig. 2, lane 12) . no detection of the m protein was found in cells expressing either the m protein or mycactin alone (fig. 2, lanes 10 and 11) . these results confirm that the ibv m protein could indeed interact with actin. to map the region in the m protein responsible for the interaction with actin by yeast two-hybrid screening, five deletion and one mutation constructs were made (fig. 3a) . in figure 3a , md1 contains the amino acid sequence from 104 to 159 (with deletion of amino acids 160 to 225); md2 contains the deletion of amino acids 104-159; md3 contains the amino acid sequence from 104 to 192 (with deletion of amino acids 193 to 225); md4 contains the deletion of amino acids 155-162; and md5 contains the deletion of amino acids 159-160 (fig. 3a) . the mutant construct mm1 contains the mutation of amino acids 159 and 160 from alanine and lysine to proline and glutamic acid, respectively (fig. 3a) . introduction of individual deletion and mutant constructs into the yeast stain ah109 together with pact-actin showed growth of wild type and md3 constructs on sd-trp/-leu/-his/ -ade selective plates (fig. 3b) . however, none of the other deletion and mutant constructs could grow on the same plate (fig. 3b) . western blot analysis showed similar expression levels of wild type and mutant constructs in yeast (data not shown). as md5 contains the minimal deletion of two amino acids, this study maps the actinbinding site on the m protein to the region containing amino acids a159 and k160. co-immunoprecipitation was then carried out by expressing the myc-tag wild type m (c-terminal domain from amino acid 104 to 225, myc-m) and myc-mm1 in cells. western blot analysis with anti-myc monoclonal antibody showed the expression of myc-m and myc-mm1 at a similar level (fig. 3c, upper panel, lanes 1 and 2) . the same cell lysates were then subjected to immunoprecipitation with anti-actin antibody, and western blot analysis of the precipitates with anti-myc antibody showed the presence of myc-m but not myc-mm1 (fig. 3c, lower panel, lanes 1 and 2) . the detection of myc-m in the immunoprecipitates was greatly enhanced by co-expression of myc-m and actin (fig. 3b, lane 4) . colocalization of the m protein with actin in cells treated with cytochalasin d as a type iii membrane protein with three transmembrane domains, the ibv m protein is mainly localized to the golgi apparatus in virus-infected cells and in cells expressing the m protein [8] . cytochalasin d is a cell-permeable fungal toxin. it can bind to the barbed end of actin filaments and inhibit both the association and dissociation of actin subunits, resulting in the disruption of actin filaments and inhibition of actin polymerization. to test the subcellular localization of the m protein in cells treated with cytochalasin d, h1299 cells were transfected with m construct and 12.5 mg/ml of cytochalasin d was added to the cells at 4 hours post-transfection. cells were permeabilized with 0.2% triton x-100 and stained with anti-m antibodies at 48 hours posttransfection. the same cells were also stained with alexa fluor 488 phalloidin, a specific dye for actin (molecular probe). staining of cells treated with dmso and alexa fluor 488 phalloidin showed a normal three-dimensional network (fig. 4, upper panels) . phalloidin showed that the regular cell actin filaments were destroyed (fig. 4, lower panels) , displaying random distribution of actin dots in cells without the m protein expression. in cells expressing the m protein, both m protein and actin were found mainly in the golgi area with fairly well overlapping of the two staining patterns (fig. 4, lower panels) . effects of deletion and mutation of amino acid residues a159 and k160 on the replication, budding and release of ibv two approaches were used to test the effect of deletion and mutation of amino acid residues a159 and k160 in the m protein on the replication, budding and release of ibv. first, the a159-k160 deletion and mutation were introduced into a full-length ibv infectious clone, and the effect of this minimal deletion and mutation on the replication of viral rna and the recovery of infectious ibv was analyzed. introduction of full-length transcripts derived from wild type (wt), the a159-k160 deletion construct (md5) and a159-p/k160-e mutation construct (mm1) into vero cells by electroporation showed efficient recovery of infectious ibv from cells transfected with wild type transcripts. however, it was consistently observed that no infectious virus could be recovered from cells transfected with either md5 or mm1 transcripts. as no infectious virus was recovered from cells transfected with md5 and mm1 transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in the transfected cells. the primer pair (59-14931 gcttatc-cact agtacatc 14949 -39 and 59-15578 cttctcgcacttc-tgcactagca 15600 -39) was chosen to amplify the negative strand ibv sequence from nucleotides 14931 to 15600 by the rt-pcr. if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 5a , the 670 bp rt-pcr fragments were obtained from cells transfected with both wild type and mutant transcripts at 24 and 72 hours posttransfection, respectively (upper panel, lanes 2-7). interestingly, the amounts of the fragment detected from cells transfected with md5 and mm1 transcripts were increased at 72 hours posttransfection (fig. 5a , upper panel, lanes 3-4 and 6-7), demonstrating the replication of the transfected mutant transcripts. no rt-pcr fragment was detected in cells without transfection (fig. 5a, upper panel, lane 1) . rt-pcr amplification of subgenomic mrnas was then carried out to check if subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts at 24 and 72 hours postelectroporation. the forward primer (59-acttaagataga-tatta a-39) used in this reaction corresponds to the leader sequence from nucleotides 26-46 in the genomic rna and the downstream primer (59-ctctggatccaataacctac-39) covers the ibv sequence from nucleotides 24784 to 24803. if transcription of subgenomic mrnas occurs, a 415 bp pcr product corresponding to the 59-terminal region of the subgenomic mrna4 would be detected. as shown in fig. 5a , a dominant 415 bp was observed in cells electroporated with wild type full-length transcripts at two time points (lower panel, lanes 2 and 5). in cells transfected with md5 and mm1 transcripts, a much weaker band was detected in cells transfected with the two mutant transcripts at 24 hours post-transfection (fig. 5a, lower panel, lanes 3-4) . similarly to the results described above, the amounts of the subgenomic rna 4 fragment detected from cells transfected with md5 and mm1 transcripts were increased approximately 3 to 5 fold, respectively, at 72 hours posttransfection based on real time pcr assay (fig. 5a, lower panel, lanes 3-4 and 6-7) . as a negative control, no detection of the amplified fragments was obtained from a mixture of rna templates containing the in vitro transcribed rna and total rna extracted from mock infected cells (fig. 5a, lower panel, lane 1) . in the second approach, plasmids containing wild type m (pibvm) and the a159-k160 deletion m sequences (pmd5) were transfected into h1299 cells using the vaccinia/t7 recombinant antibodies, respectively (fig. 5b) . in cells transfected with both wild type and md5 constructs with or without ibv infection, detection of similar amounts of m protein was observed (fig. 5b, top two panels, lanes 1 and 2) , suggesting that both constructs were expressed at similar efficiencies. however, much more n protein was detected in the infected cells transfected with wild type m construct than that in cells transfected with the md5 mutant (fig. 5b, middle panel, lanes 1 and 2) . as a loading control, similar amounts of actin were detected in cells transfected with both constructs (fig. 5b, bottom panel) . these results confirm that expression of the a159-k160 deletion m protein significantly reduces the infectivity of ibv, suggesting that expression of this mutant protein could inhibit the production of infectious virus in a dominant-negative manner. to rule out the possibility that the deletion and mutation may render detrimental effect on the overall structure of the m protein and its interaction with other viral proteins that are essential for virion assembly, interaction of the mm1 mutant with the e protein was tested by co-immunoprecipitation. as shown in fig. 5c , western blot analysis with anti-ibv e protein antibodies showed the presence of e protein in cells expressing the protein on its own or together with wild type or mm1 mutant protein (top panel). immunoprecipitation of the same lysates with anti-m antibodies and subsequent analysis of the precipitates by western blot with anti-m antibodies led to the detection of wild type or the mm1 mutant proteins in cells expressing m forms either alone or together with the e protein (fig. 5c, middle panel) . western blot analysis of the same precipitates with anti-ibv e antibodies, however, showed the presence of e protein only if it was coexpressed with either wild type or the mm1 mutant protein (fig. 5c, bottom panel) . these results confirm that the a159-p/ k160-e mutations did not affect the interaction between ibv m and e proteins. to define more precisely the stage of the viral replication cycle that is facilitated by the interaction between m protein and actin, the effects of disrupting actin filaments by cytochalasin d on the replication, budding and release of ibv were tested by detailed time-course experiments. confluent monolayers of vero cells in six-well plates were infected with ibv at a multiplicity of infection of approximately 3 pfu/cell. at 0, 4, 8, 12 and 16 hours postinfection, either 12.5 mg/ml of cytochalasin d or equal volume of dmso was added to the cells. the supernatants and cell pellets were separately harvested at 24 hours post-infection and the presence of ibv n protein was checked by western blot with anti-n antibodies. typical n protein profiles, including the full-length and posttranslationally modified forms of the protein, were detected in the total cell lysates (fig. 6, top panel) . in cells treated with cytochalasin d, slightly less amounts of the n protein were detected when cytochalasin d was added to the cells at 0, 4, 8 and 12 hours post-infection, respectively (fig. 6, top panel, lanes 1-4) . no obvious difference in the expression of n protein was seen when cytochalasin d was added at 16 hours post-infection (fig. 6 , top panel, lane 5). western blot analysis of actin was included as a loading control (fig 6, middle panel) . analysis of the n protein in the clarified supernatants showed the detection of a single n protein species that represents the n protein incorporated into the virion particles in cells treated with dmso alone (fig. 6, bottom panel, lanes 6-10) . in supernatants collected from cells treated with cytochalasin d, much reduced amounts of the n protein were detected in cells treated with the reagent at 12 and 16 hours postinfection, respectively (fig. 6, bottom panel, lanes 4 and 5) . no n protein was detected in supernatants harvested from cells treated with cytochalasin d at 0, 4 and 8 hour post-infection, respectively (fig. 6, bottom panel, lanes 1-3) . coronavirus m protein plays essential roles in virion assembly and budding [18, 19] . the protein itself cannot bud. however, with the help of e protein, vlps may be formed at the ic membranes. as coronaviruses do not contain a matrix protein that underlines the membrane, the m protein may serve as a matrixlike protein. it is proposed that the formation of coronavirus envelope is dominated by lateral interaction between m molecules that form a two-dimensional lattice in intracellular membranes [20] . the m-m interaction is essential but not sufficient for coronavirus envelope assembly. free energy is needed to generate and stabilize membrane curvature, suggesting that interaction of m protein with host proteins would be a must [21] . to support this speculation, the results present in this study demonstrate that interaction of the ibv m protein with b-actin is essential for virion assembly and budding. the interacting region in the m protein was pinpointed by deletion and mutation studies. as md3 is able to interact with actin, but md1 and md2 could not, it was reasoned that the domain responsible for this interaction may be located in the region covering amino acids 159-160. this region was chosen for further deletion and mutational analysis, confirming that it is indeed involved in the interaction with actin. however, the two amino acids are not conserved in other coronavirus m proteins. at present, we do not know whether interaction between coronavirus m protein and actin is a general phenomenon for all coronaviruses, or is just ibv-specific. available evidence suggests that the host cytoskeleton, especially actin, is involved in the budding process of several animal viruses [22] . actin has been identified in many enveloped viruses, such as rous sarcoma virus, mouse mammary tumor virus, sendai virus and measles virus. in the case of measles virus, actin was originally thought as a cellular contaminant, but was later demonstrated to be an internal component of the virus [23] [24] [25] . actin was found in the purified paramyxovirus particles and actin filaments were seen in association with the budding virions by electron microscopy [24] [25] [26] . polymerized actin was also found in hiv preparations [27, 28] . the functional implication for the incorporation of actin into these virus particles remains unclear. one possibility is that actin polymerization serves as an additional force for membrane bending during budding [1, 29] . as an illustrative example, actin was shown to control the movement of measles virus envelope proteins on the surface of infected cells [30] . interaction of actin with viral components was also reported in several other viral systems. one example is the interaction of the nuclocapsids of murine mammary tumor virus with actin, which was required for extruding the virus particles from virus-infected cells [31] . the m protein of newcastle disease virus could interact with actin in vitro [1, 32] . the sars-cov n protein could aggregate elongation factor 1a through direct interaction, leading to the inhibition of protein translation and cytokinesis by blocking f-actin bundling [33] . it was also reported that the sars-cov n protein could induce apoptosis and actin reorganization in mammalian cells under stress conditions [34] . the results shown in this study demonstrate that actin could be co-purified with ibv virions and that the ibv m protein is associated with actin, suggesting that actin is likely to be incorporated into ibv virions through its interaction with the m protein. however, more conclusive data, such as immunogold labeling of highly purified virions, are currently lacking. it is interesting that ibv replication cycle was disturbed in cells treated with cytochalasin d. cytochalsin d could prevent actin from polymerization, leading to malfunction of actin. when actin was disrupted, much less ibv virions were released from the cells into the culture medium, demonstrating that actin plays a very important role in the ibv replication cycle. two possible mechanisms were considered. first, actin may be involved in the formation of the m lattice that may promote and stabilize its curving, resulting in the enhancement of some late processes, such as virion budding and release. second, the mhv m protein was shown to be recycled from the golgi complex back to early compartments, such as the er and ic, which are functionally important in coronavirus infected cells [21] . therefore, interaction of actin with m protein may help the retrograde transport of m protein from golgi apparatus to some early compartments and provide more m protein for virion assembly and budding. the actin-binding proteins are grouped into 60 distinct classes based on primary structures [35] . the sequence of the actinbinding sites ranges from 10-30 residues, which shows no obvious homology among different classes [36] . for example, aldolase, a glycolytic enzyme, binds actin filaments to concentrate enzyme and substrate [37] . the sequence 32-adestgsiakrlqsig-tente-52 of aldolase has been identified as the actin-binding motif [38] . furthermore, carcinoembryonic cell adhesion molecule 1 (ceacam1) was found to bind f-actin. the actin binding site is flhfgktgssgplq, which is not similar to other existing actin-binding sequences [39] . coronavirus m protein is an essential and predominant component of the virions. it plays pivotal roles in virion assembly and budding by interaction with other viral components. firstly, the monomers of m could interact with each other particularly via the transmembrane domains [21] . besides, the m-n interaction region was narrowed to the 16 residues adjacent to the carboxy terminus of tgev m protein [40] . however, the specific amino acids on m protein responsible for m-s and m-e interactions are still enigmatic. in the present study, deletion or mutation of amino acids a159 and k160, which are essential for the interaction of m protein with actin, is detrimental to the recovery of ibv from an infectious ibv clone due to a defect in late stages of the ibv replication cycle. although, the deletion or mutation of amino acids a159 and k160 could also interact with another structural protein e, we cannot exclude the possibility that this region is essential for maintaining the integrity of the m protein and involved in the interactions with other viral components. nevertheless, it lends support to the conclusion that interaction of the ibv m protein with actin is essential for the completion of the ibv infection cycle. more evidence is derived from the study using cytochalasin d. similar amount of viral proteins were detected in the total cell lysates but were not detected in supernatants when cytochalasin d was added early in the viral replication cycle suggests that actin filaments are essential for some early events, such as virion assembly and budding, during the assembly and maturation processes of ibv. as ibv proteins can be efficiently detected in the supernatants when cytochalasin d was added at 12 and 16 hours post-infection, respectively, it suggests that actin filaments are unlikely involved in the release of the ibv particles. instead, our data support that actin filaments may be involved in the virion assembly and budding process of the coronavirus replication cycles. as coronavirus m protein could bind to the viral genomic rna through packaging signal [41] and rna replication in the cytoplasm would rely on the support of actin 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hemagglutinin at the surface of chronically infected cells the mechanisms of appearance of viral glycoproteins at cell surface membrane interaction of cellular tubulin with sendai virus m protein regulates transcription of viral genome the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors xin repeats define a novel actin-binding motif structural relationships of actinbinding proteins opposite effects of alpha-actinin and of fructose 1,6 -bisphosphate aldolase on the microfilament network. the role of orthophosphate revisited identification of an actin binding region in aldolase carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal cellular factors in the transcription and replication of viral rna genomes: a parallel to dna-dependent rna transcription key: cord-317587-rrx2r4n2 authors: fan, wensheng; tang, ning; dong, zhihua; chen, jiming; zhang, wen; zhao, changrun; he, yining; li, meng; wu, cuilan; wei, tianchao; huang, teng; mo, meilan; wei, ping title: genetic analysis of avian coronavirus infectious bronchitis virus in yellow chickens in southern china over the past decade: revealing the changes of genetic diversity, dominant genotypes, and selection pressure date: 2019-09-26 journal: viruses doi: 10.3390/v11100898 sha: doc_id: 317587 cord_uid: rrx2r4n2 the high mutation rates of infectious bronchitis virus (ibv) pose economic threats to the poultry industry. in order to track the genetic evolutionary of ibv isolates circulating in yellow chickens, we continued to conduct the genetic analyses of the structural genes s1, e, m, and n from 64 ibv isolates in southern china during 2009–2017. the results showed that the dominant genotypes based on the four genes had changed when compared with those during 1985–2008. based on the s1 gene phylogenetic tree, lx4-type (gi-19) was the most dominant genotype, which was different from that during 1985–2008. the second most dominant genotype was ldt3-a-type, but this genotype disappeared after 2012. new-type 1 (gvi-1) isolates showed increasing tendency and there were four aa (qkep) located in the hypervariable region (hvr) iii and one aa (s) insertion in all the new-type 1 isolates. both the analyses of amino acid entropy and molecular evolutionary rate revealed that the variations from large to small were s1, e, m, and n. purifying selection was detected in the s1, e, m, and n gene proteins, which was different from the positive selection during 1985–2008. six isolates were confirmed to be recombinants, possibly generated from a vaccine virus of the 4/91-type or ldt3-a-type and a circulating virus. the estimated times for the most recent common ancestors based on the s1, e, m, and n genes were the years of 1744, 1893, 1940, and 1945, respectively. bayesian skyline analysis revealed a sharp decrease in genetic diversity of all the four structural genes after 2010 and since late 2015, the viral population rapidly rose. in conclusion, the ibvs circulating in southern china over the past decade have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines to counteract the emerging lx4-type and new-type ibvs. infectious bronchitis (ib) is one of the major viral diseases affecting the poultry industry globally. the causative agent avian infectious bronchitis virus (ibv) is a member of the genus gammacoronaviruses, subfamily coronavirinae, family coronaviridae and is prone to mutate. there are multiple genotypes and serotypes of ibv isolates identified worldwide and limited cross-protection confers between serotypes of ibvs [1] [2] [3] [4] [5] [6] , which poses great challenge to the control of ib by vaccination. the ibv has a single-stranded rna genome of approximately 27.6 kb in length [7] and encodes four structural proteins: the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins [8, 9] . the s protein is cleaved into subunits s1 and s2 by proteases [10, 11] . the function of the four structural proteins has been extensively reviewed by others [12] [13] [14] . the genetic analysis based on the s1 gene has become the primary method of classifying ibv strains because of variability and functional importance [15] . however, those previous ibv molecular characterizations that were merely focused on the analysis of the s1 gene or partial s1 gene sequence could not explain the changes in the serotypes and pathotypes of ibv variants [16] . sometimes, single gene analysis even is misleading. therefore, it is necessary to analyze all the structural protein-coding genes s1, e, m, and n simultaneously in order to obtain comprehensive genetic information and molecular mechanism of variation of circulating ibv isolates. a variety of ibv genotypes and variants are distributed globally. so far, a total of seven genotypes comprising 35 distinct viral lineages have been defined worldwide based on the complete s1 gene sequences [17] . ibv strains within a certain country or region are unique even though many countries share some common antigenic types. for example, two distinct lineages that fall in two different genotypes-gi-21 and gii-1-were identified as unique to europe. gi-9, gi-27, and giv-1-genotypes have been implicated in widespread disease disseminations and persistent virus infections in north american [16, 18, 19] . gi-1, gi-11, and gi-16 are currently circulating in south american flocks [20] . lx4-type (qx-type or which was firstly isolated in china in 1996 spread westward invading russia, middle east, and europe, and then becoming the most prevalent genotype in many countries, such as korea, russia, iran, italy, uk, malaysia, sudan, and so on [7, 8, [21] [22] [23] [24] [25] . nowadays, lx4-type and ck/ch/lsc/99i-type (gi-22) appear to be the dominant viruses based on the s1 gene in china [18] . because of differences in breeding variabilities and feeding patterns of chickens in china, the characteristics of circulating ibvs at different times and in different regions are variable. hence, it is necessary to conduct long-term tracking of the ibv circulating isolates in specific geographic regions or countries for the effective control of ib [4, 18, 26] . the appearance of ibv variants was related to high mutation and recombination rates, which result in the generation of genetic diversity and phenotypic heterogeneity. however, ibv evolution is not driven by genetic drift alone. evolution involves two fundamental steps-i.e., generation of genetic diversity and selection [27] . the selection process was affected by multiple factors, such as immune responses, the microenvironment of infected hosts, physical and biosafety conditions [16, 27] . vaccines not only give rise to new variants through recombination, but also impose selection pressure on the evolution of field strains [28] [29] [30] . it is essential for appropriately controlling and prevention of the disease to understand the evolution of ibv [27] . southern china is the major production region of yellow chickens (4.0 billion in 2018 and made a proportion of 37% of the total chickens) in the country. guangxi province, located in southwest of china, has the biggest production of local breeds of chickens (0.75 billion birds in 2018) [31] , and most of the birds are free-range and raised for a longer time (about 120-day-old) at a rather high density, and the chicks are raised in relatively closed environment and lack of ventilation during brooding [32, 33] . also, flocks of various companies, with different chicken breeds and differing ages and vaccination programs located in the same areas are common [32] . these situations surely increase the odds of multiple-infection of several ibv strains including the vaccine strains or/and field strains in flocks of birds [34] . despite the widespread use of mass-type (massachusetts genotype) (h120, h52, ma5, m41, and w93), 4/91, ltd3-a live vaccines and inactivated vaccines, ib has been a continuing problem in vaccinated flocks in these regions [35] . our previous studies found the serotype and genotype diversity of guangxi ibvs from 1985 to 2008 and the important role of vaccine strains in the emerging of new ibv strains via recombination [1, 2, 4] . however, the comprehensive genetic information of circulating ibv strains in this region was unavailable over the past decade. hence, we continued to carry out the genetic analysis of 64 ibv isolates during 2009-2017. the aim was to track the genetic evolutionary trends of ibv field strains circulating over the past decade and their possible causes through relatively new and comprehensive analyses of virus genes, and then provide valuable reference and countermeasure against ibv field breakouts in southern china. sixty-four ibv strains isolated from flocks of yellow chickens during 2009-2017 were analyzed in the present study (table 1 ). all ibv field isolates were obtained from the birds of previously vaccinated flocks with h120, ldt3-a and/or 4/91 vaccines and experienced clinical signs of the ibv infection. all ibvs were isolated and propagated as previously described [4] . nanning jx292005 jx273219 jx014368 kj872794 gx-gl11077 2011 17 guilin jx291992 jx273212 jx273186 kj940507 gx-gl11078 2011 11 guilin jx291993 jx273209 jx273187 kj940508 gx-gl11079 2011 n/a guilin jx291994 jx273208 jx273188 kj940509 gx-nn-4 2011 32 nanning jx291983 jx273199 jx273176 kj940515 gx-nn-5 2011 77 nanning jx291984 jx273200 jx273177 kj940517 gx-nn-6 2011 18 nanning jx291985 jx273207 jx273178 kj940519 gx-nn-8 2011 27 nanning jx291986 jx273201 jx273179 kj940520 gx-nn-9 2011 38 nanning jx291987 jx273202 jx273180 kj940521 gx-nn-11 2011 45 nanning jx291988 jx273203 jx273182 kj940523 gx-nn-13 2011 39 nanning jx291989 jx273204 jx273183 kj940525 gx-nn-14 2011 34 nanning jx291990 jx273205 jx273184 kj940526 gx-nn-15 2011 15 nanning jx291991 jx273206 jx273185 kj940527 gx-nn11034 2011 12 nanning jx291999 jx273223 jx273191 kj940535 gx-yl11072 2011 15 yulin jx291995 jx273210 jx273189 kj940544 gx-yl11073 2011 14 yulin jx291996 jx273211 jx273190 kj940545 gx-nn1201 2012 14 nanning jx436331 jx567014 jx567012 kj940532 gx-nn120079 table 1 . cont. days of age locations a genbank accession numbers s1 n m e gx-nn150019 2015 20 nanning mk887049 mk887118 mk887095 mk887072 gx-qz150024 2015 13 qinzhou mk887057 mk887126 mk887103 mk887080 gx-yl150028 2015 20 yulin mk887060 mk887129 mk887106 mk887083 gx-lz150619 2015 19 liuzhou mk887046 mk887115 mk887092 mk887069 gx-yl150727 2015 73 yulin mk887061 mk887130 mk887107 mk887084 gx-lz160322 2016 10 liuzhou mk887047 mk887116 mk887093 mk887070 gx-yl161022 2016 15 yulin mk887063 mk887132 mk887109 mk887086 gx-yl161015 2016 100 yulin mk887062 mk887131 mk887108 the entire s1, e, m, and n genes were amplified for each ibv strain and the primers used were designed as previously described [4, 36] . the anticipated amplification segments for the s1, e, m, and n genes are 1760 bp, 633 bp, 750 bp, and 1300 bp in lengths, respectively. 2.3. rna extraction and amplification of s1, e, m, and n genes viral rna was extracted and the first cdna strand was synthesized as previously described [4] . the pcr conditions for the s1, m, and n gene amplification were the same as previously described [4] . the pcr conditions for the e gene amplification were 95 • c for 5 min, 35 cycles of 95 • c for 30 s, 52 • c for 30 s, and 72 • c for 30 s, followed by 72 • c for 6 min. the pcr products were analyzed on 1.0% agarose-gel electrophoresis. the pcr products of s1, e, m, and n genes were sequenced by beijing genomics institute (bgi) (shenzhen, china) after cloning. the open reading frames of 64 ibvs were determined and their nucleotide sequences were submitted to genbank database and assigned accession numbers ( table 1) . sequences of 43 reference ibv strains (with the exception of 42 strains for e gene) retrieved from the genbank database were used (supplementary table s1 ). the 27 ibvs isolated during 1985-2008 in guangxi [4] were also analyzed together in order to get a general profile of ibv evolution. the nucleotide and deduced amino acid (aa) sequences of the s1, e, m, and n genes obtained from the ibv isolates were aligned using the editseq program in the lasergene package (dnastar inc., madison, wi, usa) and compared to those of ibv reference strains representing the main well-established lineages and genotypes using the megalign program in the same package. to ensure the scientificity and reliability of the results, we extracted the s1, e, m, and n genes from the complete genome sequences of reference strains. phylogenetic trees based on the aa sequences of s1, e, m, and n genes were constructed using mega version 6.06 according to previous description with nodal support values obtained by posterior probabilities and 1000 bootstrap replicates [37] . aligned nucleotide sequences of s1, e, m, and n genes were subjected to the recombination detection program (rdp 4 version 4.95) to detect potential recombination events by seven algorithms (rdp, geneconv, bootscan, maxchi, chimera, siscan and 3seq) in rdp 4.95 [4] . the detection of recombination breakpoints by at least four of these methods were considered as confirmation of any putative recombination event. the potential recombination events and breakpoints were further verified by similarity plots (simplots) analysis in simplot version 3.5.1 [7, 14] . entropy of amino acid sequences within the s1, e, m, and n proteins of ibv isolates was calculated by bioedit version 7.1.11.0 in order to understand the variation degree of these four structural protein genes [4] . positive selection and positively selected sites within the s1, e, m, and n proteins were analyzed by the single-likelihood ancestor counting (slac), fixed effects likelihood (fel), and internal fixed effects likelihood (ifel) methods of datamonkey version (http://www.datamonkey.org/26/08/2019) [26] to detect whether these proteins have undergone positive selection. the recombinants were excluded in order to reduce a false detection of positive selection. the potential n-glycosylation sites were predicted within the s1, e, m, and n proteins. the analysis was performed using netnglyc server 1.0 software available at http://www.cbs.dtu.dk/services/ netnglyc [38] . the results of aligned sequences were computed by multiple alignment with fast fourier transformation (mafft) [39] . the nucleotide substitution process was modelled independently for each partition with gtr (general time reversible) + g (gamma distribution with a discrete) + i (proportion of invariant sites) based on aic by the jmodel test 2.1.7. bayesian tree reconstructions were performed in beast version 1.8.2. a bayesian skyline coalescent model and strict molecular clock were selected. the bayesian markov chain monte carlo (mcmc) chains of the s1, e, m, and n genes were run for 300 million, 20 million, 100 million, and 200 million generations, respectively. results were analyzed using tracer version 1.5 and confirmed convergence of mcmc chains with 10% of each chain discarded as burn-in and sampled every 10,000 steps [30] . statistical uncertainty (reflected calculating 95% high probability density (hpd) values) and convergence (reflected calculating effective sample size) in parameter estimates were evaluated in tracer version 1.5 program. the posterior sets of trees were summarized as a maximum clade credibility (mcc) tree using tree annotator version 1.8.2 with 10% burn-in and then displayed the created mcc tree using figtree version l.4.3 [40] . the mutation rates and the most recent common ancestor (tmrca) of the aligned sequences were estimated. the change of effective population size over time was inferred by bayesian skyline plots (bsp). the nucleotide and deduced aa sequence similarities of the s1, e, m, and n genes among the 64 isolates during 2009-2017 were 63.2-100% and 57.2-100%, 80.1-100% and 78.3-100%, 85.7-100% and 86.7-100%, and 84.6-100% and 88.3-100%, respectively. compared with h120, the isolates gx-qz150024, gx-lz160322, and gx-nn171125 have higher amino acid sequence similarities of 97.4-99.8%, 93.6-100%, 99.1-100%, and 94.4-100% in the s1, e, m, and n genes, respectively. within the s1 gene, there were 11 different nucleotide lengths (from 1590 to 1638 bp), and the most common lengths were 1620 bp (35/64 isolates, 54.69%) and 1626 bp (18/64 isolates, 28.13%). there were eight types of s protein cleavage site motifs found: rrfrr (22/64), hrrrr (18/64), rrlrr (9/64), rrsrr (7/64), hrrkr (5/64), hrikr (1/64), hrskr (1/64), and rkrkr (1/64) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s1 genes of six isolates (gx-nn09032, gx-nn120079, gx-nn120084, gx-nn120089, gx-qz130064, and gx-qz130065), respectively (supplementary figure s1 ). the s1 gene phylogenetic tree showed that the ibv isolates during 2009-2017 were divided into eight distinct groups (figure 1a ). counts of 17, 14, 7, 7, and 6 out of 64 ibv isolates during 2009-2017 belonged to lx4-type (qx or gi-19), ldt3-a-type (gi-28), 4/91-type (gi-13), mass-type (gi-1), and ck/ch/lsc/99i-type (gi-22), respectively. five isolates (gx-nn1014, gx-nn130059, gx-nn170502, gx-nn170829, and gx-nn171123) in recent years were grouped with taiwan reference strains tw2575/98 as taiwan-i-type (gi-7). six isolates (gx-nn09032, gx-nn120079, gx-nn120084, gx-nn120089, gx-qz130064, and gx-qz130065) and two isolates (gx-nn130021 and gx-yl150727) showed considerable low similarities (57.2-68.1%, 58.8-67.3%) with the above genotypes and belonged to two separate groups new-type 1 (gvi-1) and new-type 2 (gvii-1). viruses 2019, 11, x for peer review 6 of 24 (7/64), hrrkr (5/64), hrikr (1/64), hrskr (1/64), and rkrkr (1/64) among the isolates. there were four aa (qkep) (located in the hypervariable region (hvr) iii) and one aa (s) insertion found in the s1 genes of six isolates (gx-nn09032, gx-nn120079, gx-nn120084, gx-nn120089, gx-qz130064, and gx-qz130065), respectively (supplementary figure s1 ). the s1 gene phylogenetic tree showed that the ibv isolates during 2009-2017 were divided into eight distinct groups (figure 1a ). counts of 17, 14, 7, 7, and 6 out of 64 ibv isolates during 2009-2017 belonged to lx4-type (qx or gi-19), ldt3-a-type (gi-28), 4/91-type (gi-13), mass-type (gi-1), and ck/ch/lsc/99i-type (gi-22), respectively. five isolates (gx-nn1014, gx-nn130059, gx-nn170502, gx-nn170829, and gx-nn171123) in recent years were grouped with taiwan reference strains tw2575/98 as taiwan-i-type (gi-7). six isolates (gx-nn09032, gx-nn120079, gx-nn120084, gx-nn120089, gx-qz130064, and gx-qz130065) and two isolates (gx-nn130021 and gx-yl150727) showed considerable low similarities (57.2-68.1%, 58.8-67.3%) with the above genotypes and belonged to two separate groups new-type 1 (gvi-1) and new-type 2 (gvii-1). the phylogenetic trees of e, m, and n genes of the 64 isolates were segregated into six, four, and six unique groups, respectively (figure 1b-d). and their phylogenetic trees exhibited considerably different topology compared with that of the s1 gene. no obvious geographic differences were found among the 64 isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s2-s5) . the percentages of different genotypes based on s1, e, m, and n genes of ibv isolates in different years were summarized in figure 2 . based on the s1 gene, the ck/ch/lsc/99i-type was the predominant genotype during 1985-2008, but the lx4-type was the predominant genotype circulating in the field during 2009-2017. the ldt3-a-type was the second most dominant genotype, but this genotype disappeared after 2012. based on the e and m genes, the ck/ch/lsc/99i-type was the predominant genotype during 1985-2008, while the ck/ch/lsc/99i-type and lx4-type were the predominant genotypes during 2009-2017. based on the n gene, the ck/ch/lsc/99i-type and the lx4-type were the predominant genotypes during 1985-2008, but only the lx4-type was the predominant genotype during 2009-2017. therefore, our results demonstrated that the ck/ch/lsc/99i-type isolates were the predominant ibvs according to the phylogenetic study of the s1, e, m, and n genes from 1985 to 2008. thereafter, the proportion of lx4-type, ldt3-a-type, and the phylogenetic trees of e, m, and n genes of the 64 isolates were segregated into six, four, and six unique groups, respectively (figure 1b-d) . and their phylogenetic trees exhibited considerably different topology compared with that of the s1 gene. no obvious geographic differences were found among the 64 isolates, while there was a high degree of sequence identity among the isolates in the same period of time (supplementary tables s2-s5) . the percentages of different genotypes based on s1, e, m, and n genes of ibv isolates in different years were summarized in figure 2 . based on the s1 gene, the ck/ch/lsc/99i-type was the predominant genotype during 1985-2008, but the lx4-type was the predominant genotype circulating in the field during 2009-2017. the ldt3-a-type was the second most dominant genotype, but this genotype disappeared after 2012. based on the e and m genes, the ck/ch/lsc/99i-type was the predominant genotype during 1985-2008, while the ck/ch/lsc/99i-type and lx4-type were the predominant genotypes during 2009-2017. based on the n gene, the ck/ch/lsc/99i-type and the lx4-type were the predominant genotypes during 1985-2008, but only the lx4-type was the predominant genotype during 2009-2017. therefore, our results demonstrated that the ck/ch/lsc/99i-type isolates were the predominant ibvs according to the phylogenetic study of the s1, e, m, and n genes from 1985 to 2008. thereafter, the proportion of lx4-type, ldt3-a-type, and new-type strains increased over time. recombination events of the s1, e, m, and n genes of 64 isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s1 gene of six isolates ( figure 3 and table 2 ). gx-nn1011 was derived from recombination between the two lx4-type strains gx-nn6 (major parent) and gx-nn-9 (minor parent). gx-nn1201 was derived from recombination between lx4-type strain qx (major parent) and ck/ch/lsc/99i-type strain saibk (minor parent). gx-nn130048 was derived from recombination between lx4-type strain gx-hc1006 (major parent) and 4/91 strain (minor parent). gx-nn120091 was found to be a recombinant between the new-type 1 isolate gx-nn120089 (major parent) and the vaccine strain ldt3-a (minor parent). gx-nn130003 was found to be a recombinant isolate formed by a major parent isolate gx-yl11072 (ldt3-a-type) and a minor parent isolate gx-yl170717 (ck/ch/lsc/99i-type). the potential parents of gx-yl170805 were proved to be a major parent isolate gx-nn1014 (tw2575/98-type) and a minor parent isolate gx-nn10 (4/91-type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s1 subunit gene in all of recombinant isolates except gx-yl170805 (supplementary figure s2) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure 4) . therefore, the dominant recombinants were the lx4-type isolates from 2009 to 2017. recombination events of the s1, e, m, and n genes of 64 isolates were examined using the rdp in the study. the results showed that recombinant events were found in the s1 gene of six isolates ( figure 3 and table 2 ). gx-nn1011 was derived from recombination between the two lx4-type strains gx-nn6 (major parent) and gx-nn-9 (minor parent). gx-nn1201 was derived from recombination between lx4-type strain qx (major parent) and ck/ch/lsc/99i-type strain saibk (minor parent). gx-nn130048 was derived from recombination between lx4-type strain gx-hc1006 (major parent) and 4/91 strain (minor parent). gx-nn120091 was found to be a recombinant between the new-type 1 isolate gx-nn120089 (major parent) and the vaccine strain ldt3-a (minor parent). gx-nn130003 was found to be a recombinant isolate formed by a major parent isolate gx-yl11072 (ldt3-a-type) and a minor parent isolate gx-yl170717 (ck/ch/lsc/99i-type). the potential parents of gx-yl170805 were proved to be a major parent isolate gx-nn1014 (tw2575/98-type) and a minor parent isolate gx-nn10 (4/91-type). in addition, it was found that a-t rich hotspot sequence atttt (t/a) was near the breakpoint site of the s1 subunit gene in all of recombinant isolates except gx-yl170805 (supplementary figure s2) . in order to confirm the results of the rdp analysis, genomic sequences analyses of the six ibv isolates were carried out by the simplot software, and the results were consistent with those of the rdp analysis ( figure 4) . therefore, the dominant recombinants were the lx4-type isolates from 2009 to 2017. the entropy of the amino acid sequences of s1, e, m, and n genes showed that there were many high entropy amino acid sites within the s1 gene, but a few high entropy amino acid sites were scattered within the e, m, and n genes (supplementary figure s3) . the average entropy values sorted from large to small as follows: s1 (0.4047), e (0.2453), m (0.1149), and n (0.1094). the percentages of entropy value which was bigger than 0.4 in s1, e, m, and n genes were 37.23% (207/556), 24.17% (29/120), 10.57% (24/227), and 11.25% (46/410), respectively. therefore, the largest variation was observed in the s1 gene, and the e gene was also more variable than the m and n genes. the selection profile of s1, e, m, and n proteins of totally 80 (with 11 deleted recombinant strains) ibvs were showed in table 3 . the dn/ds ratio of s1, e, m, and n proteins of eighty isolates were 0.283, 0.266, 0.146, and 0.147, respectively, indicating that the s1, e, m, and n proteins of these ibv isolates had evolved under purifying selection (table 3) . however, a few positively selected sites were detected although most sites were under neutral selection and purifying selection (table 3 and supplementary table s6 ). aa residues 19, 39, 106, 119, 155, and 503 of the s1 protein were consistently highlighted by positive selection models (slac, fel, and ifel) as positive selection sites. similarly, residue 119 of the e protein, residues 12, 16, 17, 47, and 151 of the m protein, residues 7, 10, 235, 342, and 345 of the n protein were identified as positively selected sites. in addition, all of the positively selected sites within s1, e, m, and n genes had high entropy values of larger than 0.6 (supplementary table s6 ). the results showed that there were 14-22, 1-3, 1-2, 1-3 potential glycosylation sites within the s1, e, m, and n proteins (except that gx-nn120084 strain did not have a glycosylation site in the n protein). the comparison of the estimated n-glycosylation sites of s1 protein between the isolates and the reference strains showed that all the strains presented one n-glycosylation site at the residue 239/240 (nfsd) (except ck/ch/lsc/99i-type (gi-22) strains). and at the residue 427/428 (nitl), all the strains presented one n-glycosylation site (except taiwan-type (gi-7) and new-type 1 (gvi-1)). similarly, all the strains presented one n-glycosylation site at e protein residue 11/12 (ngsf) and residue 5/6 (nktl) (except ck/ch/lsc/99i-type and conn-type), all the strains presented one n-glycosylation site at m protein residue 3/4/6 (nctl) and at n protein residue 32 (nasw) (except gx-nn120084 strain) (table 4 and supplementary figure s4 -s7). the mean substitution rates for the s1, e, m, and n genes of the epidemic isolates during 1985-2017 were calculated to be 4.6 × 10 −3 , 4.3 × 10 −3 , 3.9 × 10 −3 , and 3.7 × 10 −3 substitutions/site/year (s/s/y), respectively ( table 5 ), indicating that s1 gene is the most easily mutated and n gene is the most stable among the four structural genes. the estimated times for most recent common ancestor (tmrca) of s1, e, m, and n genes were before 1744.97, 1896. 7, 1940.3, and 1945 .92, respectively ( figure 5 ). based on s1, genotypes of ck/ch/lsc/99i (gi-22), new 1 (gvi-1), mass (gi-1), lx4 (gi-19), 4/91 (gi-13), taiwan (gi-7), ldt3-a (gi-28), and new 2 (gvii-1) were dated back to 1914. 5, 1954.33, 1971.15, 1971.82, 1983.26, 1985 hpd, highest probability density. viruses 2019, 11, x for peer review 14 of 24 the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s1, e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between 1970s and 2010s. a more obvious decrease was observed between 1990s and 2000s base on the s1 gene ( figure 6a ). remarkably, a sudden and sharp decline in the effective population size was observed after 2010s base on the s1, e, m, and n genes. however, since late 2015, the viral population rapidly rose ( figure 6 ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s1, e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between 1970s and 2010s. a more obvious decrease was observed between 1990s and 2000s base on the s1 gene ( figure 6a ). remarkably, a sudden and sharp decline in the effective population size was observed after 2010s base on the s1, e, m, and n genes. however, since late 2015, the viral population rapidly rose ( figure 6 ). the reconstruction of population history was assessed using a bayesian skyline plot coalescent model. results showed that the effective population size of s1, e, m, and n genes of ibvs was featured by a continuous and slow reduction in viral population size between 1970s and 2010s. a more obvious decrease was observed between 1990s and 2000s base on the s1 gene ( figure 6a ). remarkably, a sudden and sharp decline in the effective population size was observed after 2010s base on the s1, e, m, and n genes. however, since late 2015, the viral population rapidly rose ( figure 6 ). southern china is the largest region of yellow chickens produced in china. despite extensive vaccination, ib continues to be a serious problem. in the present study, 64 strains of ibv were isolated from diseased chicken flocks in southern china during 2009-2017 and the genetic properties of the entire s1, e, m, and n genes analyzed. to increase our insight into the comprehensive epidemiological situation and evolutionary trend of ibv in southern china, 27 ibv isolates from 1985 to 2008 in southern china were also used to analyze together. the 43 representative reference strains included the live vaccine strains commonly used and the prevalent strains in china and other countries worldwide, which represented the main well-established lineages and genotypes in recent years in china [17] . our results indicated that there was a remarkable change in genetic diversity, dominant genotypes, and selection pressure of ibv strains in southern china over the past decade compared with the previous period of 1985-2007. the co-circulation of multiple ibv genotypes and the increasing of ibv variants have resulted in great challenges for the controlling ib through vaccination. in our study, multiple ibv genotypes were also identified in guangxi over the past decade. a total of eight, six, four, and six genotypes were identified based on the s1, e, m, and n genes, respectively. therefore, there is still ongoing genetic diversity of ibvs in southern china, which is the same as previous epidemics in this region and other parts of china [4, 15, 35] . interestingly, the predominant genotype changed from ck/ch/lsc/99i-type lx4-type was the most dominant genotype in our study, which agreed with many previous descriptions [15, 30, 35, 41] . surprisingly, the second most dominant genotype of ibv circulating in southern china was ldt3-a-type, which was a pandemic type and frequently isolated from chicken flocks in china [15, 42, 43] . ldt3-a commercial live vaccine has been issued by the official authority in china since 2011 [6] . the re-isolation of vaccine strain is possible when ldt3-a live vaccine strain is extensively used. we noticed that among the 14 ldt3-a type isolates during 2009-2017, four, eight and two strains were isolated in 2010, 2011, and 2012 respectively. however, only one ldt3-a type strain was isolated during 1985-2008. we are not sure whether or not the 10 strains of ldt3-a type isolated between 2011 and 2012 are re-isolation of vaccine strains, but it seems that the ldt3-a type vaccine could provide sufficient protection against the circulating homologous field strains in southern china because none of ldt3-a genotype strains were isolated after 2012. at the same time, we found the e genes of most ldt3-a type isolates belonged to the ck/ch/lsc/99i-type, and the m and n genes of most ldt3-a type isolates belonged to lx4 (qx)-type. these results implied that ldt3-a-type might isolate the recombinants from ldt3-a vaccine strain and other circulating ibv strains. considering that the ldt3-a types were occasionally isolated in other regions of china recently [43] , the ldt3-a type isolates in southern china still need to be further monitored. the taiwan-type ibvs were firstly isolated in taiwan in 1990s and have been divided into the taiwan-i and taiwan-ii subgroups [44, 45] . more studies reported that an increasing number of taiwan-type strains have been isolated in southern china in recent years [15, 35, 46, 47] . taiwan-ii-type strains (gx-xd and gx-g) were isolated in 1988 and no taiwan-ii-type strain was isolated in guangxi after that [4] , but five taiwan-i-type strains were isolated during 2010-2017. in our study, 12.5% (1/8), 9.1% (1/11), and 20% (3/15) of taiwan-i-type strains were isolated in 2010, 2013, and 2017, respectively. it seemed that the taiwan-i-type strains were increasing in recent years. evidences proved that the taiwan-i-type strains were widely spread from taiwan to guangdong, guangxi, fujian, sichuan hunan, zhejiang, yunnan province and so on [15, 46, [48] [49] [50] , and the increasing isolates demonstrated that the currently used vaccines were lacking protective efficacy against the taiwan-i-type strains. the recombinants between taiwan-i-type and lx4 (qx) causing severe economic losses have been reported [51] . how the taiwan-type strains spread from taiwan to mainland china remains unknown. recently, wild birds have been of concern as natural ibv carriers, since infected birds have been showed to carry the viruses over long distances [8, 23, 52, 53] . therefore, monitoring of wild birds should be needed in further. of course, ibv live vaccines and poultry products will also need to be followed. the earliest strain of new-type 1 (gvi-1) was isolated in 2007 [54] and has in recent years spread to many chicken flocks [5, 15, 30, 43, 55] . surprisingly, majorities of the new-type 1 strains from other studies were isolated from south china [2, 5, 15, 55] . recently, another novel genotype vii (gvii-1) was reported [17] . both these two new-type viruses were identified in our study and the total number of their strains showed new-type was the third most dominant genotype. we found that there was a four aa (qkep) insertion located in the hvr iii and one aa (s) insertion in all the new-type 1 isolates. interestingly, the four aa (qkep) were predicted b cell epitopes by the bepipred /iedb tool and half of the new-type viruses' serotype were different from that of 4/91 or other vaccine strains [56] . the aa mutation in the hvr of the s1 gene maybe lead to the occurrence of new serotype and immunity escape. the origin of new-type 1 viruses in china remains unknown. interestingly, the "novel" isolates were shown to undergo recombination in our study. the new-type 1 isolate gx-nn120089 was a major parent to the recombinant ldt3-a-type isolate gx-nn120091. to the best of our knowledge, this first report of a recombination event needs to be further investigated. analyzing the complete-genome and identifying the antigenicity and pathogenicity of new-type 1 isolates will be further studied. it is uncertain whether the new-type 1 would become the predominant type in china in further, but it continues circulating in the field suggests that the new-type 1 ibvs are still endemic in china and more attention should be paid to them. high rates of recombination result in ibv variability. many recombinants were confirmed. some recombination occurred between field and vaccine viruses [5, 14, 15, 26] , some occurred between field isolates [26, 35, 43] . six recombinants were identified in this study and three had a major parent of the lx4-type, and two with minor parents of 4/91-type genotype. our previous studies confirmed five recombinants, which were between the vaccine strain 4/91 and the ck/ch/lsc/99i-type field strain gx-yl2 during 2006-2007 [4] . therefore, we have identified 11 recombinants so far and 7 recombinants were derived from the 4/91-type strain during 1985-2017. high frequencies of recombination between vaccine and field strains have been reported frequently worldwide [4, 5, 15, 22, 57, 58] . the 4/91 vaccine strain has been commonly used in china for a long time, so it is not surprising that 4/91-type recombinants were found in the field. however, there were more recombinants involved in the 4/91-type vaccine strain when compared to the mass-type vaccine strains' recombinants according to our and other previous reports [4, 14, 15, 18, 59] , although both mass-type and 4/91-type vaccine strains were widely used in china. the phenomenon may have the following three explanations. firstly, incomplete protection of 4/91-type vaccine strain against heterologous strains might result in co-infection of vaccine and heterologous strains in the same birds and eventually led to recombination. secondly, the 4/91-type vaccine strain recombinants may be more likely to escape vaccine immunization than mass-type vaccine strain recombinants. thirdly, the 4/91-type vaccine strain persisted longer in the immunized birds than the mass-type vaccine strain, increasing the likelihood of co-infection with the latter infected strain/strains, as a previous report showed that 4/91 vaccine could persist in birds for 40 days [60] . the complete genome, pathogenicity, and immunogenicity of 4/91-derived recombinants should be assessed in further studies. the isolation ratio of 4/91 (gi-13) genotype strains was relatively stable recently. therefore, in order to reduce the recombination occurred between field and vaccine viruses, the multi-valent live vaccine combined with 4/91 strain or other new vaccines (such as ldt3-a strain, lx4 strain, and so on) should be used with caution and thoughtful consideration. in order to understand the variation degree of the s1, e, m, and n genes and the correlation between gene variation and selection pressure, the analyses of entropy of amino acid sequences, molecular evolutionary rate, and positive selection were carried out in our study. the results showed that mean substitution rates were between 3.7 × 10 −3 and 4.6 × 10 −3 substitutions per site per year in different genes, which is comparable with previous description [58] . both the average entropy and mean substitution rates sorted from large to small as follows: s1, e, m, and n genes, and the dn/ds ratios of s1, e, m, and n genes were 0.283, 0.266, 0.146, and 0.147, respectively. the dn/ds ratios of s1, e, m, and n genes were less than 1, meaning that the analyzed region were under purifying selection. therefore, our results indicated that the degree of variation sorted from large to small as follows: s1, e, m, and n genes and there was a positive correlation between selection pressure and gene variation. it is known that s1 genes had the largest variation, but the mutation of e gene has not been paid enough attention. a recent study reported that the e gene evolved at the fastest rate among four structural protein-coding genes [30] . the e gene mutation needs to be focused on in the future. to our knowledge, it is the first report to analyze systematically the variation degree of the four structural genes of ibvs and correlation between gene variation and selection pressure. as mentioned above, the s1, e, m, and n genes of guangxi ibvs during 2009-2017 were under purifying selection. despite that the four structural genes underwent purifying selection, there were six, one, five, and five positively selected sites within the s1, e, m, and n genes respectively. the dn/ds value and positively selected sites of guangxi isolates were different from those of isolates from other countries and regions [26, 30, 38] . in addition, all of the positively selected sites in s1, e, m, and n genes had high entropy values of bigger than 0.6. the accumulation of amino acid variation will have an important effect on the gene characteristics and evolutionary direction of viruses [4] . therefore, more attention should be paid to those positively selected sites with high entropy values. in the present study, positive selection was not detected in the s1, e, m, and n proteins of ibv isolates in southern china during 2009-2017 although there were high number of mutations. zhao et al. also found that purifying selection was the main evolutionary pressure in the protein-coding regions in china over the past two decades [30] . however, positive selection was detected in the s1, m, and n proteins of ibv isolates in southern china during 1985-2008 according to our previous investigation [4] . therefore, positive selection in the s1, m, and n proteins in different periods in southern china was changeable. this phenomenon was also seen in some other previous reports [61] [62] [63] . the change in selection pressures may be due to immune responses induced by multiple types of vaccines, the microenvironment of infected hosts, or physical and biosafety conditions [16] . molecular evolutionary rate analysis can accurately estimate the molecular dating and it was used to determine successfully the timing of ancestors of classical swine fever virus (csfv) [64] , sars virus [65] , and ibv [30, 58] . according our previous investigation [1] , the first ibv strain was isolated in 1985, demonstrating that the ibv was already present at that time. this study further backdated the ibvs emergence of more than 30 years (during 1985-2017) and indicated that the most recent ancestor of guangxi ibv isolates based on the genes s1, e, m, and n existed in the early 1744, 1893, 1940, and 1945, respectively. different structural protein genes of the ibvs had different time of the most recent ancestor. this phenomenon was also reported in other viruses [51, 66, 67] . the data of tmrca estimated from our study suggested ibv has been already circulating in field for a period before its first outbreak. interestingly, the evolution rates sorted from large to small as follows: s1, e, m, and n genes; the similarity plot data showed high genetic divergence and recombination was found in the s1 gene of ibv isolates, which agreed with the dates of most recent ancestor. although ibv was discovered in 1930s, it is thought that it may have a long history as a species. the similar results were reported in other viruses [66, 67] . in our early study, maybe unavailability of advanced diagnostic and sequencing technologies, the backyard poultry farming, inefficient surveillance systems, and limited financial resources resulted in the non-detection of ibv isolates for such a long time in guangxi. bayesian skyline analysis of s1, e, m, and n genes of ibvs revealed that a persistent and slow decrease in relative genetic diversity between 1970s and 2010s, mirroring the implementation of mass-or 4/91-based vaccine was effective in controlling the virus in field conditions to some extent, although not being able to eradicate the pathogen. analysis of s1 showed a more obvious decrease between 1990s and 2000s. the live vaccine h120 has been used in china for many years and the 4/91-like live vaccine was also commonly used in china since 1990s even without official authorization. therefore, the 4/91-like vaccine was effective between 1990s and 2000s. surprisingly, a sudden and sharp decrease in genetic diversity base on the s1, e, m, and n genes was observed after 2010, mirroring the implementation with a newly registered ldt3-a-based vaccine. in our study, the ldt3-a-type disappeared after 2012. ldt3-a-like strains were also rarely isolated in other recent studies [30, 43] . it was reported that the change of viral population size had a strong association with the vaccine administration/withdrawal [68, 69] . therefore, it can be speculated that the ldt3-a vaccine could provide a better protection against the circulating strains in china. however, since late 2015, a sharp increase was observed in viral population size, which may suggest the implementation of current vaccines could not provide higher protection against the circulating strains. the lx4-type isolates had become the predominant ibvs since 2011 in our study. the proportion of lx4-like genotype strains increased over time in china [30] . increasing of lx4 genotype viruses over time agreed with the rapid rise in the viral population since late 2015. our previous study showed that the h120, 4/91, or ldt3-a vaccines could not provide complete protection against the prevalent local strains of ibv (such as lx4 genotype isolates) in southern china [6] . therefore, it is predicted that the epidemic trend of lx4 type strain will not decline in the next few years and will become more and more intense. therefore, new lx4 type vaccine was urgently needed. lx4 live vaccine was officially authorized in china last year, but has not yet come into the market. lx4 commercial vaccine urgently needs to go to the market in china. taken together, our results of genetic analyses of the four structural protein genes s1, e, m, and n clearly demonstrated that there has been a remarkable genetic change of ibv in southern china over the past decade. the coexistence of multiple genotypes of ibv, the changes of dominant genotypes in different periods, the emergence of new genotypes, the recombination of vaccine strains with the field strains, the rapid evolving rate of ibv, the ever-changing of viral population size, and the presence of multiple positively selected sites in the structural proteins suggest a complexity of ibv epidemic strains in southern china. this epidemiological complexity may be caused by multiple factors: simultaneous vaccination of multiple live vaccines, illegal use of live vaccines, large number of avian varieties, raising of chickens at a rather high densities and the patterns of raising (free-range style and lack of biosecurity), etc. the complexity also suggests the importance of molecular epidemiological investigation and permanent monitoring of circulating strains as well as more serious challenges for ib prevention and control. based on the present results, it is predicted that lx4-type will remain to be dominant genotype in near future and new-type ibv isolates will be more and more prevalent in the future. therefore, it is necessary to choose and/or develop new vaccines to counteract the ibvs of these two genotypes. in addition, multivalent new vaccines should be developed and rational modified vaccination strategies and the strict biosafety should be observed. to our knowledge, this is the first report showing the changes of genetic diversity, dominant genotypes, and selection pressure of ibv strains. the present study extends our knowledge about past and present ibv variability in southern china, providing valuable reference and countermeasures against ibv field breakouts in china, also emphasizing the importance of constant dynamic surveillance of the circulating isolates. in conclusion, our results indicate that the ibv strains in southern china have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines which counteract the emerging lx4-type and new-type ibvs and should be resistant to recombination and mutation. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/10/898/s1, table s1 : ibv reference strains used in this study; table s2 : estimates of evolutionary distances over sequence pairs between groups of s1 gene; table s3 : estimates of evolutionary distances over sequence pairs between groups of e gene; table s4 : estimates of evolutionary distances over sequence pairs between groups of m gene; table s5 : estimates of evolutionary distances over sequence pairs between groups of n gene; table s6 : the selection profile and entropy values of s1, e, m, and n proteins of guangxi ibv isolates; figure s1 : alignment of the deduced amino acid sequences of the s1 subunit of the spike protein of 6 ibv isolates (new-type 1 or gvi-1) and other strains. the insertion of critical amino acids 303, 304, 305, 306, and 394 are highlighted in red; figure s2 : the sequences were aligned using the clustal w method in the bioedit version 7.1.11.0 package. the hotspot sequence atttt(t/a) (green underline) was found near the breakpoint site (red box) of the s1 subunit gene in all of recombinant isolates except gx-yl170805. the gx-yl170805 isolate hotspot sequence and breakpoint site were marked in blue box; figure s3 : entropy plot of amino acid of s1 (a), e (b), m (c), and n (d) protein genes of ibvs. x-axis shows the amino acid sites of s1, e, m, and n genes; y-axis shows the entropy of each amino acid site; figure s4 : the location of positive selection sites and putative glycosylation sites of the s1 subunit of 91 ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s5 : the location of positive selection sites and putative glycosylation sites of the e subunit of 91 ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s6 : the location of positive selection sites and putative glycosylation sites of the m subunit of 91 ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately; figure s7 : the location of positive selection sites and putative glycosylation sites of the n subunit of 91 ibv isolates. the positively selected sites and n-glycosylation site are highlighted in red and blue separately. serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in guangxi continuous evolution of avian infectious bronchitis virus resulting in different variants co-circulating in southern china complete genome sequences of two chinese virulent avian coronavirus 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and genomics of hepatitis b virus subgenotype c2 strain predominant in the chronic patients in bangladesh continued use of ibv 793b vaccine needs reassessment after its withdrawal led to the genotype's disappearance effect of different vaccination strategies on ibv qx population dynamics and clinical outbreaks this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflicts of interest. key: cord-343893-sophqqne authors: chu, victor c; mcelroy, lisa j; aronson, jed m; oura, trisha j; harbison, carole e; bauman, beverley e; whittaker, gary r title: feline aminopeptidase n is not a functional receptor for avian infectious bronchitis virus date: 2007-02-26 journal: virol j doi: 10.1186/1743-422x-4-20 sha: doc_id: 343893 cord_uid: sophqqne background: coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (sars), and have the continued potential for emergence from animal species. a major factor in the host range of a coronavirus is its receptor utilization on host cells. in many cases, coronavirus-receptor interactions are well understood. however, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (ibv). feline aminopeptidase n (fapn) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (fipv), canine coronavirus, transmissible gastroenteritis virus (tgev), and human coronavirus 229e (hcov-229e). a recent report has also suggested a role for fapn during ibv entry (miguel b, pharr gt, wang c: the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review. arch virol 2002, 147:2047–2056. results: here we show that, whereas both transient transfection and constitutive expression of fapn on bhk-21 cells can rescue fipv and tgev infection in non-permissive bhk cells, fapn expression does not rescue infection by the prototype ibv strain mass41. to account for the previous suggestion that fapn could serve as an ibv receptor, we show that feline cells can be infected with the prototype strain of ibv (mass 41), but with low susceptibility compared to primary chick kidney cells. we also show that bhk-21 cells are slightly susceptible to certain ibv strains, including ark99, ark_dpi, ca99, and iowa97 (<0.01% efficiency), but this level of infection is not increased by fapn expression. conclusion: we conclude that fapn is not a functional receptor for ibv, the identity of which is currently under investigation. the family of coronaviridae is composed of group 1-3 coronaviruses (covs) [1] . these viruses are able to infect human, canine, feline, murine, bovine, porcine, rat, and avian species. the etiological importance and zoonotic characteristics of coronaviruses have received much attention since the discovery of the newly emerged severe acute respiratory syndrome associated coronavirus (sars-cov) in 2003 [1, 2] . coronaviruses have a high frequency of viral genome recombination and polymerase infidelity, which may have contributed to the increase of viral pathogenesis, inter-species transmission, and tissue tropism [3] [4] [5] . in the case of sars-cov, its ancestral origin remains undetermined, but some evidence suggests that chinese horseshoe bats may be the natural reservoirs, while himalayan palm civets harbor and support inter-species transmission to humans [5, 6] . other examples of extended tissue tropisms can also be found in some group 2 covs. it is speculated that the acquisition of hemagglutinin esterase (he) activity from influenza c virus gives rise to the ability of sialic acid recognition and the extended tissue tropism and pathogenesis for some group 2 covs [7] [8] [9] . furthermore, bovine coronavirus (bcv) is thought to have jumped to human hosts, possibly by recombining with influenza c virus, thus giving rise to human coronavirus-oc43 (hcov-oc43) around 1890 [4, 8] . receptor interaction between the virus and its host is the first step leading to a successful entry and productive replication. viruses increase fitness by adapting to environmental pressure through mutation and recombination. in contrast to other families of viruses that utilize a universal receptor to gain entry into host cells, members in the coronavirus family use a variety of cellular proteins and/or cofactors. group 1 covs -including human coronavirus-229e (hcov-229e), feline infectious peritonitis virus (fipv), transmissible gastroenteritis virus (tgev) and canine coronavirus (ccv) -utilize human, feline, porcine, and canine aminopeptidase n (apn) as functional receptors during virus entry [10] [11] [12] [13] . the only notable exception is hcov-nl63, which utilizes angiotensin-converting enzyme 2 (ace2). in group 2 cov, mouse hepatitis virus (mhv) of group 2a and sars-cov of group 2b independently utilize carcinoembryonic antigen-cell adhesion molecule (ceacam1) and ace2 to mediate infection [14, 15] . however, other group 2a covs, including hcov-oc43 and bcov recognize n-acetyl-9-oacetylneuraminic acid as a functional receptor [9] . while the cellular receptors for both groups 1 and 2 covs have been identified and independently confirmed, group 3 cov receptors remains undetermined. the avian covs, such as turkey cov and infectious bronchitis viruses (ibv), have been classified in group 3, with ibv the most extensively studied. recently, winter and colleagues suggested that sialic acids are responsible for ibv strain massachusetts 41 entry [16] . however, group 3 covs lack he as a key viral protein regulating sialic acid binding, and the use of sialic acid would not explain the dependence on chicken cells for infection. therefore ibv is unlikely to use sialic acids as a functional entry receptor, but rather as a non-specific attachment factor. heparan sulfate may also serve as an attachment factor for the ibv strain beaudette (ibv_bdtt) [17] . ibv_bdtt is a highly chicken embryo-adapted strain [18, 19] , which has an extensive tropism in cell culture and efficiently infects various cell types, including bhk-21 cells [17, 19, 20] . in contrast, clinical isolates and field strains of ibv typically only infect chicken cells. in the effort to identify the receptor for group 3 covs, feline apn (fapn) was reported to allow entry of the ibv strain arkansas 99 (ibv_ark99) [21] . this could therefore be the first indication of a more universal receptor for the cov family. apn belongs to a family of metalloproteases [22] . it is a type ii membrane-bound glycoprotein, and it is expressed on a variety of cell types, including granulocytes, monocytes, and fibroblasts. apn can also be found on the synaptic membranes of the central nervous system neurons, and on epithelial cells in the proximal convoluted tubules, intestinal brush border, and respiratory tract [11, 23] . for coronaviruses in general, there is a crossspecies restriction that permits cells of a certain species to be infected only by its own complimentary cov. however, several studies on fipv and hcov-229e, ccv and tgev have identified fapn as a universal entry receptor for group 1 coronaviruses [11] [12] [13] 23] . the demonstration that fapn can allow infection by the ibv strain ark_99 prompted us to examine both prototype and field isolates of ibv and test them for the potential use of fapn as a receptor. in this study, we first verified the use of the expressed fapn as a receptor for fipv and tgev by transient and constitutive expression of fapn in non-permissive bhk-21 cells. we also cultured seven strains of ibv, including arkansas 99, arkansas_dpi, california 99, connecticut 46, holland 52, iowa 97, and massachusetts 41 (designated as ark99, ark_dpi, ca99, conn46, h52, iowa97, and mass41) as candidates to test for fapn utilization by group 3 avian covs. surprisingly, expression of fapn did not increase viral infection in any of the strains tested. as a consequence, we conclude that fapn is not a functional receptor during ibv entry. the authentic receptor is still under investigation. in order to determine fapn receptor usage by ibv, our goal was to rescue ibv infection in non-permissive cells by expressing fapn on the cell surface. to authenticate fapn expression, we first transiently transfected bhk-21 cells with fapn/pcdna3.1d/topo plasmid dna for 24 hours. samples were then subjected to immunofluorescence staining to detect fapn protein expression (fig. 1a) . we found that nearly 40% of the bhk-21 population were efficiently transfected and stained positive for fapn expression. next, we verified the fapn expression on the bhkexp.fapn cell line that constitutively expresses fapn and its negative control cells, bhkexp.pcineo ( fig. 1b and 1c ). 100% of bhkexp.fapn cells also stained positive, while the negative control showed no fapn expression ( fig. 1) . to verify the functionality of fapn as a coronavirus receptor, we first tested its ability to rescue fipv-1146 and tgev infection of non-permissive cells, as reported in previous studies [11] . feline kidney crfk cells and canine fibroblast a72 cells are able to support fipv-1146 and tgev infection respectively (dr. edward dubovi, cornell university, personal communication). therefore, these cell lines served as positive controls for fipv-1146 and tgev infection. we observed that fipv-1146 and tgev efficiently infected crfk and a72 host cells respectively, but they were unable to mediate infection in bhk-21 cells ( fig. 2a and 2b), presumably due the lack of a functional receptor on the cell surface. however, transient transfec-tion of fapn in bhk-21 cells rescued fipv and tgev infection to 34% and 21% respectively (fig. 2c) . we therefore confirm that fapn can function as an entry receptor for both fipv-1146 and tgev. group 3 covs such as infectious bronchitis viruses (ibv) can infect primary chicken kidney cells at high efficiency, but they generally restrict interspecies infections [19] . however, previous studies have shown that feline kidney cells (fek) can support ibv_ark99 infection [21] , suggesting the presence of a feline receptor on the fek cell surface that enables ibv_ark99 entry. since ibv_mass41 has served as a prototype virus to study cov entry [24, 25] , we first examined the potential utilization of a feline receptor by ibv_mass41. we found that whereas bhk-21 cells were resistant to ibv_mass41 infection, and ibv_mass41 infected ck cells at 45% efficiency, ibv_mass41 was able to also infect feline crfk cells at 6% efficiency ( fig 3b) . as indicated by miguel et al. [21] , the ibv_mass41 infection found in crfk cells suggests the potential use fapn as a receptor. to determine the potential role of fapn as a receptor for the prototype ibv (mass_41), we transiently transfected fapn into bhk-21 cells. both untransfected and fapntransfected bhk-21 cells failed to show any detectable infection with ibv_mass41, whereas primary ck cells were efficiently infected (fig. 4) . to rule out the potential inhibitory effect of viral infectivity caused by transient transfection, we also examined ibv infection in cell lines constitutively expressing fapn. we found that expression of fapn in bhkexp.fapn cells could not rescue ibv_mass41 infection (fig. 4a and 4b ). to examine if fapn might act in a strain specific manner, seven field ibv isolates: ark99, ark_dpi, ca99, conn46, h52, iowa97, and mass41 were obtained and cultured in 10-day old specific pathogen free (spf) chicken embryonic eggs. to verify virus infectivity, monolayers of ckc were individually incubated with all seven strains of ibv for 12 h. figure 5 shows that ckc monolayers were found to be efficiently infected by all seven field strains of ibv tested. to determine the role of fapn as a functional receptor for entry of these seven fields strains of ibv, as well as the prototype mass 41, were inoculated onto bhkexp.pcineo or bhkexp.fapn cell monolayers in triplicate. we found that bhk cells expressing empty vector alone (bhkexp.pcineo) were completely resistant to infection by ibv strains conn46, h52 and mass41. however, ibv strains ark99, ark_dpi, ca99, and iowa97 showed limited infection in the same cells, with a level of infection of less than 0.01% (fig. 6a) . however, fapn expression did not significantly enhance ibv infectivity in any of the virus strains tested (fig. 6b) . consequently, our data show that ibv does not utilize fapn as a functional receptor during virus entry. viruses make use of a variety of receptors to gain entry into their target cells. the ability to recognize sialic acids that are ubiquitously present on the cell surface gives influenza viruses the ability to indiscriminately infect varied tissue or cell types. in contrast, retroviruses require more specialized receptor interactions between viral glycoprotein 120 (gp120) and cd4 as well as other chemokine receptors on the t-helper lymphocytes during invasion [26] . despite some subtle differences among different species or strains of viruses in the same family, they may still retain a trace of evolutionary similarity in terms of receptor utilization. however, receptor usage appears to have very little consensus among coronaviruses across different groups and species. coronaviruses seem to be able to devise various in order to gain a more comprehensive understanding of ibv receptor utilization, we cultured several clinical strains of ibv for this study. surprisingly, we found that the bhk-21 cells are in fact, weakly permissive to ibv_ark99, ibv_dpi, ibv_ca99, and ibv_iowa97 infections. however, fapn expression on bhk-21 cell surface did not increase viral infectivity for any ibv strain. we therefore conclude that fapn could not be a functional receptor for ibv entry. it is important to note that one, highly chick-embryo-adapted, ibv strain (ibv beaudette) gives efficient infection in bhk cells [20] ; combined with the data presented here, that a low level of infection of bhk cells could be obtained with some clinical strains, this suggests that there are no post-entry restrictions to ibv replication and gene expression that might account for a lack of infection of bhk cells. how do we explain the discrepancy between these data and previous studies that show that fapn is the receptor for ibv_ark99 entry? according to our observations, ibv_ark99 viruses were able to infect bhk-21 cells to a limited degree (<0.01%), although the infection profile was not always consistent among samples. the relevance of this very limited infection of hamster cells is unclear. as a consequence, previous studies may have shown only localized and under-represented populations of cells, which were infected by ibv_ark99, since there was no further quantification of viral infectivity. in contrast, our present study was performed in a quantitative manner by measuring infection frequency of nearly 10 6 cells and we provide numerical virus infection data between fapnexpressing and non-expressing cells. various studies have shown several potential receptor candidates for group 3 coronaviruses, which include fapn, sialic acids, and heparan sulfate [16, 21] . however, there is still little consensus on which of these might be the functional entry receptor in vivo. sialic acid utilization as a functional entry receptor by ibv remains controversial since ibv generally has a highly restricted species tropism, and does not possess a he glycoprotein that would normally be responsible for sialic acid destruction and hence efficient virus spread. unlike influenza viruses that are established to use cell surface sialic acids for entry into a wide variety different cells, ibv is generally considered only to infect cells of chicken origin. the infected chickens often show lesions or lymphocyte infiltration in the ciliated epithelia cells, mucus secreting cells, and sub-epithelial cells along the respiratory tract [27] . ibv also infects lung, ovary, and kidney tissues [24] . in cell culture, ibv_mass41 infects ckc but not chick embryo fibroblast cells or the chicken df-1 fibroblast cell line (data not shown), suggesting possible cell type or tissue specificity for ibv infection. feline apn has been previously reported to serve as a functional receptor for ibv_ark99 entry, a conclusion based in part on the ability of the virus to infect feline kidney cells (crfk, also known as fek) [21] . we have inde-ibv strain mass41 does not utilize fapn as an entry receptor pendently confirmed the ability of feline kidney cells to support ibv_mass41 infection (fig. 3a) with relatively high efficiency. as a consequence, cats could potentially serves as an intermediate host during an extension of coronavirus tissue tropism. in the case of sars-cov, viral transmission is thought to originate from chinese horseshoe bats to himalayan palm civet cats before jumping to humans [5, 6] . the use of a feline receptor for ibv could potentially be another example of coronaviruses using feline cells as an interspecies reservoir to cross species boundaries. the reason why cats may serve as such an intermediate host is unclear, although it may be noteworthy that the endogenous feline coronavirus (fecv) can undergo distinct recombination events within feline cells that result in the emergence of the clinically different virus fipv [28] . also of potential relevance is the suggestion that the sars-cov s gene may be a mosaic of feline and avian sequences [29] . we conclude here that ibv cannot utilize fapn as a functional receptor. it is however important to note that although ibv does not utilize fapn as an entry receptor, there is still a possibility that chicken apn (capn) may serve as the key receptor. future work will address the potential usage of capn, as well as other chicken cell-specific proteins, as ibv receptors. ibv conn46, iowa97, h52, and mass41 were obtained from dr. benjamin lucio-martinez, unit of avian health, cornell university, and ibv ark_dpi was obtained from dr. mark w. jackwood, university of georgia. ibv ark99 and ca99 were obtained dr. shankar p. mondal (university of california, davis). viruses were propagated in 11 day-old embryonated chicken eggs and purified as described previously [20] . virus stocks were titered by infection of primary chicken kidney cells, followed by immunofluorescence microcopy with monoclonal anti-s antibodies after 8-12 h of infection. typically, the highest titer stocks were able to infect between 50% and 70% of the ck cells present, and were used at this concentration for all infections, unless otherwise indicated. fipv-1146 and tgev were obtained from dr. edward j. dubovi, animal health diagnostic center, cornell university, and were propagated in crfk (feline kidney cell) and a72 (canine fibroblast cells) respectively. bhk-21 cells were purchased from atcc and propagated twice weekly in dmem, supplemented with 10% fetal bovine serum (fbs) 1% penicillin-streptomycin, and 10 mm hepes (cellgro). crfk and a72 were grown in l:m media. bhkexp.fapn or and bhkexp.pcineo cells were kindly provided by dr. kathryn v. holmes (university of ibv field isolates efficiently infect primary chicken kidney cells figure 5 ibv field isolates efficiently infect primary chicken kidney cells. monolayers of ck cells were separately infected with ibv strain ark99, ark_dpi, ca99, conn46, h52, iowa97, and mass41 for 12 h. cell monolayers were labeled with monoclonal α-s1 or m (15:88, 13:18, or 9:19) antibodies to detect viral infection. fapn is not a functional receptor for field strains of ibv figure 6 fapn is not a functional receptor for field strains of ibv. bhkexp.pcineo (a) or bhkexp.fapn (b) cells were independently infected with ibv strain ark99, ark_dpi, ca99, iowa97, conn46, h52, and mass41 for 12 h. cell monolayers were labeled with monoclonal α-s1 or m (15:88, 13:18, or 9:19) antibodies to detect and determine viral infections. viral infectivity was determined by counting approximately 10 6 cells in three separate experiments. primary chicken kidney cells (ckc) were prepared as follows: in short, spf white leghorn chicks (11-14 days) were sacrificed in a co 2 chamber, and kidneys were removed via aseptic techniques. kidney cells were washed in 50 ml sterile phosphate buffer saline (pbs) twice via gentle stirring to remove red blood cells. the cells were then treated with 25 ml of trypsin-edta (cellgro) for two minutes to separate large chunks of kidney tissues. cell supernatant was mixed with 1 ml of fbs and centrifuged at 1,000 rpm for 2 min. media were removed and kidney cells were resuspended in 25 ml of m20 media. cell population was determined on a hemocytometer. for plating, 1-1.5 × 10 6 cell/ml was prepared in m25 media (m20 media with 5% fbs) and incubated in 5% co 2 at 37°c. for infection, 60% confluent cell monolayers were inoculated with virus stocks and incubated for 8, 12, or 24 hours before fixation. fapn/pcdna3.1d/topo and hapn/pcineo plasmid dnas were provided by dr. kathryn v. holmes (university of colorado health sciences). for standard transfection, 0.6 µg of plasmid dna were premixed with 2 µl of lipofectamine 2000 (invitrogen) in 300 µl of opti-mem (gibco) at room temperature according to manufacture's instructions. cell monolayers were transfected at 37°c for 24 hours before fixation or virus infection. infection and immunofluorescence microscopy were essentially performed as described previously [30] except with methanol fixation for detecting viral antigens. for fapn staining, cell monolayers were fixed in 3% paraformaldehyde at room temperature and labeled with monoclonal antibody r-g-4 (provided by dr. tsutomu hohdatsu, kitasato university, towada, aomori, japan). for viral antigen staining, anti-s1 monoclonal antibody 15:88 was used for ibv massachusetts 41 and iowa 97. the anti-s1 monoclonal antibody 13:18 was used for ibv arkansas_dpi. the anti-m monoclonal antibody 9:19 was used for ibv arkansas 99, connecticut 46, california 99, and holland 52. monoclonal antibody 17b71 and rabbit polyclonal antibody 367 were provided by dr. edward dubovi, cornell university, and were used detect fipv and tgev infection respectively. the secondary antibodies alex-fluor 488 goat anti-mouse igg and alex-fluor 488 goat anti-rabbit igg were purchased from molecular probes. cell nuclei were counter-stained with hoechst 33258 (molecular probes). cells were viewed on a nikon eclipse e600 fluorescence microscope. images were captured with a spot rt monochrome camera and spot advanced v.4.0.9 software and processed with adobe photoshop v.7. virus taxonomy: family coronaviridae wet markets--a continuing source of severe acute respiratory syndrome and influenza? lancet high-frequency rna recombination of murine coronaviruses complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats sequence of mouse hepatitis virus a59 mrna 2: indications for rna recombination between coronaviruses and influenza c virus the hemagglutinin/esterase gene of human coronavirus strain oc43: phylogenetic relationships to bovine and murine coronaviruses and influenza c virus sialic acids as receptor determinants for coronaviruses human aminopeptidase n is a receptor for human coronavirus 229e feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n is a receptor for all group i coronaviruses spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus heparan sulfate is a selective attachment factor for the avian coronavirus ibv beaudette. avian diseases cultivation of the virus of infectious bronchitis new jersey agricultural experiment station studies on avian infectious bronchitis virus (ibv). ii. propagation of ibv in several cultured cells the avian coronavirus infectious bronchitis virus undergoes direct low-ph-dependent fusion activation during entry into host cells the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review families of zinc metalloproteases identification of residues critical for the human coronavirus 229e receptor function of human aminopeptidase n infectious bronchitis fluorescence dequenching assays of coronavirus fusion retrovirus receptors feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses mosaic evolution of the severe acute respiratory syndrome coronavirus influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis we thank (in alphabetical order) dr. joel d. baines, dr. edward j. dubovi, dr. tsutomu hohdatsu, dr. kathryn v. holmes, dr. mark w. jackwood, dr. benjamin lucio-martinez, and dr. shankar p. mondal for their generous support on providing various reagents possible for this study. we thank a. damon ferguson for her skillful technical assistance, and all members of the whittaker laboratory for helpful suggestions. this work was supported by grant r03 ai060946 from the national institutes of health. the author(s) declare that they have no competing interests. vc participated in the design of the study, carried out the experiments presented, and drafted the manuscript. lm, jm, to and ch carried out preliminary experiments on which the manuscript is based. bb contributed to the conception and design of the experiments. gw conceived of the study, participated in its design and coordination, and wrote the final manuscript. all authors read and approved the final manuscript. key: cord-353310-19kzb6ag authors: quinteros, josé a.; markham, philip f.; lee, sang-won; hewson, kylie a.; hartley, carol a.; legione, alistair r.; coppo, mauricio j. c.; vaz, paola k.; browning, glenn f. title: analysis of the complete genomic sequences of two virus subpopulations of the australian infectious bronchitis virus vaccine vics date: 2015-04-01 journal: avian pathol doi: 10.1080/03079457.2015.1022857 sha: doc_id: 353310 cord_uid: 19kzb6ag although sequencing of the 3′ end of the genome of australian infectious bronchitis viruses (ibvs) has shown that their structural genes are distinct from those of ibvs found in other countries, their replicase genes have not been analysed. to examine this, the complete genomic sequences of the two subpopulations of the vics vaccine, vics-v and vics-del, were determined. compared with vics-v, the more attenuated vics-del strain had two non-synonymous changes in the non-structural protein 6 (nsp6), a transmembrane (tm) domain that may participate in autocatalytic release of the 3-chymotrypsin-like protease, a polymorphic difference at the end of the s2 gene, which coincided with the body transcription-regulating sequence (b-trs) of mrna 3 and a truncated open reading frame for a peptide encoded by gene 4 (4b). these genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. phylogenetic analysis of the whole genome showed that vics-v and vics-del did not cluster with strains from other countries, supporting the hypothesis that australian ibv strains have been evolving independently for some time, and analyses of individual polymerase peptide and s glycoprotein genes suggested a distant common ancestor with no recent recombination. this study suggests the potential role of the tm domain in nsp6, the integrity of the s2 protein and the b-trs 3, and the putative accessory protein 4b, as well as the 3′ untranslated region, in the virulence and replication of ibv and has provided a better understanding of relationships between the australian vaccine strain of ibv and those used elsewhere. infectious bronchitis is a highly contagious and widespread disease of chickens and is a major ongoing problem in all countries with an intensive poultry industry (sapats et al., 1996; casais et al., 2003) . the aetiological agent is infectious bronchitis virus (ibv), a member of the family coronaviridae (beaudette & hudson, 1937; tyrell, 1968) , subfamily coronavirinae and genus gammacoronavirus (group 3; cavanagh et al., 1994) . while ibv has been detected in other avian species, including guinea fowl (numida meleagridis), partridge (alectoris sp.), peafowl (pavo cristato) and the blue-winged teal (anas sp.), clinically detectable disease has only been observed in chickens (liu et al., 2005; cavanagh, 2007; sun et al., 2007) . ibv has a positive sense, single-stranded rna genome approximately 27.6 kb in length (tannock, 1973; watkins et al., 1975; schochetman et al., 1977; jackwood & de witt, 2013) with the general organization 5′ untranslated region (utr) -1 a, ab (or polymerase genes) -s -3 a, b, c (e) -m -4 b, c -5 a, b -n -6b -3′ utr (cavanagh, 2007; ammayappan et al., 2008) . the genes encoding the structural proteins are s (spike glycoprotein), e (envelope glycoprotein), m (membrane glycoprotein) and n (nucleocapsid protein) (ignjatovic et al., 1997; cavanagh, 2007) , while the 3a, 3b, 4b, 4c, 5a, 5b and 6b genes encode accessory proteins (liu et al., 1991; liu & inglis, 1992a, b; cao et al., 2008; hewson et al., 2011; bentley et al., 2013) , the functions of which are still unknown (liu et al., 2008) . the s glycoprotein is a virion surface, rod-shaped protein that is post-translationally cleaved into two subunits, s1 and s2 (cavanagh, 1981; stern & sefton, 1982; cavanagh, 2007) . during ibv infection, virions bind to the target cell receptors and release the viral genome into the cytoplasm of the host cell (ziebuhr, 2005; knoops et al., 2008) . the polyproteins 1a and 1ab (pp1a and pp1ab) are then translated, and the papain-like (plp) and 3-chymotrypsin-like (3cl) proteases are released from these polyproteins after an autocatalytic process. these proteases initiate the cleavage in trans of the 15 peptides contained in pp1a/pp1ab. these peptides, probably together with some cellular proteins, form the replication-transcription complex. this initiates the transcription of a series of 3′ nested subgenomic rnas that are translated into the viral structural and accessory proteins (gorbalenya et al., 1989; liu & brown, 1995; tibbles et al., 1996; liu et al., 1998; ziebuhr et al., 2001; ziebuhr, 2005) . in australia, three genotypic subgroups of ibv have been distinguished (hewson et al., 2012) . subgroup 1 or "classical" strains were isolated between 1962 and 1984 and include the subtype b vaccines, which were classified using serological methods (wadey & faragher, 1981; ignjatovic & galli, 1995) and vaccine-related strains. subgroup 2 or "novel" strains emerged between 1988 and 1994, while subgroup 3 strains, which emerged around 2002, are recombinants derived from subgroup 1 and 2 strains (ignjatovic et al., 2006; hewson et al., 2012) . four different ibv strains are included in the commercially attenuated vaccines available for the control of infectious bronchitis in australia. three of these, vics, i (inghams) and s (steggles), belong to the same serotype (subtype b), while a3 (armidale) is serotypically distinct (subtype c; ignjatovic et al., 2006; hewson et al., 2012) . previous studies (hewson et al., 2012) have demonstrated that the vics vaccine contains two subpopulations, referred to as vics-v (the predominant subpopulation in the vaccine) and vics-del, which has a 40-nucleotide deletion in the 3′ utr and multiple single-nucleotide differences in the structural protein genes compared with vics-v (hewson et al., 2012) . these viral subpopulations differ in their ability to replicate in vitro and in their pathogenicity in vivo. in vics-v inoculated groups, all the birds exhibit clinical signs, including head shaking and eye scratching/irritation, from day 2 to day 8 post-inoculation, whereas in the vicsdel inoculated group the clinical signs are less severe and only seen between days 6 and 9 post-inoculation. birds inoculated with vics-v also have significantly more severe tracheal lesions than those inoculated with vics-del. over the last few years, a number of complete genome sequences of many ibvs have been determined and the sequences of more than a hundred are now available in genbank (national center for biotechnology information, ncbi). however, the complete genome of an ibv isolated in australia has not been determined. as the pathogenicity and replication of the two ibv subpopulations, vics-v and vics-del, in the vics vaccine, differ in vivo and in vitro under laboratory conditions, the complete genome sequences of these two variants were compared in order to identify potential novel molecular determinants of pathogenicity and replication in ibv. virus. the vics-v and vics-del strains of ibv, purified previously in our laboratory, were propagated in specific pathogen-free embryonated hen eggs (australian spf services, woodend, victoria, australia), as described previously (hewson et al., 2009) . after 8-9 days of incubation, the allantoic cavities of the specific pathogen-free embryonated eggs were inoculated with one of the two strains. after 48 h of incubation, the eggs were chilled for 12-18 h at 4°c and the allantoic fluid collected and stored at -80°c. viral purification and nucleic acid extraction. the allantoic fluid was clarified as previously described (lougovskaia et al., 2002) , with slight modifications. the gradients were fractioned into 1 ml volumes, and the fractions containing the highest concentration of virus were determined by pcr using primers and a protocol described previously (hewson et al., 2009) . after centrifugation at 90,000 â g for 1 h at 4°c the viral pellets were resuspended in 250-350 µl of tris-buffered saline (ph 7.4; 0.02-m tris-hcl, 0.15-m nacl) and stored at -80°c. viral rna was extracted using rneasy™ extraction kits (qiagen pty. ltd., chadstone, victoria, australia), following the manufacturer's protocol, and then stored at -80°c. complete genome sequencing. complete genome sequencing of the vics-v and vics-del strains of ibv was performed using the pgm ion torrent™ platform (life technologies australia pty. ltd., mulgrave, victoria, australia). rna was fragmented using rnase iii (life technologies) and purified using magnetic beads. to construct the libraries, the rna was reverse-transcribed using superscript™ iii (life technologies). the cdna was purified and amplified following the manufacturer's protocols and then sequenced using the 314 chip, 200 base sequencing ion onetouch™ kit v2. all the reads that matched to chicken ribosomal rna and mitochondrial genome sequences were discarded. for vics-v, the remaining reads were mapped to the genomes of the beaudette strain [genbank accession number (gan) nc_001451], two vaccine strains from usa (conn46 1996 and massachusetts, gan fj904716 and gq504724, respectively) and two strains from china (saibk and sc021202, gan dq288927 and eu714029, respectively) using geneious™ version 6.1.4 (biomatters ltd., auckland, new zealand). because of the high level of sequence diversity in the s gene, the readings were also mapped to the previously determined sequence of structural protein genes from the vics vaccine (gan jn176213). for vicsdel, the readings were mapped to the genome of vics-v and the previously determined sequence of the structural protein gene region of vics-del (gan jn983807). all gaps and ambiguous sequences were corrected using sanger sequencing and the big dye™ terminator v3.1 sequencing protocol (life technologies), following the manufacturer's instructions. as well as three turkey coronavirus (tcov) sequences and sequences of coronaviruses isolated from a duck and a peafowl in china (gan eu022525, tcov-540; eu022526, tcov-atcc; gq427174, tcov/tx-gl/01; jf705860, duck cov; and ay641576, peafowl, respectively), were used for phylogenetic analyses. the sequences were aligned using clustal-omega (sievers et al., 2011; www.ebi.ac.uk) and the phylogenetic tree constructed using geneious™ 6.1.4, using the nearest neighbour interchange maximum likelihood heuristic method with 100 bootstrap replications and the general time-reversible (gtr) substitution model, gamma distributed with invariant sites (g+i). the gtr+g+i substitution model was selected as the ideal model for these data using the "find best dna/protein model" tool, available in mega5.2 (tamura et al., 2011) . phylogenetic trees were also constructed for individual peptides encoded by the polymerase genes, the main proteinase, also referred to as 3cl, the helicase (hel), the plp and the rna-dependent rna polymerase (rdrp), and also for the s1 glycoprotein. the sequences were aligned with clustalw (geneious™ 6.1.4) and the phylogenetic trees constructed as described above. in these phylogenetic trees, only the vics-v sequence was included (except for the s1 glycoprotein tree, where both vics-v and vicsdel were included), as the vics-v and vics-del sequences were highly similar (99.8%), and vics-v is the main component of the vics vaccine (hewson et al., 2012) . to assess the extent of potential recombination between viruses, a network tree was constructed using the same complete genome alignments as described above and splitstree 4.13.1 (huson & bryant, 2006) . the distances were calculated with the uncorrected p-method and the network constructed with neighbor-net using 1000 bootstrap replicates. the sequences of the complete genomes of vics-v and vics-del have been submitted to genbank under accession numbers kf460437 and kf931628, respectively. sequencing and coverage. for vics-v and vics-del, the ion-torrent 314 chip generated 52.9 and 130.3 mb of data, comprising 503,622 and 480,037 reads, respectively, with mean read lengths of 102.9 and 124.2 nucleotides, respectively. after removal of the host-matched reads, a total of 466,235 and 456,359 reads were retained (92.57% and 95.06%), respectively. for vics-v, a total of 212,507 readings (45.6%) were matched with the complete genome sequence of the beaudette strain. the mean depth of coverage was 716 fold. a total of 98,124 reads could be mapped to the structural protein region, yielding a mean depth of coverage of 1200 fold and a minimum depth of 92 fold. for vics-del, 285,759 reads (62.6%) were matched with the complete genome sequence of the vics-v strain. the mean depth of coverage was 760 fold. a total of 95,802 reads could be mapped to the structural protein region, yielding a mean depth of coverage of 925 fold and a minimum depth of 126 fold. the vics-v and vics-del genomes were 27,610 and 27,567 nucleotides in length, with a g + c content of 38.2% and 37.8%, respectively. vics-v had the typical ibv genome organization (5′ utr -1a/1ab -s -3 a, b, c (e)-4 a, b -m -5 a, b -n -3′ utr; stadler et al., 2003; cavanagh, 2007; thor et al., 2011) , while vics-del had a truncated 4b open reading frame (orf). comparisons of the complete genomes of vics-v and the more attenuated vics-del ( figure 1 ) revealed several differences, which are summarized in table 1 . the most significant of these differences found in vics-del were the absence of a glycine (gly) in two gly-gly motifs in vicsdel and two insertions-deletions (indels) in the nucleotide sequence of the non-structural protein 6 (nsp6) gene, which would lead to a change in 11 amino acids. this frameshift introduced two positively charged amino acids, changing the region from predominantly hydrophobic, and resulted in the loss of a third gly-gly motif. there was also a single-nucleotide polymorphism (snp) in the s gene at nucleotide 3475, close to the 3′ end of the gene, with approximately equal numbers of vics-v genomes having either a g or a u, while in vics-del most genomes had a u in this position. finally, a frameshift mutation resulting in the truncation of orf 4b and a 40-nucleotide deletion in the 3′ utr region were recognized in the genome of vics-del. the snp in the s gene generated two non-synonymous codons, gaa (glutamate) or uaa (a premature stop codon), nine codons prior to the usual s gene stop codon. this snp also lies within the body transcription (b-trs) for mrna 3, which encodes the accessory proteins 3a and 3b, and the structural protein e. in vics-v, 48.4% of the reads (595/ 1222) contained the codon gaa and 51% contained the uaa stop codon. in vics-del, 5.5% of the reads contained the gaa codon and 93.6% the uaa codon (table 1) . a second gly-gly motif, which has been described as a plp cleavage site (ziebuhr, 2005) , was identified in the vics-v sequence between nucleotides 7306 and 7311. nucleotide alignment and phylogenetic analyses. phylogenetic analysis assigned most of the genomes into two main clusters (figure 2 ). the first major cluster (a) included predominantly usa strains, except for the ck/ch/ ldl/97i (china) and peafowl (china) strains. the peafowl (china) isolate lay within the beaudette subcluster and has been described previously as very similar to a mass 41 strain isolated in 1985 (liu et al., 2005) . the usa cluster can be subdivided into the mass 41 vaccine, beaudette and holte subclusters. the second cluster (b) contained only strains of chinese origin, including a strain isolated from a duck, and these were subdivided in two subclusters (b1 and b2 in figure 2 ). tcov strains lay in a separate cluster, closely related to usa ibv strains. there was a high level of similarity between the ita/90254/2005 (italy) and ck/swe/0658946/10 (sweden) strains, which have been described previously as european qx-like strains, a chinese origin genotype widely distributed in europe (ducatez et al., 2009; abro et al., 2012) . the strains ck/ch/lsd/05i (china) and tw2575/98 (taiwan) also shared a cluster. the vics-v and vics-del strains (australia) form their own cluster, while ibadan (nigeria) and snu8067 (south korea) strains did not fall into any cluster. in the phylogenetic trees of the 3cl, hel, plp and rdrp genes (figure 3) , the chinese and usa clusters were conserved. however, vics-v grouped within the chinese cluster in the 3cl and plp gene phylogenetic trees and within the usa cluster in the hel and rdrp gene phylogenetic trees. in the s glycoprotein gene trees ( figure 4 ) the isolates fell into distinct chinese and usa clusters. in these trees, vics-v, vics-del and ibadan lay closer to the usa cluster, while the south korean, taiwanese and european strains were positioned closer to the chinese cluster. the georgia 1998 and delaware 072 strains were distinct from all other strains, and were located between the ibv and tcov groups. a phylogenetic tree inferred using the amino acid sequences had the same cluster organization ( figure 5 ). network tree analysis. in the analysis shown in figure 6 , the boxes indicate the likelihood of recombination events between strains of ibv. the tree has the same cluster organization seen in the phylogenetic tree using whole genome sequences (figure 2 ). the vics-v and vics-del branch splits from all clusters close to the centre of the tree, suggesting limited and historically distant recombination with other strains. although there has been considerable investigation of ibv and of coronaviruses in general, the determinants of their virulence and their capacity to replicate in vivo remain uncertain. as recent studies have suggested that the virulence determinants may not be located in the structural protein region of some ibvs (armesto et al., 2009) , it can be inferred that these determinants may be encoded within the 1a and 1ab polyproteins. therefore, studies of complete genome sequences could provide useful information about the position and characteristics of potential virulence determinants. in this study, the genome sequences of the strains vics-v and vics-del were completed. the 1a and 1ab polyproteins of ibv are post-translationally cleaved by virus-encoded proteinases into 15 peptides (nsp2 to nsp16). nsp1, which is found in all other coronaviruses, is absent in ibv, but nsp2 is considerably larger (liu et al., 1998; snijder et al., 2003; stadler et al., 2003; gorbalenya et al., 2004; ziebuhr, 2005; fang et al., 2007; sawicki et al., 2007; carbajo-lozoya et al., 2012) . in order to determine the location of the 15 peptides encoded by the orfs in the 1a and 1ab polyproteins, we used the annotations of the beaudette strain (gan nc_001451). we complemented these annotations with those of ziebuhr (2005) . the locations of these peptides within orfs 1a and 1ab and their first and last amino acid residues are summarized in table 2 . as discussed above, vics-v exhibits higher rates of replication in vitro and causes more severe clinical signs and tracheal lesions in vivo than vics-del. as the genomes of these two viruses have very high levels of nucleotide sequence identity, it can be inferred that any differences in their genomic sequences may influence these differences in virulence and replication. as described previously, gly-gly is the cleavage motif for plp (lim et al., 2000; ziebuhr, 2005) . vics-del lacks three gly-gly motifs seen in vics-v that are located in the middle of nsp2, nsp6 and nsp14. these motifs may result in additional proteolytic processing of these three nsps, and the absence of them in vics-del could alter their function. nsp6 and nsp14 are reported to contain transmembrane (tm) and exonuclease domains (ziebuhr, 2005) , while the function of nsp2 remains undetermined. highly conserved gly-gly motifs were also found between nucleotides 2044 and 2049 (within nsp2), 3667 and 3672, 4426 and 4431 (within nsp3), 9199 and 9204, and 9391 and 9396 (within nsp5) in both vics-v and vics-del and in other ibvs. these motifs may also be further processed by plp, although the previously characterized plp cleavage sites (between nsp2 and 3, and nsp3 and 4) have an extended motif of lys-ala-gly-gly, which has been identified in all the complete genome sequences analysed in this study; this extended motif is not seen at these additional gly-gly sites. positions were based on 1ab from ibv beaudette strain (accession number nc_001451), and modified according to ziebuhr (2005) , with position 1 the methionine at the beginning of orf 1a and 1ab. b based on phillips et al. (2012) . the mutations in nsp6 of vics-del (table 1 ) may have implications for the replication of this virus, which would be consistent with data from previous in vivo studies (hewson et al., 2012) . during infection 3cl, the main protease, is auto-catalytically released (ziebuhr et al., 2001; ziebuhr, 2005) . there may be an interaction between the 3cl (nsp5), the tm2 (nsp4) and the tm3 (nsp6) hydrophobic domains (ziebuhr, 2005) , which are also referred to as mp1 and mp2 (tibbles et al., 1996) , during this proteolytic process. the importance of tm3/mp2, the transmembrane domain downstream of the 3cl proteinase, in the autocatalytic processing of 3cl has been suggested by in vitro assays (tibbles et al., 1996) . in this experiment, the further processing of a 66-kda immature protein product into smaller, post-translationally cleaved products of 59, 42, 35 and 24 kda (with the 35-kda peptide the size predicted for the fully processed form of 3cl protease) did not occur if these two hydrophobic domains were not associated with microsomal membranes. changes in tm3/ mp2, such as those detected in vics-del, could affect the membrane association of this domain and thus the conformation of the protein, and the autocatalytic release of 3cl, which is essential for the initial stages of replication (ziebuhr, 2005) . the snp detected at the end of the s gene is also notable. the difference in the proportions of each of the nonsynonymous codons between the vics-v and vics-del populations, with a greater prevalence of the truncation phenotype in the vics-del population, suggests that truncation of the s2 protein may be related to the difference in virulence between these strains. the carboxyl terminal end of s2 anchors the s protein to the virion and also assists in membrane fusion with target cells (cavanagh, 2007) . alternatively, the differing b-trs sequences, cugaa-caa and cuuaacaa, which have both been identified previously in ibv (mondal & cardona, 2007; woo et al., 2010) , may differ in their efficiency in regulating replication and transcription and thus may influence production of mrna 3, which encodes the e protein. in the middle east respiratory syndrome coronavirus the deletion of the e protein results in a replication-competent, propagationdefective virus (almazán et al., 2013) . a nine amino acid truncation, resulting from the nucleotide change from u to g, has been described previously in an m41 strain variant, m41h (kusters et al., 1989) . while these alternative codons have also been found in other ibv strains, the methods used for sequence determination would have precluded identification of differing subpopulations within these strains, as cloned, single cdna molecules were used for sequencing. the orfs 4b and 4c have been described in ibv (hewson et al., 2011 (hewson et al., , 2012 thor et al., 2011; phillips et al., 2012; bentley et al., 2013) , as well as in tcov (cao et al., 2008) , and have been synthesized in vitro (bentley et al., 2013) , but the products of these orfs are yet to be identified in infected cells, and their function remains unknown. both these orfs could be identified in the vics-v genome, but in vics-del, 4b was divided into two smaller orfs due to a singlenucleotide deletion. the vics vaccine is known to induce some clinical signs after vaccination. hewson et al. (2012) showed that vics-v caused more severe vaccine reactions than vics-del, had a higher replication rate in the trachea, kidneys and caecal tonsils, and induced more severe clinical signs. the putative 4b and 4c proteins have two interesting features: their high content of positively charged amino acids (lys and arg) and the presence of phosphorylation motifs, suggesting a possible role in the packaging of the genome, which may influence the rate of viral replication and therefore pathogenicity. phylogenetic analyses showed that the australian strains of ibv are distinct from strains isolated elsewhere in the world and that the most commonly used australian vaccine strain is very distinct from other ibv vaccine strains across the entire genome. this divergence has been shown previously in the structural genes (yu et al., 2001; bochkov et al., 2007) , but this is the first study to show that the divergence can be seen consistently throughout the genome. however, most of the fully sequenced ibv genomes are of strains with an origin in the usa or china, as only a few qx-type and qx-like strains (which now predominate in europe) have been fully sequenced, and this bias could have influenced the topology of the tree. these european strains were represented in this tree by strains from italy (ita/90254/2005) and sweden (ck/ swe/0658946/10). the phylogenetic tree constructed with the nucleotide sequences of the s1 protein ( figure 4 ) includes more qx-type and qx-like strains, including ck/ uk/kg3p/10 and ck/uk/3355/09 (uk), ck/ger/gb1835/ 10 (germany), ck/swe/0658946/10 and ck/swe/082066/ 10 (sweden), ita/90254/2005 (italy) and ck/sp/170/09 and la/sp/116/09 (spain). phylogenetic trees of individual orfs (figures 3, 4 and 5) indicate that different regions of the vics-v genome varied in their similarity to the two major clusters, suggesting historical recombination events. however, this seems very unlikely to have happened recently, based on the results from the network tree analysis ( figure 6 ). vics-v diverged and evolved as a distinct cluster, with the length of the vics-v branches, especially in the 3cl, hel and rdrp phylogenetic trees, suggesting more rapid evolution than most other strains. in conclusion, full genome analysis of vics-v and vicsdel has shown that the australian ibv vaccine vics has been evolving independently of strains elsewhere in the world for some time. the differences in the replication rate and pathogenicity of these two viral subpopulations may be attributable to the mutation in the tm3 domain in nsp6 and the snp found at the end of the s glycoprotein gene, described in this study for the first time. these newly described differences are additional to the previously described frameshift/truncation of the orf 4b and the 40nucleotide deletion in the 3′ utr in vics-del (hewson et al., 2012) . further analyses of the complete genome sequences of other australian ibv strains may reveal additional information about relationships between these distinct vaccine strains and ibvs within the usa and chinese clusters. additional investigation of the effect of the differences between the vics-v and vics-del strains described in this study may shed more light on the replication and pathogenicity of ibv and may also indicate whether the naturally attenuated strain vics-del and other strains with similar mutations have potential as novel vaccine candidates for the control of ibv infections. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate complete genomic sequence analysis of infectious bronchitis virus ark dpi strain and its evolution by recombination cultivation of the virus of infectious bronchitis identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and 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of poultry 13th edn sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum phylogeny of antigenic variants of avian coronavirus ibv identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products characterisation and mutational analysis of an orf 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus a polycistronic mrna specified by the coronavirus infectious bronchitis virus proteolytic processing of the polyprotein encoded by orf1b of the coronavirus infectious bronchitis virus (ibv) isolation of avian infectious bronchitis 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coronavirus group 2 lineage sars-beginning to understand a new virus coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins a massachusetts prototype like coronavirus isolated from wild peafowls is pathogenic to chickens mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods the nucleic acid of infectious bronchitis virus recombination in avian gamma-coronavirus infectious bronchitis virus characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus australian infectious bronchitis viruses: identification of nine subtypes by a neutralisation test the ribonucleic acid of infectious bronchitis virus coronavirus genomics and bioinformatics analysis. viruses characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the coronavirus replicase the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond key: cord-331020-lyxje82u authors: m. najimudeen, shahnas; h. hassan, mohamed s.; c. cork, susan; abdul-careem, mohamed faizal title: infectious bronchitis coronavirus infection in chickens: multiple system disease with immune suppression date: 2020-09-24 journal: pathogens doi: 10.3390/pathogens9100779 sha: doc_id: 331020 cord_uid: lyxje82u in the early 1930s, infectious bronchitis (ib) was first characterized as a respiratory disease in young chickens; later, the disease was also described in older chickens. the etiology of ib was confirmed later as being due to a coronavirus: the infectious bronchitis virus (ibv). being a coronavirus, ibv is subject to constant genome change due to mutation and recombination, with the consequence of changing clinical and pathological manifestations. the potential use of live attenuated vaccines for the control of ibv infection was demonstrated in the early 1950s, but vaccine breaks occurred due to the emergence of new ibv serotypes. over the years, various ibv genotypes associated with reproductive, renal, gastrointestinal, muscular and immunosuppressive manifestations have emerged. ibv causes considerable economic impacts on global poultry production due to its pathogenesis involving multiple body systems and immune suppression; hence, there is a need to better understand the pathogenesis of infection and the immune response in order to help developing better management strategies. the evolution of new strains of ibv during the last nine decades against vaccine-induced immune response and changing clinical and pathological manifestations emphasize the necessity of the rational development of intervention strategies based on a thorough understanding of ibv interaction with the host. infectious bronchitis virus (ibv) belongs to the family coronaviridae and order nidovirales [1] . ibv infects chickens and pheasants and induces a clinical disease known as infectious bronchitis (ib) [2] . the infected chickens may appear depressed with various levels of breathing difficulty and have ruffled feathers [3] [4] [5] [6] . younger chickens show the most severe clinical manifestations [7] . in addition, there is serological evidence that poultry workers may develop anti-ibv antibodies following exposure to infected birds, although there is no evidence of active infection in humans [8] . however, experimentally, certain ibv strains (i.e., massachusetts (mass) and gray) have been found to be capable of replicating in human cell lines [9] . the first ib case caused by the mass serotype was recorded from north dakota, usa [10] . since then, hundreds of ibvs with heterogenous genomes have been emerging continuously as a result of mutations and recombination [11, 12] . consequently, high ib-associated losses are recorded in spite of control attempts using live attenuated vaccines. different strains of ibv demonstrate varying properties with differences in pathogenesis, virulence, tissue and age tropisms and receptor figure 1 . phylogenetic tree based on the s1 nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of 21 infectious bronchitis virus (ibv) strains from different countries around the world. the ibv strain and genbank accession number are given for each strain. the sequences were aligned using clustal omega, and the phylogenetic tree was constructed using the maximum likelihood method available in raxml with 1000 bootstrap replicates for branch support (the numbers on the nodes represent the bootstrap values). the analyses were constructed using geneious ® v10.2.6 (https://www.geneious.com/). it is well established that, following the initial infection in the respiratory tract, the virus is disseminated to other tissues due to viremia [18, 57] (figure 2 ). however, the exact mechanisms by which ibv leads to viremia are not understood. recently, reddy et al. (2016) showed that a belgian nephropathogenic ibv strain, b1648, could infect blood monocytes and that these monocytes may facilitate the dissemination of ibv to visceral organs, including the kidney [50] . in agreement with this finding, another study [58] showed tropism of ibv towards monocytes by the mass 41, california phylogenetic tree based on the s1 nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of 21 infectious bronchitis virus (ibv) strains from different countries around the world. the ibv strain and genbank accession number are given for each strain. the sequences were aligned using clustal omega, and the phylogenetic tree was constructed using the maximum likelihood method available in raxml with 1000 bootstrap replicates for branch support (the numbers on the nodes represent the bootstrap values). the analyses were constructed using geneious ® v10.2.6 (https://www.geneious.com/). ibv is typically transmitted to the host by inhalation, whereupon it attaches to the respiratory epithelium and enters by receptor-mediated endocytosis [46] . once ibv enters the upper respiratory tract, it targets the epithelium, which is ciliated and includes mucus-secreting glands [30] , causing ciliostasis and mucus accumulation. the virus replication in the respiratory epithelium peaks 3-7 days post-infection (dpi) depending on the infecting ibv strain [35, 47, 48] . consequently, respiratory signs result within 2 dpi and peak around 6 dpi [49] [50] [51] . these respiratory clinical manifestations include sneezing, gasping, coughing, tracheal rales, nasal discharge, conjunctivitis, and dyspnea [3] . it is not uncommon to observe non-respiratory clinical manifestations such as depression, weight loss, lethargy and huddling together [32, 50, 52] . the morbidity and mortality associated with respiratory tract ibv infection depend on the age of infection; young chickens are severely affected compared to adult chickens [7, 53] . at post-mortem examination, hemorrhages and the accumulation of caseous, serous and catarrhal exudates in the trachea, nasal passage and sinuses [47, 52] are evident, as well as gross changes in air sacs (i.e., the accumulation of foamy or cloudy exudates) [54] . depending on the time of sampling, histological features include deciliation and dislodgment of epithelial cells, as well as mononuclear cell infiltration. these changes are visible around 2-3 dpi. further development of the lesions, with hyperplasia and hypertrophy of the epithelium and prominent mononuclear cell infiltration in lamina propria, are visible around 4-6 dpi. these stages are followed by recovery with repopulation of the mucosa with pseudostratified ciliated epithelium and goblet cells (10-20 dpi) [47, 55, 56] . it is well established that, following the initial infection in the respiratory tract, the virus is disseminated to other tissues due to viremia [18, 57] (figure 2 ). however, the exact mechanisms by which ibv leads to viremia are not understood. recently, reddy et al. (2016) showed that a belgian nephropathogenic ibv strain, b1648, could infect blood monocytes and that these monocytes may facilitate the dissemination of ibv to visceral organs, including the kidney [50] . in agreement with this finding, another study [58] showed tropism of ibv towards monocytes by the mass 41, california (cal)99, connecticut (conn)46 and iowa97 ibv strains. using the mass and conn ibv strains, amarasinghe et al. (2017) showed that ibv could infect low numbers of respiratory tract macrophages [59] . it is also possible that ibv dissemination beyond the respiratory tract may involve the lymphatic system and infected macrophages, similar to marek's disease virus dissemination via infected lung macrophages [60] . 2017) showed that ibv could infect low numbers of respiratory tract macrophages [59] . it is also possible that ibv dissemination beyond the respiratory tract may involve the lymphatic system and infected macrophages, similar to marek's disease virus dissemination via infected lung macrophages [60] . subsequent clinical manifestations of ibv infection related to the reproductive tract depend on the infecting ibv strain. for example, the m41, aust t and qx-like strains are known to cause reproductive tract defects in long-lived chickens [15, 17, 25] , leading to low egg production and quality, whereas ibv strains such as conn and iowa609 do not cause reproductive tract abnormalities [15] . the mechanisms that lead certain ibv strains to establish reproductive tract infection are unknown. in different body systems. all the ibv strains primarily infect the respiratory tract, and based on the genotypes, the ibv infection can extend to various tissues, either persisting or leading to clinical and pathological manifestations. the solid arrows indicate paths that have been confirmed. the empty arrows indicate paths that have been suggested. the text boxes with continuous borders summarize histological changes, and the text boxes with discontinuous borders represent clinical manifestations. ses, shell-less egg syndrome; fls, false layer syndrome; git, gastrointestinal tract. subsequent clinical manifestations of ibv infection related to the reproductive tract depend on the infecting ibv strain. for example, the m41, aust t and qx-like strains are known to cause reproductive tract defects in long-lived chickens [15, 17, 25] , leading to low egg production and quality, whereas ibv strains such as conn and iowa609 do not cause reproductive tract abnormalities [15] . the mechanisms that lead certain ibv strains to establish reproductive tract infection are unknown. however, ibv replication in reproductive mucosa has been documented in several studies [15, 61, 62] . depending on the infecting ibv serotype, localization of ibv antigens in the oviduct can vary, and evidence of higher ibv replication has been observed in chickens infected at a younger age when compared to adults [15] . this age difference in ibv replication is reflected in differences in clinical and pathological outcomes in chickens. for example, chickens infected with certain strains of ibv such as mass, qx-like strain or aust t at ages of 1-14 days develop cystic oviducts without impaired ovarian functions, which leads to false layer syndrome with no egg production [15, [63] [64] [65] . such flocks with false layer syndrome do not reach peak production, with a consequence of premature culling [21, 63] . infection with ibv in laying hens can negatively influence egg production, resulting in poor-quality eggs, such as misshapen, miscolored, thin, rough-shelled or shell-less eggs and eggs with watery albumin, meat or blood spots, which can peak at around two weeks post-infection [15, 63, 66] . in addition, egg production in laying hens can drop by 35-90% [63, 67] . although production bounces back close to normal within nine weeks, there can be a 6-12% decline relative to normal production [63, 67] . ibv infection of the reproductive tract of laying hens leads to shorter, hypoglandular oviducts and regressed ovaries [15, 66] . histologically, deciliation of the epithelium and a reduction in the height of the epithelial cells, along with epithelial desquamation and degeneration, are common in infected oviducts between 7 and 21 dpi [66] with occasional follicular destruction and microbleeding [15, 68] . fibroplasia and edema of infected lamina propria are also evident [66] . in contrast to the development of false layers, chickens with patent oviducts were found to have an intact surface epithelium with localized hypoplasia of glandular areas [5] . later, following around 10 dpi, lymphoid nodules are seen in the oviduct [15] . although the consequences of ibv targeting the reproductive tracts of female chickens is known, little attention has been given to changes corresponding to the reproductive tracts of male chickens, which may cause impacted sperm production, infertility and venereal transmission of ibv [69, 70] . recent studies have found, depending on the infecting ibv strain, that ibv targets the testes and causes low sperm production and infertility [69, 71, 72] . ibv replication in the efferent duct epithelium and the formation of epididymal stones lead to low sperm production and infertility [69] . gallardo and colleagues observed ibv replication in cells of the seminiferous tubule (i.e., sertoli cells) infected with arkansas (ark) and mass ibv strains; moreover, they demonstrated ibv transmission to layers via infected semen, indicating the possibility of venereal transmission of ibv [70] . ibv-induced changes including mononuclear cell and heterophil infiltration, necrosis and microbleeding of testes have been documented [68] . the nephropathogenic ibv strains include b1648, aus t, qx-like, 4/91, holte and gray [6, 24, 25, 32, 50] . the ibv b1648 strain is known to spread from the respiratory tissues via blood monocytes [50] . following dissemination, ibv strains could infect ciliated cells of the nephron, including proximal, distal and collecting tubules, depending on the infecting ibv strain [3, 32, 47, 48] . ibv strains that target the kidney have received increased attention due to their higher virulence in young chickens when compared to other ibv strains [13, 32, 73] . apart from the general signs associated with ibv infection, nephropathogenic ibv strains result in weight loss, watery droppings, increased water consumption and an increase in the incidence of mortality [6, 50] . the nephropathogenic ibv pathogenesis also varies according to the breed of the chickens; for example, the clinicopathological manifestations of nephropathogenic ibv are more severe in rhode island red chickens when compared to white leghorn chickens [32] . at post-mortem examination, ibv-infected kidneys are pale, discolored and enlarged [32, 47] . urate deposits are also commonly observed with tubular distention [3, 32, 50] . histologically, tubules develop degenerative changes, ureters become distended with cellular debris and urate crystals are seen in the tubules; in addition, mononuclear cell recruitment in interstitial tissues in the medulla and cortex has been observed [3, 32, 50] . although ibv has been isolated from cloacal swabs, there is no indication that ibv is transmitted via the fecal-oral route. it is possible that gastrointestinal infection follows respiratory infection, subsequent infection of monocytes and macrophages and the spread of ibv via the blood or lymphatics [50, 59] . ibv strains such as qx-like strains, 793/b (4/91) and moroccan g are known to infect the gastrointestinal tissues, leading to clinical and pathological manifestations [35, [74] [75] [76] . the qx-like strains are capable of targeting the proventriculus and ileum, leading to proventriculitis and, occasionally, diarrhea [56, 76] . moroccan g ibv has also been shown to target the gastrointestinal tissues, such as the esophagus, jejunum, ileum and rectum [74] . other experimental studies that used moroccan g ibv indicated that the virus targets the epithelial covering of the tips of villi of the ilium and rectum, leading to atrophy of the villi and desquamation of epithelial cells, with lymphocyte, macrophage and heterophil infiltration in the mucosa [3, 35] . ibv 793/b (i.e., 4/91) has also been shown to replicate in the esophagus and ileum, leading to enteritis in young broiler chickens [35] . in the early 1990s, broiler chickens infected with ibv strain 793/b were found to develop bilateral pectoral myopathy [34] . the disease was characterized by edema, due to a gelatinous material, followed by facial hemorrhage and mild separation of muscle fibers [34] . although bilateral myopathy could not be reproduced with ibv 793/b, mild gross changes were observed with no indication of histological changes or muscle damage [35] . further, a study that collected samples with bilateral pectoral myopathy from a slaughter plant in brazil could not establish an association of this condition with ibv infection, although ibv was detected in muscle tissues using molecular techniques [77] . consequently, the muscle lesions were suggested to be caused by type iii hypersensitivity involving the deposition of immune complexes in the capillary walls of pectoral muscles, rather than lesions induced by viral replication [35] . depending on the ibv strain, the virus can persist in the tissues of chickens for an extended period [40, 74, 78] . for example, ibv (i.e., aust t strain) can persist, particularly in the cecal tonsils and kidney, for more than seven months [40] . the mass ibv strain can persist in the cecal tonsils, spleen and kidney for about a month [78] . although the period of ibv persistence is influenced by the age at infection [42] , the length of persistence does not depend on the systemic anti-ibv antibody concentration [4] . the implications of ibv persistence in chickens are twofold. first, persistently infected chickens are a source of infection for naïve chickens [4] . second, persistent ibv infection promotes viral evolution [4] . the ibv-induced potential immune-suppressive mechanisms are summarized in figure 3 . ibv strains 4/91, qx-like, strain g and mass infect various immune organs, such as the cecal tonsils [35] , spleen [45] , harderian gland and bursa of fabricius [3, 79] . it is not known whether the tropism of ibv for these immune organs depends on the virulence of the infecting ibv strain or whether ibv replication in these immune organs impacts immune functions. it is very well documented that avian viruses that replicate in immune organs, such as marek's disease virus [80] , chicken anemia virus [81, 82] and infectious bursal disease virus [83] , are immunosuppressive, impacting vaccine-mediated immune response and resulting in secondary bacterial infections [84] . however, recent investigations provided molecular and cellular evidence that ibv, in fact, directly interferes with the host's innate response at various levels, potentially impacting the elicitation of adaptive host response. the toll-like receptors (tlrs) 3 and 7 are innate receptors and have a role in detecting ibv-associated molecular patterns, such as double-stranded (ds) and single-stranded (ss) ribonucleic acid (rna), respectively [85] . certain brazilian strains of ibv are capable of inhibiting tlr7 signaling, leading to decreased proinflammatory cytokines and decreased mrna expression linked to the development of cell-mediated immune response, leading to increased pathology in the kidney [86] . similarly, a conn strain of ibv has been shown to downregulate mrna expression of tlr3, interleukin (il)-1β and interferon gamma (ifn-γ), leading to increased ibv genome accumulation and more severe pathology in the respiratory tissues [87] . certain strains of ibv can inhibit the expression of pathogen recognition receptors such as toll-like receptors (tlrs) or their signaling pathway, leading to reduced expression of proinflammatory cytokines, which eventually interferes with the innate immune response and induction of the adaptive immune response. ibv is also capable of replicating in respiratory tract macrophages, inhibiting their functions and inducing apoptosis. ibv is capable of incorporating cd59 molecules into its envelop during egress from the host cells, shielding it against lysis via complement-and antibody-dependent mechanisms. protection against ib is mediated by both antibody-and cell-mediated immune responses [102, 103] . although the antibody-mediated immune response predominantly depends on a response , enabling it to persist in the host for a longer period. epithelial cells are the primary target sites of ibv replication, which results in deciliation and destruction, leading to inhibition of the mucociliary escalator mechanism. certain strains of ibv can inhibit the expression of pathogen recognition receptors such as toll-like receptors (tlrs) or their signaling pathway, leading to reduced expression of proinflammatory cytokines, which eventually interferes with the innate immune response and induction of the adaptive immune response. ibv is also capable of replicating in respiratory tract macrophages, inhibiting their functions and inducing apoptosis. ibv is capable of incorporating cd59 molecules into its envelop during egress from the host cells, shielding it against lysis via complement-and antibody-dependent mechanisms. one of the immune cell types that bridges innate and adaptive host responses is the macrophages, and the available data show that certain ibv serotypes (i.e., mass and conn) target respiratory tract macrophages and replicate within them, thus leading to a productive infection [59, 88] . although the impact of immune cell targeting of ibv has not been studied completely, ibv replication in macrophages could decrease type 1 ifns activity [59] , similar to ibv's ability to hinder type 1 ifns response in epithelial and fibroblast cells [89] . type 1 ifns are the main antiviral molecules synthesized in the host in response to viral replication, and, previously, it has been shown that ibv is sensitive to the antiviral activity of type 1 ifns given as a treatment to prevent ib in chickens [90] . although it is not known how ibv inhibits the function of type 1 ifns in macrophages, one potential explanation is that the accessory proteins of ibv could play a role in this immune evasion strategy. in agreement with this view, the ability of ibv accessary proteins 3a and 5b to interfere with type 1 ifns production in target cells other than macrophages has been shown [89, 91] . other than this interference with the production of type 1 ifns, ibv is capable of inhibiting the downstream signaling of type 1 ifns, minimizing the expression of interferon-stimulating genes (isgs) by preventing the functioning of signal transducer and activator of transcription 1 (stat1) [89] . another implication of ibv replication in macrophages is the destruction of macrophages due to apoptosis. ibv could induce programmed cell death in avian macrophages via intrinsic and extrinsic routes involved in apoptosis [88] . although the mechanisms of the destruction of macrophages by ibv require further investigation, evidence has shown that macrophage numbers increase at 24 h and then decline in the trachea and lungs in response to ibv infection [87] . it is important to understand whether this decline is related to the apoptosis of ibv-infected macrophages. the complement system functions as a part of the innate arm of the immune response and also plays a role in adaptive immune functions. the components of the complement pathway aid in the host immune response via complement-and antibody-mediated lysis of viruses [92] [93] [94] [95] . since host cells are shielded from lysis via this strategy due to the expression of cd59 in the host cell membrane, ibv is capable of incorporating cd59 into its envelop during exit from the host cells, negating lysis via complement-and antibody-dependent mechanisms [96] . previous studies showed that ibv is vulnerable to attack by this strategy [97] . ibv replication impacting the respiratory tract brings forth a defective clearance mechanism by the deciliation of the respiratory epithelium, thereby increasing the vulnerability of respiratory surfaces to secondary bacterial infections [98] . the co-infection of respiratory pathogens with ibv causes complications and worsens the clinical and pathological manifestations [18, 99] . the infection of chickens with mycoplasma gallisepticum followed by ibv infection can result in coryza, tracheitis and airsacculitis in the host [100] . in addition, secondary infections with pathogenic escherichia coli can lead to prominent lesions in respiratory surfaces, pericarditis and death [18] . in addition, the co-infection of haemophilus paragallinarum with ibv could result in severe lesion development and increased mortality rates [101] . protection against ib is mediated by both antibody-and cell-mediated immune responses [102, 103] . although the antibody-mediated immune response predominantly depends on a response to the ibv s1 protein, the main cell-mediated response, cd8+ cytotoxic t cell response is elicited by the ibv n protein [103] . following ibv infection, memory b [104] and t cells [105] are formed and are present in peripheral blood and the spleen. following ib vaccination, the number of b and t cells increases in the harderian gland [106] . increased recruitments of cd4+ and cd8+ t cells in the trachea have also been shown following application of live ib vaccines [107] . vaccination is the primary choice for the control of ib in the poultry industry. the chickens receive multiple ib vaccinations, and the frequency of vaccination depends on the expected production life of the chicken. for broilers, day-old vaccination in the hatchery is commonly practiced followed by a booster vaccination at 2-3 weeks of age [99] . multiple vaccinations with live attenuated vaccines are usually given to layers and breeders followed by a killed vaccine just before the onset of the laying period [108, 109] . the live attenuated vaccines are administered via drinking water, eye drop, or coarse spray and inactivated vaccines are given parenterally. for example, in eastern canada, the layer and breeder pullets are vaccinated with various combinations of live attenuated ib vaccines starting at one day of age and then, at two, five and nine weeks followed by an inactivated ib vaccine given at fourteen weeks of age. during the lay, the chickens are not vaccinated. the goal of such layer and breeder vaccination strategies is to ensure the transfer of maternal antibodies to the offspring [110] as well as provide the extended protection during the lay [111, 112] . however, vaccination of day-old chickens is controversial for three reasons. first, maternal antibodies could be protective against potential ibv infection during the first few days of life while early vaccination could enhance the decay of the maternal antibodies [110] . second, early vaccination (i.e., day 1 of age) induces poor b cell and t cell responses when compared to later vaccination (i.e., day 7 of age) [106] . third, ib vaccination at the day of age can induce severe vaccine reactions [113] . despite these reasons, live attenuated ib vaccination in the hatchery can be critical for the prevention of severe respiratory illness and cystic lesions in the oviduct that results from very early exposure to virulent ibvs [65, 114] . since the introduction of vaccination against ib using live attenuated (mass serotype) vaccines, different vaccine serotypes such as 4/91, ark, conn, d274 and d1466 have been developed and made available commercially [115] . based on the prevalence of the various ibv serotypes, and the availability of licensed vaccines in a geographical area, different areas within countries use different combinations of live attenuated and inactivated vaccines. in certain regions in europe, vaccines against mass and 4/91 serotypes are widely used, while, in certain states within usa, vaccines against mass, ark and conn serotypes are extensively used [108] . the serological response to ibv is mainly induced by the s1 protein [116, 117] and, consequently, cross-protection induced by ib vaccines against heterologous strains varies widely. the study of cook and colleagues indicated that the use of more than one vaccine serotype can produce better cross-protection against challenge with heterologous ibv serotypes [118] . therefore, priming with more than one heterologous live vaccines and boosting with inactivated or live attenuated vaccines are practiced particularly by layer and breeder industries [65] . it has also been shown that vaccination protocols that involve more than one serotype induced better immune cell recruitment in the respiratory mucosa when compared to vaccination with a single serotype vaccine [107] . although control of ib relies on vaccination [113] , several limitations of ib vaccination have been observed. the steady increase of ibv variants leading to frequent outbreaks in vaccinated flocks has become a concern increasingly [99, 119, 120] . in addition, attenuated ibv strains used for vaccination can spread among individual birds within flocks [121] , and changes in virulence during bird-to-bird passage can lead to production problems [120, 122] . coinfection of host cells with live attenuated ib vaccines and wild type ibv can also result in genomic recombination contributing to the virus evolution [11, 123] as well as mutations that can occur under the effect of immune pressure [124] [125] [126] . although the global poultry industry practices hatchery vaccination against ib, day 1 may not be the optimum time for vaccination in terms of inducing systemic and mucosal immune responses against ib [106] because of the developing immune system [127] . the availability of a limited number of licensed vaccines to choose in a geographical area is also a constraint. for example, in canada, various ibv variants including mass, conn, 4/91, ca1737 and dmv/1639 [11, 23, [128] [129] [130] are circulating in poultry flocks and only live attenuated vaccines developed against mass and conn serotypes and inactivated vaccines developed against mass and ark serotypes are available for optimizing on farm ib vaccination strategies. given the limited efficacy of existing ib vaccination strategies, it is critical to establish an ibv surveillance system that characterizes the different ibv strains circulating in various geographical areas and that we understand the antigenic and/or genetic similarities between the circulating ibvs and the available ib vaccines. these data will lead to the optimization of ib vaccination strategies to prevent vaccine breaks. if the existing vaccines are not useful in optimizing vaccination strategies, it would also worth developing autogenous vaccines (inactivated) using characterized unique ibv isolates prevalent in a given geographical area in order to include in the existing vaccination regimes. given the available scientific evidence against ib vaccination at the hatchery [106, 110] , it may also be appropriate to postpone the first ib vaccination to a barn vaccination done at seven days of age relying on maternal antibody response to protect the chickens during the first week of life [110] . there are numerous issues surrounding the use of live attenuated ib vaccines [11, 99, [119] [120] [121] [122] [123] [124] [125] [126] , and relying on inactivated vaccines can be an option, but they inherently lack the ability to induce mucosal immune response, which is critical for the control of ib [131] [132] [133] . it is possible that inactivated vaccines can be used for the induction of mucosal immune response when combined with various nanoparticles [132] . after decades of research into ibv and numerous studies on vaccine efficacy for the control of ib, this disease is still a major economic concern globally. although many studies have been conducted on ibv pathogenesis, there is little specific information on ibv receptors that determine macrophage tropism and tissue tropism, mechanisms that allow ibv dissemination from the respiratory tract to secondary tissues, ibv persistence in the cecal tonsils and kidney and the immunopathogenesis and immunosuppressive mechanisms of different strains of ibv. novel and sensitive assays and other necessary tools are now available for in-depth investigations of the mechanisms involved in these pathogenesis events. given the concern of vaccine breaks and the emergence of heterogeneous ibv strains, investigations leading to an in-depth understanding of the pathogenesis of ibv are necessary. studies of host-ibv interaction lead to the understanding of tropism of ibv for various body systems, severity of lesions produced in each of these tissues and ways the virus is shed to the environment [3, 51, 64, 134] . for example, certain ibv variants impact tissues such as kidney and reproductive tract in addition to the respiratory tract [13, 23, 32, 33] . we are not aware if the current vaccination protocols induce adequate mucosal immune responses in each of these tissues to minimize consequences of ibv replication. it is critical to optimize ib vaccination strategies 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infectious bronchitis strains from different pathological conditions this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-355703-l9l4ybfn authors: zhao, fei; han, zongxi; zhang, tingting; shao, yuhao; kong, xiangang; ma, huijie; liu, shengwang title: genomic characteristics and changes of avian infectious bronchitis virus strain ck/ch/ldl/97i after serial passages in chicken embryos date: 2014-08-29 journal: intervirology doi: 10.1159/000365193 sha: doc_id: 355703 cord_uid: l9l4ybfn background: we previously attenuated the infectious bronchitis virus (ibv) strain ck/ch/ldl/97i and found that it can convey protection against the homologous pathogenic virus. objective: to compare the full-length genome sequences of the chinese ibv strain ck/ch/ldl/97i and its embryo-passaged, attenuated level to identify sequence substitutions responsible for the attenuation and define markers of attenuation. methods: the full-length genomes of ck/ch/ldl/97i p5 and p115 were amplified and sequenced. the sequences were assembled and compared using the megalign program (dnastar) and a phylogenetic tree was constructed using mega4 software. results: the ck/ch/ldl/97i virus population contained subpopulations with a mixture of genetic mutants. changes were observed in nsp4, nsp9, nsp11/12, nsp14, nsp15, nsp16, and orf3a, but these did not result in amino acid substitutions or did not show functional variations. amino acid substitutions occurred in the remaining genes between p5 and p115; most were found in the s region, and some of the nucleotide mutations resulted in amino acid substitutions. among the 9 nsps in the orf1 region, nsp3 contained the most nucleotide substitutions. conclusions: sequence variations in different genes, especially the s gene and nsp3, in the genomes of ck/ch/ldl/97i viruses might contribute to differences in viral replication, pathogenicity, antigenicity, immunogenicity, and tissue tropism. coronaviruses (covs) infect a variety of animals and can cause respiratory, enteric, hepatic and neurological diseases of varying severities. covs were recently divided into four genera based on genotypic and serological characterizations: alpha, beta, gamma, and delta. infectious bronchitis virus (ibv) is a gammacoronavirus of the subfamily coronavirinae , family coronaviridae , order nidovirales [1] . ibv is distributed worldwide, it is highly infectious, and extremely difficult to control because of its extensive genetic diversity, short generation time, and high mutation rate. although live-attenuated infectious bronchitis (ib) vaccines have been used worldwide since the 1950s, ibv continues to cause disease in chickens, even in vaccinated birds, because the virus is constantly changing and evolving to avoid the host immune response. ibv can cause respiratory disease in chickens of all ages [2] . some strains are nephropathogenic, resulting in renal-induced mortality of 25-80% in susceptible flocks [3] . other strains can replicate in the oviduct and cause permanent damage in immature females or pullets resulting in limited egg production in mature hens [2] . covs are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, single-stranded, positive-sense rna genome of 28-32 kb, with a 5 ′ cap and a 3 ′ polya tail [4] . like all covs, the proximal two thirds of the ibv genome encode two overlapping open reading frames (orfs), 1a and 1b, which are proteolytically cleaved by two virus-encoded replicase proteins, the papain-like and 3c-like proteinases, into at least 15 nonstructural proteins (nsp216) [4] . replication of the cov rna genome and transcription of a nested set of 5-8 subgenomic rna species are carried out by these proteinases. however, the exact functions of individual replicases remain largely unknown. the remaining third of the ibv genome encodes four major structural proteins: the spike (s) glycoprotein, the small envelope (e) protein, the membrane (m) glycoprotein, and the nucleocapsid (n) protein. two small accessory genes, genes 3 and 5, have been described in ibv, which express accessory proteins 3a, 3b and 5a, 5b, respectively [3, 5, 6 ] . in addition, the 5 ′ and 3 ′ untranslated regions (utrs) in the ibv genome, like those of other covs, usually harbor important structural elements involved in replication and/or translation [7] [8] [9] [10] [11] . ib is mainly controlled by the use of live-attenuated vaccines derived from virulent viruses prevalent in a particular country or region by multiple serial passages in 9-to 11-day-old embryonated chicken eggs [12] [13] [14] [15] [16] . as a consequence of this process, the virus becomes more adapted and more highly pathogenic to the embryo, with concomitant attenuation in mature chickens. the ibv strain beaudette was attenuated after several hundred passages in embryonated hen's eggs [17] resulting in loss of virulence and also potential loss of immunogenicity. a similar result was observed in ibv strain ck/ch/ lhlj/04v, the immunogenicity of which was substantially decreased after 110 passages in embryonated hen's eggs, resulting in loss of virulence to chickens [16] . however, most attenuated ib vaccine strains, such as h120, maintain their immunogenicity after adaptation to embryonated chicken eggs [12] [13] [14] [15] , though the genomic mutations in passaged strains associated with attenuation of pathogenicity, immunogenicity, tissue tropism, and replication capacity in chicken tissues are unknown and variable, leading to differing efficacies associated with different vaccines. a novel type of ibv, designated as ck/ch/ldl/97i, emerged in the mid-1990s and circulated among both vaccinated and non-vaccinated chicken flocks in china, causing 100% morbidity and about 10% mortality in young infected chicks. in recent years, this type of ibv has also been found in taiwan [18] and regions of the middle east, resulting in severe losses to both the layer and broiler industries [19] . both experimental and field studies suggested that vaccination using available commercial vaccines provided poor protection against this type of virus, emphasizing the need for the development of an efficacious, live-attenuated vaccine against ck/ch/ldl/97i to prevent and control this type of ibv [20] . we previously attempted to develop an ib vaccine by serial passage of the ibv strain ck/ch/ldl/97i in embryonated eggs [21] . although the vaccination challenge test showed that the attenuated p115 virus could provide complete protection against the homologous pathogenic parent virus p5, in terms of clinical response and virus recovery in the trachea and kidney [20, 21] , the genomic changes in the pathogenic parental ck/ch/ldl/97i strain and its attenuated virus, and the genomic changes associated with attenuation of pathogenicity, immunogenicity, tissue tropism, and replication capacity after serial passages remain unknown. although it is possible to study the evolution of viruses and the subsequent impact on viral characteristics by comparing genomic sequences between heterologous strains, analysis of homologous strains provides a unique opportunity to elucidate the specific genes or gene sites involved in viral characteristics, such as pathogenicity, immunogenicity, and tissue tropism. to identify specific sequence changes responsible for adaptation of the field ibv isolate to chicken embryonic tissue and subsequent attenuation, the whole viral genome of the ibv p5 pathogenic parental ck/ch/ldl/97i strain was sequenced and compared with the attenuated, p115 level virus, to provide a better understanding of the relationship between the genomic differences and related characteristics of the pathogenic and attenuated viruses. fertile white leghorn specific pathogen-free (spf) chicken eggs were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, harbin, china. the pathogenic ibv strain ck/ch/ldl/97i (p5), previously characterized as belonging to the ck/ch/ldl/97i type [21] , and its embryo-passaged, attenuated virus (p115) [20] were used in this study. ck/ch/ldl/97i was isolated in 1997 from the swollen proventriculi of broiler chickens and propagated following standard procedures at 37° in the chorioallantoic cavities of 9-to 11-day-old spf embryonated eggs. this attenuated virus can provide complete protection against challenge of the parental ck/ch/ldl/97i strain according to clinical response and virus recovery in tracheas and kidneys [20, 21] . the virus stocks of ck/ch/ldl/97i p5 and p115 were produced by inoculating embryonated spf chicken eggs via the allantoic cavity with the virus, and by collecting the infectious allantoic fluid 72 h after inoculation, respectively, as described previously [21] . each virus was identified by electron microscopy, reverse transcriptase-polymerase chain reaction (rt-pcr), and sequencing of the entire s1 protein gene, as described previously [21] , before aliquoting and storage as virus stocks at -80°. the infectious virus titer was determined by inoculation of spf embryonated eggs with 10-fold serial dilutions and measured as embryo infectious doses of 50% (eid 50 ) following the reed and muench method [22] . viral rna was extracted from 200 μl of ck/ch/ldl/97i p5 and p115 virus stocks, respectively, using trizol reagent (invitrogen, grand island, n.y., usa) according to the manufacturer's instructions. rna was air-dried for 2-10 min, re-dissolved in 30 μl of rnase-free water, and stored at -70° for further use. the initial results (data not shown) revealed that primers n (-) contained sequences consistent with those of ck/ch/ldl/97i, and reverse transcription (rt) was therefore performed with m-mlv reverse transcriptase (invitrogen) using the primer n (-). rt procedures were performed using 40 μl of rna in an 80-μl reaction volume, as previously described [16] . each cdna fragment was pcr-amplified from the rt products, as described previously [16] . pcr products were purified from agarose gels using a dna extraction kit (boehringer mannheim gmbh, mannheim, germany) and cloned into the pmd18-t vector (takara bio, inc., dalian, china), following the manufacturer's instructions. fourteen pairs of overlapping primers were used for pcr amplification. the sequences and locations of the primers used in this study are listed in online supplementary table 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000365193). rna extraction, cdna generation, pcr amplification, and genefragment cloning and sequencing were conducted independently for each of the two passages [16] . the 3 ′ and 5 ′ ends of the viral genomes were confirmed by rapid amplification of cdna ends (race) using a 3 ′ /5 ′ race kit (takara bio, inc.), according to the manufacturer's instructions. pcr products were purified from agarose gels using a dna extraction kit (boehringer mannheim gmbh) and cloned into the pmd18-t vector (takara bio, inc.), following the manufacturer's instructions. a total of five clones of each gene fragment were selected and sequenced for each of the passages to exclude errors due to rt and pcr reactions. sequences were assembled using the megalign program (dnastar, madison, wis., usa) and edited manually to produce final sequences of the viral genomes. in addition, another 15 overlapping pairs of primers covering the full-length genome of ibv were designed (online suppl. ta-ble 1) based on the conserved genomic nucleotide consensus sequences of most published ibvs, and were used to facilitate the sequence validation of ck/ch/ldl/97i p5 and p115 by conventional one-step rt-pcr using the one step primescript tm rt-pcr kit (takara bio, inc.), according to the manufacturer's instructions. the viral stocks used in each rt-pcr assay were from independently inoculated embryos. pcr products were also purified from agarose gels using a dna extraction kit (boehringer mannheim gmbh) and cloned into the pmd18-t vector (takara bio, inc.), following the manufacturer's instructions. five clones of each gene fragment were selected and sequenced for p5 and p115. the nucleotide and amino acid sequences of the entire ibv ck/ ch/ldl/97i p5 and p115 genomes were assembled, aligned, and compared with those of other reference ibv strains using the megalign program. orfs were determined using the gene runner program version 3.00 (http://www.generunner.com) and the sequences were analyzed using lasergene dnastar version 7 (lasergene corp., madison, wis., usa). a total of 55 ibv reference strains for which the entire genomic sequences were available in the genbank database (www.ncbi.nlm.nih.gov/genbank) were selected for phylogenetic analysis using the neighboring-joining method in mega4 software (www.megasoftware.net) at a bootstrap value of 1,000 replicates. the selected avian cov reference strains and their accession numbers are shown in online supplementary table 2. in addition, accurate estimation and comparison of the nucleotide sequences of the entire genomes of ibv ck/ch/ldl/97i p5 and p115 were conducted using the clustalw algorithm (www. clustal.org). all sequences were edited manually and adjusted for errors. mutations and insertions were determined according to the results of pairwise comparisons. the genomic sequences of ibv strains ck/ch/ldl/97i p5 and p115 were submitted to the genbank database and assigned the accession numbers jx195177 (ck/ch/ldl/97i p5) and jx195178 (p115). to investigate substitutions in the nucleotide and/or amino acid sequences during passage, p5 and p115 were subjected to entire genomic amplification and sequence analysis by rt-pcr. all rt-pcr products were analyzed and a single band of the expected size was visualized after ethidium bromide staining of the products on a 1.0% agarose gel. five independent clones of each fragment from two independent rt-pcrs (in total, the entire genomes of each passage were sequenced 10 times) were selected and sequenced, thus the sequence profiles represented all (7) (7) 828 c→c (7) (7) the dominant sequences of both ck/ch/ldl/97i p5 and p115 were assembled into one contiguous sequence to represent the entire viral genome, irrespective of the subpopulations. sequences of 27,680 and 27,682 nucleotides were obtained from ck/ch/ldl/97i p5 and p115, respectively, excluding the polyadenylation tail at the 3 ′ end. p115 had two nucleotide insertions in the intergenic utr between m and gene 5, compared to p5 ( table 1 ; fig. 1 a, b) . the genomes of both viruses were similar in terms of overall coding capacity and genomic organization to those of other ibvs. the genome organization of ck/ch/ldl/97i was as follows: 5 ′ -utr-gene 1 (orf1a, 1b)-s-gene 3 (orf3a, 3b, e)-m-gene 5 (orf5a, 5b)-n-utr-3 ′ . phylogenetic reconstruction of the whole genomes of 57 ibvs using maximum likelihood and maximum parsimony produced well-supported trees ( fig. 2 ) that placed ck/ch/ldl/97i p5/p115 with the delaware-type and other ibv types isolated in the usa, which demonstrated a close genetic relationship between ck/ch/ldl/97i and the us strains. however, other chinese ibv strains, the massachusetts type, and west africa ibv isolates were classified in separate groups. subpopulations were identified in both ck/ch/ ldl/97i p5 and p115, evidenced by the variations in genomic sequences of the viruses ( table 1 ). as summarized in table 1 , no sequence substitutions were observed in nsp5, nsp6, nsp7, nsp8, nsp10, orf3b, or orf5a after 115 passages. substitutions were observed in nsp4, nsp9, nsp11/12, nsp14, nsp15, nsp16, and orf3a, but these did not result in amino acid substitutions and/or variations. amino acid substitutions occurred in the remaining genes between p5 and p115. two and three nucleotide substitutions were observed, respectively, in the 5 ′ -and 3 ′ -utrs of ck/ch/ldl/97i p115, compared to the parental strain p5. most nucleotide mutations were found in the s region between p5 and p115, and some of the nucleotide mutations resulted in amino acid substitutions. more mutations occurred in the s2 gene than in the 326 s1 gene. among the 9 nsps in the orf1 region, which showed nucleotide alterations between the pathogenic p5 and attenuated p115 viruses, nsp3 had the most. in this study, ck/ch/ldl/97i was unexpectedly grouped together with the us ibv strains, but was distinct from all other ibv strains reported so far in china. ck/ch/ldl/97i emerged in the mid-1990s as a novel ibv type in china [21, 23] and was associated with ib outbreaks on the liaotung peninsula between 1997 and 2001. this isolate seems to have high affinity for the proventriculus in chickens and is implicated in a novel pathogenicity associated with transmissible proventriculitis [23] , though few experimental proventriculitis models have been generated using this type of ibvs. the origin of this virus type remains unknown and no complete genomic sequence for it is available in the genbank database. the lack of sufficient information limits the comprehensive tracing of the origin and evolution of the ck/ ch/ldl/97i strain, but it is noteworthy that this type of ibv was also found in taiwan [18] and regions of the middle east in recent years [19] . we identified a close genetic relationship between ck/ch/ldl/97i and the delaware-type ibvs, which was isolated in the us, by entire genome analysis, suggesting a common origin or epidemiological link between the ck/ch/ldl/97i and delaware-type ibv strains. it has been suggested that minor differences near the receptor-binding domain of the s1 subunit of the spike protein could modify the affinity and virulence of covs for different cell surface glycoproteins [24] [25] [26] . consequently, the s protein has been shown to be correlated to the virulence attenuation and broadening of host or celltype ranges [27, 28] . this protein has also been reported as a determinant of cell tropism in ibv, as some amino acids contained in s protein contribute to attenuation of ibv [29] . variations in the s1 gene may reduce the ability of ck/ch/ldl/97i p115 to infect host cells after serial passage in eggs, but not its ability to infect the chorioallantoic membrane, where ibv replication initially occurs in embryonated chicken eggs. however, such conclusions should be drawn with caution, as the results of this and other studies found no identical substitutions, insertions, or deletions in s proteins between different pathogenic ibv strains and their embryo-passaged, attenuated derivatives, even though the attenuated ibv strains showed distinctly different biological features, in-cluding lack of pathogenicity to 1-day-old spf chicks compared to the pathogenic parental strains [26, 30, 31] . some findings suggested that the replicase gene, rather than the s1 or s gene, may be responsible for viral pathogenicity [32, 33] . the s2 subunit of the spike protein of covs associates non-covalently with the s1 subunit and contains the transmembrane and c-terminal cytoplasmic tail domains. the ectodomain region [33] and two heptad repeat regions [34] of subunit s2 are involved in oligomerization of the s protein and required for entry into susceptible cells [35] [36] [37] . changes in the s2 gene may affect viral entry [32] . in this study, multiple changes in the s2 gene occurred during passage of ck/ch/ldl/97i in embryonated eggs, which may have had an effect on viral entry into chicken host cells in vivo, indirectly decreasing the replication capacity. however, introduction of an s protein from a virulent ibv isolate did not confer virulence to the apathogenic strain beau-r [29] . a further study showed that replacing all the beaudette structural and accessory proteins with those from a virulent ibv isolate did not restore virulence [45] . these results suggest that ibv attenuation may not be caused by changes in the s2 gene. however, it has been speculated that changes in the s2 protein may contribute to adaptation of the field virus to chick embryonic tissue [32] . as in other covs, the ibv e protein is a small structural protein containing a single hydrophobic domain with multiple functions [38] [39] [40] [41] [42] . although information on the ibv e protein is limited [41, 43] , those from different covs perform similar functions during viral infection [44] . in the current study, we identified a mutation of nucleotide 184 (c-t) in p115, compared to p5, resulting in an amino acid substitution (ala → ser). however, this substitution may have only a minor effect on viral replication after 115 passages in eggs because only the hydrophobic domain (amino acids 12-32) of ibv e is important for efficient viral replication [41] . the cytoplasmic tail of ibv e is responsible for its interaction with ibv m [4] , and mutations in the cytoplasmic tail of mouse hepatitis virus (mhv) e by targeted rna recombination resulted in the production of elongated virions [45] . therefore, substitutions in the cytoplasmic tail of the ibv p115 e protein caused by serial passages warrant further investigations. no previous studies to date have investigated the function of the ibv intergenic utr between m and gene 5, though some sequence diversities and mutations have been found in this region among ibv strains [32, 46] . this region contains a potential orf with an initiation codon immediately downstream of the m gene stop codon in some ibv strains. this study identified a similar 273-nucleotide orf that potentially encodes a 90-amino acid product in ck/ch/ldl/97i p5 at a similar position; however, this orf was extended in p115 by two nucleotide insertions, resulting in an orf of 285 nucleotides that potentially encodes a 11-kd, 94-amino acid product. we could not predict the functions of the orf in ck/ ch/ldl/97i p5 or the potential effect of the insertions. the lack of subgenomic mrna for this potential orf, the long distance between the initiation codon and potential transcription regulatory sequence, and the loss of the potential orf in some ibv strains indicate that this orf is probably a pseudogene [32] . furthermore, removal of the intergenic utr from an apathogenic ibv had no effect on the functionality of the virus [32] . this study detected a substitution (tyr → the) in the n protein due to the mutation at nucleotide position 398, between the p5 and p115 viruses. the n protein binds to the 3 ′ end of the utr, which is essential for the synthesis of negative-strand viral rna. the change in the n gene suggests that it may have an impact on viral replication and thus on viral pathogenesis, although the sequencespecific interaction between the n gene and 3 ′ -utr remains unclear. the amino acid substitution at residue 188 (thr → ile/ala) in the m protein, which was related to antigenicity and/or virulence in ibv strains h52/h120, tw2296/95, and arkansas [30] , was not found in our ck/ch/ldl/97i strain; however, a nucleotide substitution between p5 and p115 was detected that resulted in an amino acid substitution at residue 223 (gly → ser). this substitution may affect the interaction between m and the cytoplasmic tail of ibv e [4] , and thereby viral budding. the ibv replicase gene encodes 15 nsps, some with known enzymatic functions [47] . however, the functions of these proteins in the context of pathogenesis remain poorly understood, although some nsps of other covs have been linked to the loss of virulence [32] . like its homologs in other covs, 3clpro of ibv is encoded by orf1a and resides in nsp5. this proteinase specifically cleaves polyproteins 1a and 1ab at 11 sites to produce 12 mature products (nsp5-nsp16). a cluster of small nsps, including nsp6, nsp7, nsp8, nsp9, and nsp10, is located at the c-terminal region of the 1a polyprotein. although the functional roles of these proteins in ibv are unknown, roles for these nsps in rna replication and transcription of other covs are emerging from biochemical and structural analyses [48] . in the current study, we found no differences in nucleotide sequences between ck/ch/ ldl/97i p5 and p115 in nsp5, nsp6, nsp7, nsp8, or nsp10. other groups have attenuated the virulent ark dpi 11 strain of ibv following 101 passages in embryonated eggs (ark dpi 101) and compared the complete genomes of both viruses. they identified two and one amino acid substitutions in nsp6 and nsp10, respectively, but none in nsp5, nsp7, or nsp8 [47] , as in the present study. in addition, although substitutions were found in nsp4, nsp9, nsp11/12, nsp14, nsp15, and nsp16 in our study, these substitutions did not produce amino acid substitutions due to viral subpopulation selection, other than several mutations. in contrast, apart from alterations due to viral subpopulations, three, two, and one amino acid substitutions were found in nsp1/2, nsp3, and nsp13 proteins, respectively, between p5 and p115. of the three amino acid substitutions, one similar substitution was also reported in this coding region between challenge and vaccine m41 strain, and ark dpi 11 and attenuated dpi 101 [47] . a recent report showed that nsp2 plays a role in facilitating de novo ibv protein synthesis by blocking protein kinase r phosphorylation of eukaryotic initiation factor 2 (eif-2α), which shuts down protein synthesis. in addition, nsp2 induces expression of gadd34, which dephosphorylates eif-2α [49] . the nsp3 of cov contains multiple domains including an acidic domain, an adpribose-1 ′ ′ -phosphatase, a papain-like proteinase (pl2 pro ), a y domain, and a transmembrane domain [50] . inactivation of adp-ribose-1 ′ ′ -phosphatase activity in mhv nsp3 reduced viral replication in the livers of infected mice, but did not induce liver disease [51] . two amino acid substitutions in this study were located in the viral proteinase pl2 pro and y domains, respectively, which were similar to the positions altered between ark dpi 11 and 101 [47] . the substitution in pl2 pro may restrict viral maturation or replication. however, we were unable to predict the effects of the y-domain change because the role of this domain in ibv is currently unknown. nsp13 contains an rna helicase and has an important function in viral replication. an amino acid substitution between ck/ch/ldl/97i p5 and p115 was found in the helicase domain at nucleotide position 15859, resulting in an amino acid substitution (leu → pro). this substitution was similar to that in the helicase domain of ark pdi, located at nt 15763. this substitution in the attenuated p115 strain might alter viral replication [47] . the ibv replicase gene is not usually associated with antigenicity, immunogenicity, or tissue tropism. the amino acid substitutions in the orf1a/1b proteins offer insight into putative residues that may be involved in the adaptation of chick embryonic tissue and subsequent at tenuation of the virus. it is possible that very few nucleotide substitutions within the replicase gene may cause attenuation following serial passage in embryonated eggs [32, 47] , although the role of the replicase gene in pathogenicity is not well understand. the 5 ′ -and 3 ′ -utrs in the genome of covs, like most rna viruses, usually harbor important structural elements involved in replication and/or translation [7] [8] [9] [10] [11] . in the present study, we found two and three variations in the 5 ′ -and 3 ′ -utrs of the attenuated ck/ch/ldl/97i p115 virus, respectively, compared to the pathogenic p5 parental strain. these nucleotide variations may play a role in pathogenicity by affecting the secondary structures of cis -acting elements involved in viral replication [52] . selection of a more fit virus subpopulation and mutations in the s1 gene have reportedly been associated with the replication of modified live ibv vaccine viruses in chickens [53] . s1 gene sequence analysis demonstrated that distinct virus subpopulations were selected when chickens were vaccinated with live-attenuated ibv strains [21] . further analysis confirmed that the virus subpopulations differed between microenvironments in distinct host tissues [54] . this investigation did not compare the replication capacities of ck/ch/ldl/97i before and after serial embryo passage in the oviduct of female chickens, or assess damage to the reproductive tract, egg production, or egg quality in laying chickens. however, ibv replication in the epithelium of the oviduct has been shown to result in decreased egg production and quality [55] . the damage is more severe and permanent if young chicks are affected [56] , although repeated embryo passage was shown to reduce the virulence of some ibvs in the chicken oviduct [57] . limited information is currently available regarding the pathogenicity of ck/ch/ ldl/97i-type ibv strains and embryo-passaged, attenuated p115 in the chicken oviduct. future studies will therefore focus on the effects of these ibv strains on the chicken oviduct. compared with the large genetic differences between cell-adapted covs and their parental viruses [58, 59] , few genetic changes were found between the embryo-passaged, attenuated covs and their pathogenic parental viruses [15, 16, 47] . the result is still very interesting to understanding of molecular markers of ibv attenuation, tissue tropism, and replication capacity changes, as in the case of the transmissible gastroenteritis virus, which showed that two amino acid substitutions at the n-terminus of spike protein resulted in loss of enteric tropism [60] . the current sequence analyses were unable to identify specific amino acid residues responsible for these changes because the attenuated strain differed from the parental strain at multiple positions. further investigations using reverse genetics and animal studies are therefore necessary to verify the exact functions of these changes. in addition, none of the sequence substitutions were shared by all pathogenic ibv strains or their attenuated derivatives [26, 30, 31, 47] , indicating changes in viral replication, pathogenicity, antigenicity, immunogenicity, or tissue tropism might be attributable to different genes in the viral genome. however, some of the sequence substitutions identified in this study could be used as the potential molecular markers, at least between the ck/ch/ ldl/97i-type pathogenic and attenuated strains, providing important information for the development of specific diagnostics of differentiating ck/ch/ldl/97i-type ibv infections from vaccination because the attenuated p115 can be used as a potential vaccine candidate [20] to control the circulation of ck/ch/ldl/97i-type ibvs in china and regions of the middle east in recent years [18, 19, 21, 23] . eu418975 arkdpi101-arkansas type(vaccine) eu418976 arkdpi11-arkansas type gq504720 arkansas dpi-arkansas type gq504721 arkansas vaccine-arkansas type(vaccine) ay514485 california 99-california type fj904715 cal557 2003-california type fj904714 cal 1995-california type gu393331 cal56b-california type(vaccine) vaccine-ga98 type(vaccine) fj904713 mass41 2006-mass type(united states) gu393333 fl18288-fl18288 type jf330899 ck/ch/lnm/091017-mass type jf828981 ck/ch/ldl/101212-mass type fj888351 h120-mass type(vaccine) eu714028 zj971-mass type gq504725 mass41 vaccine-mass type(vaccine) eu817497 h52-mass type fj904722 mass41 1979-mass type(united states) nc 001451 beaudette-mass type peafowl/gd/kq6/2003-mass type(china) fj904721 mass41 1972-mass type jf274479 ck/ch/lhlj/07vii-mass type(china) jf828980 ck/ch/lhlj/100902-mass type(china) ay851295 mass 41-mass type dq834384 m41-mass type gq504724 massachusetts-mass type gu393336 holte-holte type gu393337 iowa 97-iowa 97 type dq646405 tw2575/98-taiwan group i(taiwan, china) jf330898 ck/ch/lhb/100801-taiwan group ii ay338732 lx4-lx4 type(china) hm245924 cq04-lx4 type(china) hq848267 gx yl-5-lx4 type(china) hq850618 gx yl-9-lx4 type jx195176 ck/ch/lzj/111113-recombinant strain(china) hm245923 dy07-lx4 type lx4 type(china) the sequence of the genome of the coronavirus avian infectious bronchitis virus structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis genomic characteristics and changes of avian ibv strain ck/ch/ldl/97i nidovirus transcription: how to make sense da: a cis-acting function for the coronavirus leader in defective interfering rna replication stem-loop iii in the 5 ′ untranslated region is a cis-acting element in bovine coronavirus defective interfering rna replication stem-loop iv in the 5 ′ untranslated region is a cis-acting element in bovine coronavirus defective interfering rna replication a hypervariable region within the 3 ′ cis-acting element of the murine coronavirus genome is nonessential for rna synthesis but 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infectious bronchitis virus strain ck/ch/ldl/97i in the middle east evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/97i strain of infectious bronchitis coronavirus s1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives a simple method of estimating fifty percent endpoints molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity variations in the spike protein of the 793/b type of infectious bronchitis virus in the field and during alternate passage in chickens and embryonated eggs the murine coronavirus mouse hepatitis virus strain a59 from persistently infected murine cells exhibits an extended host range persistent infection promotes crossspecies transmissibility of mouse hepatitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism sequence changes of infectious bronchitis virus isolates in the 3 ′ -7.3 kb of the genome after attenuating passage in embryonated eggs characterization of a novel live attenuated infectious bronchitis virus vaccine candidate derived from a korean nephropathogenic strain the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein evidence for a coiled-coil structure in the spike proteins of coronaviruses structural characterization of the sars-coronavirus spike s fusion protein core identification of a new region of sars-cov s protein critical for viral entry role of spike protein endodomains in regulating coronavirus entry a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo the small envelope protein e is not essential for murine coronavirus replication enjuanes l: absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway the hydrophobic domain of infectious bronchitis virus e protein alters the host secretory pathway and is important for release of infectious virus the transmembrane oligomers of coronavirus protein e the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction exceptional flexibility in the sequence requirements for coronavirus small envelope protein function analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly the present of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity -a preliminary study identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi nmr structure of the sars-cov nonstructural protein 7 in solution at ph 6.5 inhibition of protein kinase r activation and upregulation of gadd34 expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virus-infected cells the sars-coronavirus plnc domain of nsp3 as a replication/ transcription scaffolding protein mouse hepatitis virus liver pathology is dependent on adp-ribose-1 ′ ′ -phosphatase, a viral function conserved in the alpha-like supergroup evidence for a pseudoknot in the 3 ′ untranslated regions of the bovine coronavirus genome avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination host intraspatial selection of infectious bronchitis virus effects of infectious bronchitis on the reproductive tracts, egg production, and egg quality of laying chickens pathogenesis of oviduct lesions in immature chickens following exposure to infectious bronchitis virus at one day old pathogenesis of two embryo passage levels of avian infectious bronchitis virus for the oviduct of young chickens of various ages selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism key: cord-326319-3538jmqd authors: yuan, yuan; zhang, zhi-peng; he, yi-ning; fan, wen-sheng; dong, zhi-hua; zhang, li-hua; sun, xin-kuan; song, li-li; wei, tian-chao; mo, mei-lan; wei, ping title: protection against virulent infectious bronchitis virus challenge conferred by a recombinant baculovirus co-expressing s1 and n proteins date: 2018-06-27 journal: viruses doi: 10.3390/v10070347 sha: doc_id: 326319 cord_uid: 3538jmqd avian infectious bronchitis virus (ibv) is the causative agent of infectious bronchitis, which results in considerable economic losses. it is imperative to develop safe and efficient candidate vaccines to control ibv infection. in the current study, recombinant baculoviruses co-expressing the s1 and n proteins and mono-expressing s1 or n proteins of the gx-yl5 strain of ibv were constructed and prepared into subunit vaccines rhbm-s1-n, rhbm-s1 and rhbm-n. the levels of immune protection of these subunit vaccines were evaluated by inoculating specific pathogen-free (spf) chickens at 14 days of age, giving them a booster with the same dose 14 days later and challenging them with a virulent gx-yl5 strain of ibv 14 days post-booster (dpb). the commercial vaccine strain h120 was used as a control. the ibv-specific antibody levels, as well as the percentages of cd4+ and cd8+ t lymphocytes, were detected within 28 days post-vaccination (dpv). the morbidity, mortality and re-isolation of the virus from the tracheas and kidneys of challenged birds were evaluated at five days post-challenge (dpc). the results showed that the ibv-specific antibody levels and the percentages of cd4+ and cd8+ t lymphocytes were higher in the rhbm-s1-n vaccinated birds compared to birds vaccinated with the rhbm-s1 and rhbm-n vaccines. at 5 dpc, the mortality, morbidity and virus re-isolation rate of the birds vaccinated with the rhbm-s1-n vaccine were slightly higher than those vaccinated with the h120 control vaccine but were lower than those vaccinated with the rhbm-s1 and rhbm-n vaccines. the present study demonstrated that the protection of the recombinant baculovirus co-expressing s1 and n proteins was better than that of recombinant baculoviruses mono-expressing the s1 or n protein. thus, the recombinant baculovirus co-expressing s1 and n proteins could serve as a potential ibv vaccine and this demonstrates that the bivalent subunit vaccine including the s1 and n proteins might be a strategy for the development of an ibv subunit vaccine. avian infectious bronchitis (ib) is a highly contagious disease of chickens caused by the infectious bronchitis virus (ibv), belonging to the gammacoronavirus genus within the coronaviridae family [1] . ibv affects chickens of all ages and types and primarily infects the respiratory and urogenital systems of chickens, causing massive economic losses [2] [3] [4] . there are dozens of ibv serotypes related to the extensive variability of the spike (s) protein. there is little or no cross-protection between different serotypes of ibvs or circulating variant viruses, frequently leading to immune failures and making it extremely difficult to control the disease [5] [6] [7] . control of the disease currently relies on conventional live-attenuated and inactivated vaccines [8] [9] [10] . however, live-attenuated vaccines can result in the occurrence of vaccine-like viruses with increased virulence and persistence due to point mutation and recombination with other vaccines or field strains [11] [12] [13] . inactivated vaccines are not only high in cost but also do not establish long-term immunity. the application of inactivated vaccines alone has frequently failed to induce strong cellular immunity, resulting in little or no protection [14, 15] . despite the widespread application of these conventional vaccines, ib continues to cause severe economic losses in many countries, underscoring the need to develop new safer and more effective candidate vaccines for the practical control of ibv. among new vaccines, subunit vaccines have been showed to elicit strong humoral and cellular immune responses [16] [17] [18] . subunit vaccines have significant advantages, such as efficient antigenic presentation, high stability and flexibility in protein or epitope selection, compared with live-attenuated and inactivated vaccines [16] . additionally, no infectious viral particles are involved in vaccine production, so they are very safe. subunit vaccines are genetically engineered and are thus easy to mass produce [19] . subunit vaccines also allow the creation of multivalent vaccines. thus, subunit vaccines may have the potential to act as safe and effective vaccine candidates. ibv consists of four structural proteins. they are the spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins [20] . the s protein is post-translationally cleaved into s1 and s2 subunits [21] . the s1 protein determines the antigenicity and immunogenicity of the virus and plays an important role in the induction of neutralizing and hemagglutination inhibition antibodies, tissue affinity and cell adsorption, all involved in the humoral immune response [22] [23] [24] . the n protein plays an extremely important role in the replication and assembly of ibv, affecting its immunogenicity and is thus involved in the cellular immune response [25] [26] [27] . hence, the s1 and n proteins are the most promising subunit vaccine candidates against ibv. therefore, the development of subunit vaccines including both the s1 and n proteins has promising prospects. the baculovirus expression system (bes) is an efficient system for gene expression because of its good safety, high expression and excellent protein processing ability compared to other expression systems. a major advantage of the bes is the ease of scaling up from the laboratory to a large-scale production system [28, 29] . in addition, baculoviruses are insect pathogens and thus non-pathogenic for vertebrates. therefore, the bes is currently widely used for protein expression and vaccine production. there have been a few studies describing the expression of ibv proteins by bes [18, [30] [31] [32] [33] but none of these studies have focused on developing and evaluating the immune response of a subunit vaccine from the baculovirus which co-expresses s1 and n proteins of ibv. this study therefore set out to measure the protective effect of the subunit vaccine co-expressing both the s1 and n proteins of ibv. moreover, we compared the immune protection offered by this vaccine with those induced by recombinant subunit vaccines expressing s1 protein or n protein alone and h120 inactivated vaccine. the aim is to obtain safer and more effective subunit vaccines, thus providing a novel subunit vaccine candidate for ibv. sf9 insect cells were purchased from qiyin biological technology co., ltd. (jiangyin, china) and cultured in serum-free sf900ii medium (gibco, grand island, ny, usa) at 27 • c. the virulent gx-yl5 strain of ibv was isolated from broilers with nephritis disease and is the representative dominant serotype (different from the h120 strain) isolate in southern china and shared heterologous serotype with the h120 strain [34] . the 50% tracheal organ culture infection dose (toc-id 50 ) of the ibv gx-yl5 strain was determined as previously described [34] . the euokaryotic expression vector pfastbac tm dual was the product of invitrogen (waltham, ma, usa). in order to increase the expression and yield of heterologous secreted proteins in insect cells, the honeybee melittin (hbm) signal peptide recognized by insect cells was introduced into the bes. the coding mature s1 (55-1621 bp) (accession number fj907238.1) and n (1-1230 bp) (accession number j548847.1) protein genes of the ibv gx-yl5 strain were amplified by reverse transcription polymerase chain reaction (rt-pcr). the insect signal peptide hbm gene was amplified by pcr using a pair of primers with a partial overlapping sequence. then the fusion genes hbm-s1 and hbm-n were obtained by fusion pcr, cloned into the peasy-t1 vector and then sub cloned into the transfer vector pfastbac tm dual ( figure 1a ) at the bamh i/pst i sites as well as the xho i/kpn i sites under the control of ph and p10 promotors, respectively ( figure 1b ,c) to obtain recombinant transposon vectors pfast-hbm-s1 and pfast-hbm-n. a 6×his tag and a tobacco etch virus (tev) protease cleavage site were added before the stop codon to facilitate the identification and purification of the expressed proteins. the hbm-n gene was then directly sub cloned into pfast-hbm-s1 to yield the recombinant transposon vector pfast-hbm-s1-n. this involved the s1 gene being inserted under the control of promotor ph and the n gene being inserted under the control of promoter p10 ( figure 1d ). all the recombinant transposon vectors were verified by pcr, restriction endonuclease digestion and sequencing. the verified recombinant transposon vectors were then transformed into dh10bac tm escherichia coli cells to generate recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n, which were identified by pcr with m13 primers. the purified recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n were obtained after several instances of screening. they were then transfected into sf9 insect cells to obtain recombinant baculoviruses through lipofectin-mediated transfection, following the manufacturer's instructions (invitrogen). the recombinant baculoviruses rhbm-s1, rhbm-n and rhbm-s1-n were identified by pcr with ibv specific primers and m13 primers the recombinant baculovirus titers were determined using end-point dilution analysis. all the primers used in this study are shown in table s1 . in order to increase the expression and yield of heterologous secreted proteins in insect cells, the honeybee melittin (hbm) signal peptide recognized by insect cells was introduced into the bes. the coding mature s1 (55-1621 bp) (accession number fj907238.1) and n (1-1230 bp) (accession number j548847.1) protein genes of the ibv gx-yl5 strain were amplified by reverse transcription polymerase chain reaction (rt-pcr). the insect signal peptide hbm gene was amplified by pcr using a pair of primers with a partial overlapping sequence. then the fusion genes hbm-s1 and hbm-n were obtained by fusion pcr, cloned into the peasy-t1 vector and then sub cloned into the transfer vector pfastbac tm dual ( figure 1a ) at the bamh i/pst i sites as well as the xho i/kpn i sites under the control of ph and p10 promotors, respectively ( figure 1b ,c) to obtain recombinant transposon vectors pfast-hbm-s1 and pfast-hbm-n. a 6×his tag and a tobacco etch virus (tev) protease cleavage site were added before the stop codon to facilitate the identification and purification of the expressed proteins. the hbm-n gene was then directly sub cloned into pfast-hbm-s1 to yield the recombinant transposon vector pfast-hbm-s1-n. this involved the s1 gene being inserted under the control of promotor ph and the n gene being inserted under the control of promoter p10 ( figure 1d ). all the recombinant transposon vectors were verified by pcr, restriction endonuclease digestion and sequencing. the verified recombinant transposon vectors were then transformed into dh10bac tm escherichia coli cells to generate recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n, which were identified by pcr with m13 primers. the purified recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n were obtained after several instances of screening. they were then transfected into sf9 insect cells to obtain recombinant baculoviruses through lipofectin-mediated transfection, following the manufacturer's instructions (invitrogen). the recombinant baculoviruses rhbm-s1, rhbm-n and rhbm-s1-n were identified by pcr with ibv specific primers and m13 primers the recombinant baculovirus titers were determined using end-point dilution analysis. all the primers used in this study are shown in table s1 . the hbm-s1 gene was sub cloned into the vector pfastbac tm dual under the control of ph promotor; (c) the hbm-n gene was sub cloned into the vector pfastbac tm dual under the control of p10 promotor; (d) hbm-s1 and hbm-n genes were sub cloned into the vector pfastbactm dual under the control of ph and p10 promotors, respectively. the resulting recombinant transposon vectors were named pfast-hbm-s1, pfast-hbm-n and pfast-hbm-s1-n. the expression of recombinant proteins s1, n and s1-n in sf9 cells were detected using indirect immunofluorescence assay (ifa) and western blot. for ifa, sf9 cells were infected with the recombinant baculoviruses rhbm-s1, rhbm-n and rhbm-s1-n at a multiplicity of infection (moi) of 5, respectively and then fixed with 4% paraformaldehyde at 72 h post-infection. mouse anti-his igg (1:2000 dilution, cwbio, beijing, china) was used as the primary antibody, while fluorescein schematic diagram of s1, n and s1-n genes baculovirus expression systems. (a) the schematic diagram of the transfer vector pfastbac tm dual; (b) the hbm-s1 gene was sub cloned into the vector pfastbac tm dual under the control of ph promotor; (c) the hbm-n gene was sub cloned into the vector pfastbac tm dual under the control of p10 promotor; (d) hbm-s1 and hbm-n genes were sub cloned into the vector pfastbactm dual under the control of ph and p10 promotors, respectively. the resulting recombinant transposon vectors were named pfast-hbm-s1, pfast-hbm-n and pfast-hbm-s1-n. the expression of recombinant proteins s1, n and s1-n in sf9 cells were detected using indirect immunofluorescence assay (ifa) and western blot. for ifa, sf9 cells were infected with the recombinant baculoviruses rhbm-s1, rhbm-n and rhbm-s1-n at a multiplicity of infection (moi) of 5, respectively and then fixed with 4% paraformaldehyde at 72 h post-infection. mouse anti-his igg (1:2000 dilution, cwbio, beijing, china) was used as the primary antibody, while fluorescein isothiocyanate (fitc)-labeled goat anti-mouse antibody igg (1:2000 dilution, cwbio, beijing, china) was used as the secondary antibody. specific fluorescent signals in sf9 cells were observed using fluorescent microscopy. at 72 h post-infection, cell culture supernatants and cell lysates infected with the recombinant baculoviruses rhbm-s1, rhbm-n and rhbm-s1-n were collected for the analysis of recombinant proteins using western blot. protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), transferred onto polyvinylidene fluoride (pvdf) membranes and blocked in 5% skim milk with phosphate buffer solution tween-20 (pbst) buffer. mouse anti-his igg (1:2000 dilution, cwbio, beijing, china) was used as the primary antibody and horseradish peroxidase (hrp)-labeled goat anti-mouse antibody igg (1:2000 dilution, cwbio, beijing, china) was used as the secondary antibody. normal sf9 cells and sf9 cells infected with the same amount of wild-type baculovirus were set as negative controls. the proteins were visualized using a diaminobenzidine (dab) substrate kit as recommended by the supplier. the specific-pathogen-free (spf) chickens used in this study were hatched from fertilized white leghorn spf eggs (beijing merial vital laboratory animal technology co., ltd., beijing, china) in our facilities and the chickens were housed in separate isolation units until reaching 14 days of age. fourteen-day-old spf chickens were assigned randomly into six groups (n = 10 chickens/group). each bird in the rhbm-s1-n, rhbm-s1and rhbm-n groups was injected subcutaneously with 0.5 ml (50 µg) s1-n, s1 and n recombinant proteins, respectively. these proteins were emulsified in freund's complete adjuvant on day 14 and boosted with the same dose of proteins emulsified in freund's incomplete adjuvant on day 28. birds in the h120 group, set as a positive control, were injected with the h120 inactivated vaccine. the wt-b group and cell group, serving as negative controls, were set up by replacing the recombinant proteins with emulsified wild-type baculovirus and normal sf9 cell lysates, respectively. at 14 days post-booster (dpb), all the birds from each group were challenged with 10 5 toc-id 50 of the ibv gx-yl5 strain in 0.2 ml by the nasal-ocular route. they were monitored daily for clinical signs, morbidity and mortality and they were then euthanized at 5 days post-challenge (dpc). any birds that died during the observation period were immediately necropsied. any remaining birds were euthanized at the end of the observation period. the trachea and kidney samples were collected from each bird aseptically for the re-isolation of ibv. these animal experiments were approved by the animal care & welfare committee of guangxi university (approval number gxu2016-011, 25th november 2016) and were performed in accordance with animal ethics guidelines and approved protocols. blood samples were collected from the wing vein of 10 birds in each group prior to prime immunization (0 day) and at 7, 14, 21 and 28 days post-vaccination (dpv) for the detection of the ibv-specific igg antibody using commercial enzyme-linked immunosorbent assay (elisa) kits (idexx laboratory, inc., westbrook, me, usa) according to the manufacturer's recommendations. peripheral blood samples were collected from the wing vein of 10 birds in each group at 0, 7, 14, 21 and 28 dpv. peripheral blood lymphocytes were isolated and adjusted to 1 × 10 6 cells/ml, stained with 1 µl mouse anti-chicken cd4-pe and cd8a-fitc antibodies (wuhan amyjet scientific inc., wuhan, china) and analyzed for the percentages of cd4+ and cd8+ t lymphocytes by a bd accuritm c6 flow cytometer (becton, dickinson and company, minneapolis, mn, usa). all birds were euthanized at 5 dpc. trachea and kidney mixtures were collected for virus re-isolation through inoculation into 10-day-old spf chicken embryos via the allantoic cavity route. allantoic fluid was harvested at 72 h post-inoculation. three passages in embryonated eggs were conducted. viral rna in allantoic fluid was extracted using the easypure rna purification kit (transgen biotech, beijing, china) and subjected to a reverse transcriptase polymerase chain reaction targeting the m gene of ibv. the absence of detectable virus in the trachea and kidney was considered to be correlated with the protection of the subunit vaccines against the challenge. all data were shown as mean values ± standard deviation (mean ± sd) and were compared by a one-way analysis of variance (anova) and student's t-test using spss 18.0 (spss inc., chicago, il, usa) biostatistics software. p-values less than 0.05 were regarded as significant and those less than 0.01 were regarded as highly significant. the results of pcr, restriction enzyme digestion analysis ( figure s1 ) and sequencing showed that recombinant transposon vectors pfast-hbm-s1, pfast-hbm-n and pfast-hbm-s1-n were successfully constructed. the recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n were obtained by transforming the verified recombinant transposon vectors to dh10bac tm escherichia coli cells and identified using pcr. the results of pcr identification showed that the recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n were generated ( figure s2 ). the purified recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n were transfected into sf9 insect cells to obtain the recombinant baculovirus. the recombinant baculovirus rhbm-s1, rhbm-n and rhbm-s1-n were identified using pcr with ibv specific primers and m13 primers. the sizes of the amplified bands matched those of the predicted products ( figure s3 ). strong specific immunofluorescence signals were observed in the sf9 cells infected with the recombinant baculovirus rhbm-s1 (figure 2a) , rhbm-n ( figure 2b ) andrhbm-s1-n ( figure 2c ) at 72 h post-infection but no fluorescence was detected in the sf9 cells infected with wild-type baculovirus ( figure 2d ) and normal sf9 cells ( figure 2e ). the expected sizes of 93 ( figure 3a ) and 51 kda ( figure 3b ) proteins were detected in the culture supernatant and cell lysate infected with rhbm-s1 and rhbm-n, respectively. two specific bands of 93 and 51 kda ( figure 3c ) proteins were detected in both the culture supernatant and cell lysate infected with rhbm-s1-n. no bands were observed in the culture supernatant or cell lysate infected with wild-type baculovirus and normal sf9 cells. the results showed that the sizes of expressed recombinant proteins are consistent with the sizes of native s1 or n proteins and these recombinant proteins retained their antigenicity. at 14 dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s1-n, rhbm-s1, rhbm-n and inactivated vaccine strain h120 started to rise (p > 0.05). the antibody levels of the vaccinated groups increased notably at 21 and 28 dpv (i.e., 7 and 14 dpb) and were significantly higher than those of the negative control groups (p < 0.01) (figure 4) . the highest ibv antibodies in each vaccinated group were observed at 7 dpb. the antibody levels in the rhbm-s1-n group were always higher than those in the rhbm-s1 and rhbm-n groups but the difference was not significant (p > 0.05). similarly, the antibody titers in rhbm-s1 groups were always slightly higher than those in the rhbm-n group (p > 0.05). chickens in the h120 group developed the at 14 dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s1-n, rhbm-s1, rhbm-n and inactivated vaccine strain h120 started to rise (p > 0.05). the antibody levels of the vaccinated groups increased notably at 21 and 28 dpv (i.e., 7 and 14 dpb) and were significantly higher than those of the negative control groups (p < 0.01) (figure 4) . the highest ibv antibodies in each vaccinated group were observed at 7 dpb. the antibody levels in the rhbm-s1-n group were always higher than those in the rhbm-s1 and rhbm-n groups but the difference was not significant (p > 0.05). similarly, the antibody titers in rhbm-s1 groups were always slightly higher than those in the rhbm-n group (p > 0.05). chickens in the h120 group developed the at 14 dpv, ibv-specific antibody levels in birds immunized with subunit vaccines rhbm-s1-n, rhbm-s1, rhbm-n and inactivated vaccine strain h120 started to rise (p > 0.05). the antibody levels of the vaccinated groups increased notably at 21 and 28 dpv (i.e., 7 and 14 dpb) and were significantly higher than those of the negative control groups (p < 0.01) (figure 4) . the highest ibv antibodies in each vaccinated group were observed at 7 dpb. the antibody levels in the rhbm-s1-n group were always higher than those in the rhbm-s1 and rhbm-n groups but the difference was not significant (p > 0.05). similarly, the antibody titers in rhbm-s1 groups were always slightly higher than those in the rhbm-n group (p > 0.05). chickens in the h120 group developed the highest antibody titers throughout the experimental period but the difference was not significant (p > 0.05) compared with those in the rhbm-s1-n, rhbm-s1 and rhbm-n groups. viruses 2018, 10, x for peer review 7 of 14 highest antibody titers throughout the experimental period but the difference was not significant (p > 0.05) compared with those in the rhbm-s1-n, rhbm-s1 and rhbm-n groups. the results showed that the percentages of cd4+ and cd8+ t lymphocytes in vaccinated birds were rising at 7 dpv ( figure 5 ). at 21 and 28 dpv (i.e., 7 and 14 dpb), the percentages of cd4+ and cd8+ t lymphocytes in the rhbm-s1-n and rhbm-n groups were significantly higher than those of the negative groups (p < 0.01). the percentages of cd4+ t lymphocytes in the rhbm-s1 group were significantly higher than those of the negative controls (p < 0.05) and the percentages of cd8+ t lymphocytes were significantly higher (p < 0.01) compared with the negative controls. based on percentages of cd4+ and cd8+ t lymphocytes in the vaccinated groups at 21 and 28 dpv (i.e., 7 and 14 dpb), the groups sorted from highest to lowest as follows: the rhbm-s1-n group, the rhbm-n group, the h120 group and the rhbm-s1 group. there was a significant difference between the rhbm-s1-n and rhbm-s1 groups (p < 0.05), while no significant difference was noted between other groups (p > 0.05). the results showed that the percentages of cd4+ and cd8+ t lymphocytes in vaccinated birds were rising at 7 dpv ( figure 5 ). at 21 and 28 dpv (i.e., 7 and 14 dpb), the percentages of cd4+ and cd8+ t lymphocytes in the rhbm-s1-n and rhbm-n groups were significantly higher than those of the negative groups (p < 0.01). the percentages of cd4+ t lymphocytes in the rhbm-s1 group were significantly higher than those of the negative controls (p < 0.05) and the percentages of cd8+ t lymphocytes were significantly higher (p < 0.01) compared with the negative controls. based on percentages of cd4+ and cd8+ t lymphocytes in the vaccinated groups at 21 and 28 dpv (i.e., 7 and 14 dpb), the groups sorted from highest to lowest as follows: the rhbm-s1-n group, the rhbm-n group, the h120 group and the rhbm-s1 group. there was a significant difference between the rhbm-s1-n and rhbm-s1 groups (p < 0.05), while no significant difference was noted between other groups (p > 0.05). the data are shown as mean values ± standard deviation (mean ± sd) in each group. statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01) (n = 10 chickens/group). at 4 dpc, chickens began to show clinical signs or died. morbidity, mortality, virus re-isolation rate and protection rate at 5 dpc were summarized in table 1 . most of the chickens in the two negative control groups (wt-b and cell groups) showed typical signs of ibv infection, such as coughing, sneezing, gasping, tracheal rale and wet droppings. in these groups, there was a 60-70% morbidity rate and a 30-40% mortality rate. in contrast, 20%, 30%, 40% and 10% morbidity rates as well as 0%, 0%, 20% and 0% mortality rates were observed in the rhbm-s1-n, rhbm-s1, rhbm-n and h120 groups, respectively. birds in the four vaccinated groups exhibited milder clinical signs and gross lesions compared with those in the two negative control groups. in the necropsy at 5 dpc, the kidneys of infected birds were swollen and pale and they exhibited a white sludge indicating urate deposition. some birds showed trachitis with exudates. in order to further evaluate the level of protection, the collected trachea and kidney from the challenged birds at 5 dpc were inoculated into 10-day-old spf chicken embryos for three passages for virus re-isolation. viral rna was detected from the infected allantoic fluid of the embryonated eggs using qrt-pcr. the virus re-isolation rates for the birds in the rhbm-s1-n, rhbm-s1, rhbm-n and h120 groups were 40%, 50%, 60% and 20% respectively. those in the wt-b and cell groups had virus re-isolation rates of 80% and 100% respectively (table 1) . birds in the rhbm-s1-n, rhbm-s1 and rhbm-n groups developed higher levels of protection (i.e., 60%, 50% and 40%, respectively) than those in the wt-b and cell groups (i.e., 20% and 0%, respectively) but lower levels of protection than that in the h120 group (80%). no protection was observed in the cell group and a low level of protection (20%) was observed in the wt-b group. the data are shown as mean values ± standard deviation (mean ± sd) in each group. statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01) (n = 10 chickens/group). at 4 dpc, chickens began to show clinical signs or died. morbidity, mortality, virus re-isolation rate and protection rate at 5 dpc were summarized in table 1 . most of the chickens in the two negative control groups (wt-b and cell groups) showed typical signs of ibv infection, such as coughing, sneezing, gasping, tracheal rale and wet droppings. in these groups, there was a 60-70% morbidity rate and a 30-40% mortality rate. in contrast, 20%, 30%, 40% and 10% morbidity rates as well as 0%, 0%, 20% and 0% mortality rates were observed in the rhbm-s1-n, rhbm-s1, rhbm-n and h120 groups, respectively. birds in the four vaccinated groups exhibited milder clinical signs and gross lesions compared with those in the two negative control groups. in the necropsy at 5 dpc, the kidneys of infected birds were swollen and pale and they exhibited a white sludge indicating urate deposition. some birds showed trachitis with exudates. in order to further evaluate the level of protection, the collected trachea and kidney from the challenged birds at 5 dpc were inoculated into 10-day-old spf chicken embryos for three passages for virus re-isolation. viral rna was detected from the infected allantoic fluid of the embryonated eggs using rt-pcr. the virus re-isolation rates for the birds in the rhbm-s1-n, rhbm-s1, rhbm-n and h120 groups were 40%, 50%, 60% and 20% respectively. those in the wt-b and cell groups had virus re-isolation rates of 80% and 100% respectively (table 1) . birds in the rhbm-s1-n, rhbm-s1 and rhbm-n groups developed higher levels of protection (i.e., 60%, 50% and 40%, respectively) than those in the wt-b and cell groups (i.e., 20% and 0%, respectively) but lower levels of protection than that in the h120 group (80%). no protection was observed in the cell group and a low level of protection (20%) was observed in the wt-b group. rhbm-s1-n 20 (2/10) 0 (0/10) 40 (4/10) 60 (6/10) rhbm-s1 30 ( ib is a highly contagious and acute viral disease of chickens which causes massive economic losses. live-attenuated and inactivated vaccines are commonly used to control this disease. however, live-attenuated vaccines may lead to the occurrence of new serotypes or variants of ibv due to mutation and recombination [11] [12] [13] . also, inactivated vaccines often fail to induce strong cellular immunity and have the disadvantage of high manufacturing costs [14, 15] . hence, there is an urgent need to develop new, safe and effective vaccines to control the disease. recombinant subunit vaccines have been shown to elicit strong humoral and cellular immune responses and are very safe [16] [17] [18] . therefore, subunit vaccines may serve as potential vaccine candidates in the future. in the current study, significantly higher antibody levels, higher percentages of cd4+ and cd8+ t lymphocytes and higher protection rates were demonstrated when using the subunit vaccines rhbm-s1-n, rhbm-s1 and rhbm-n compared to the wt-b and cells at 7 and 14 dpb. therefore, the recombinant subunit vaccines rhbm-s1-n, rhbm-s1 and rhbm-n could induce the immunized chickens to produce humoral and cellular immunity to resist and eliminate the ibv infection, indicating that the recombinant bacculovirus vaccines expressing the s1 and n proteins of ibv may be effective subunit vaccines for future use. the immune efficacy of subunit vaccine rhbm-s1 containing the s1 protein was stronger than that of the subunit vaccine rhbm-n containing the n protein in the generation of humoral immune responses. however, the immune efficacy of subunit vaccine rhbm-n was stronger than that of rhbm-s1 in the generation of cellular immune responses. the results confirmed that the s1 protein and n protein play a major role in humoral and cellular immune responses, respectively, which is in agreement with the previous investigations [23, 26, 35] . the immune efficacy of subunit vaccine rhbm-s1-n was better than that of subunit vaccines rhbm-s1 or rhbm-n in generating humoral and cellular immune responses. the reason for this was that rhbm-s1-n expressed both the s1 and n proteins of ibv and stimulated both the humoral and cellular immune responses simultaneously. therefore, the subunit vaccine rhbm-s1-n performed better in the activation of virus-specific immune responses. although the s1 subunit is the major inducer of neutralizing antibodies, vaccination with the s1 protein did not confer adequate protection against challenge [17] . a previous study even found that the antigenicity of the n protein is better than that of the s1 protein [36] . in another study, the cell-mediated immune response induced by the n protein was higher than that induced by either the s1 protein or h120 [37] . these results and ours indicate the importance of generating a subunit vaccine that contains both the s1 and the n genes. a previous study showed that the bacmam virus ac-cmv-s1, which expresses the s1 glycoprotein of ibv-m41, was deficient in the induction of ibv-specific antibody compared with that induced by the inactivated vaccine [18] . in our study, the antibody levels in the groups rhbm-s1-n, rhbm-s1 and rhbm-n were slightly lower than that of the h120 group at 7 and 14 dpb, which agreed with the previous study [18] . a possible reason for this was that the h120 vaccine was derived from the whole virus and was comprised of almost all the epitopes of ibv. another reason may be related to the fact that the coated antigen of the elisa kit used in the study was the mass-type virus strain. based on the percentages of cd4+ and cd8+ t lymphocytes, the groups ordered from highest to lowest are the rhbm-s1-n group, rhbm-n group, h120 group and rhbm-s1 group. it was surprising that the cellular immune response in the rhbm-n group was higher than that in the h120 group, although the difference was not found to be statistically significant. a previous study showed that ac-cmv-s1 induced a significantly better cellular immune response in spf chickens compared with that induced by the inactivated vaccine [18] , which was consistent with our results. the cell-mediated immune response induced by rhbm-n was higher than that induced by either h120 or rhbm-s1. one possible explanation was a more efficient presentation of n peptides by antigen-presenting cells following effective uptake, processing, or presentation on mhc receptors of these cells [37] . therefore, the necessity of co-expressing the s1 and n proteins is again stressed. in the present study, chickens in the h120 group did not obtain 100% protection against the ibv gx-yl5 strain challenge. this was possibly because the strain used as the h120 vaccine was heterologous to the gx-yl5 strain which was used as the challenge strain [34] . this was consistent with previous studies [38] [39] [40] . another reason could be the absence of live priming for the inactivated h120 vaccine [14, 41] . in addition, the protection rates of subunit vaccines rhbm-s1-n (60%), rhbm-s1 (50%) and rhbm-n (40%) were lower than that of the traditional vaccine h120 (80%). a previous description reported 55% protection against the virulent m41 challenge from the constructed baculovirus expressing the s1 protein of the ibv m41 strain, which was lower than that for the inactivated vaccine (72%) [42] . only 50% protection was obtained against the homologous km91 strain challenge after three immunizations with the ibv s1 glycoprotein expressed by a recombinant baculovirus [32] . a similar result was shown in another study [43] . it was surprising that the protection rate of the rhbm-s1-n vaccine (60%) against the homologous gx-yl5 strain challenge was lower than that of the h120 vaccine (80%) against the heterologous gx-yl5 strain challenge. there are some possible reasons for this. firstly, the rhbm-s1-n subunit vaccines contained only part of the whole immunogenicity of the virus. this could not lead to an immune effect that was as good as the whole-virus vaccine h120. secondly, the cell lysates were used to prepare the oil emulsion vaccines for the chickens' immunizations and it is unknown whether the complicated composition of the cell lysate had an effect on the immunized birds. thirdly, the immune dose of 50 µg/bird may be not enough. doses of 150 µg/bird were used to immunize chickens in other studies [37, 44] . finally, two immunizations may be not enough to elicit an effective immune response. a previous study pointed out that at least four immunizations with purified s1 glycoprotein were needed to induce protection against the homologous n1/62 strain challenge [45] . similarly, three immunizations were performed and only 50% protection was obtained against the homologous ibv in the previous study [32] . in this study, we only immunized the chickens twice with the recombinant proteins; this may be a reason for the low protection conferred by the subunit vaccines. live attenuated vaccines are applied from day-old to achieve early protection and are boosted with the inactivated vaccines in the case of future layers and breeders. although the subunit vaccine rhbm-s1-n could not provide complete protection against ibv infection, it conferred higher protection than rhbm-s1 and rhbm-n vaccines. thus, it still can be used as an alternative vaccine for boosting the primary vaccination with traditional vaccine/vaccines against this disease. as we know, ibv continuously evolves and there are scores of serotypes. little or no cross-protection confers between different serotypes of ibvs [6, 7] . therefore, universal ibv vaccines that protect against varying serotypes of ibv are imperative. our results should enable the generation of multivalent vaccines to prevent more serotypes of ibv, especially the newly emerging virus strains. the vaccine rhbm-s1-n can also be applied by combining it with traditional vaccines to reduce the occurrence of variants caused by virus mutations and recombination. therefore, the subunit vaccine rhbm-s1-n is an alternative to the traditional ibv vaccine. our study also provides reference and ideas for the development of subsequent new vaccines. further study is needed to determine the effectiveness of combining the subunit vaccines with traditional vaccines. in this study, a 20% protection rate was conferred following ibv challenge in the wild-type baculoviruses control group and the protection rate of the wild-type baculoviruses control group was slightly higher than that of the cell control group, which was consistent with previous reports [18, 41] . a possible reason could be that the baculovirus induced innate immunity through the toll-like receptor 9 and myd88-dependent signaling pathway and stimulates the production of various inflammatory cytokines [46] . therefore, vaccination with baculovirus alone may result in non-specific immunity, which could provide only a small amount of protection against an ibv challenge. it is critical to obtain high-level secreted expressions of recombinant proteins with native activities. hbm is an insect-derived signal peptide. it has been reported that the introduction of the hbm signal peptide could increase the expression of foreign proteins, enhance the activity of expressed proteins and achieve the secretion of foreign proteins in the baculovirus system [47] [48] [49] . there have been a few studies about expressing ibv proteins using bes [18, [30] [31] [32] [33] but none of these studies have focused on introduction of the hbm signal peptide into bes to express ibv proteins. therefore, the hbm signal peptide was introduced in the present study. the results showed that the recombinant proteins s1-n, s1 and n could be expressed both in the culture supernatant and cell lysate. thus, the introduction of the hbm signal peptide into bes can be applied to express other proteins from other pathogens. in summary, the current study showed that the constructed recombinant baculoviruses could elicit both cellular and humoral immune responses to a certain degree and the protection offered by the recombinant baculovirus co-expressing s1 and n proteins was better than that of recombinant baculoviruses containing the s1 or n protein alone. to our knowledge, this is the first report that proved that bivalent subunit vaccines, including s1 and n proteins of ibv, could induce higher immune responses and provide greater protection against infection. the recombinant baculovirus co-expressing s1 and n proteins could serve as a potential ibv vaccine and a bivalent subunit vaccine including the s1 and n proteins might be a strategy for the development of an ibv subunit vaccine. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/10/7/347/s1, figure s1 : identification of recombinant transposon vectors pfast-hbm-s1, pfast-hbm-n and pfast-hbm-s1-n by pcr and restriction endonuclease digestion. figure s2 : identification of recombinant bacmids rhbm-s1, rhbm-n and rhbm-s1-n by pcr with m13 primers. figure s3 : identification of recombinant baculovirus by pcr with ibv specific primers and m13 primers. table s1: all the primers used in this study. the authors have no conflict of interest to declare. infectious bronchitis virus variants: a review of the history, current situation and control measures coronavirus avian infectious bronchitis virus efficacy of infectious bronchitis virus vaccinations in the field: association between the alpha-ibv igm response, protection and vaccine 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undergo selection of subpopulations and mutations following vaccination evaluation of recombinant fowlpox virus expressing infectious bronchitis virus s1 gene and chicken interferon-gamma gene for immune protection against heterologous strains protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/98 current status of veterinary vaccines the protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines protection against infectious bronchitis virus by spike ectodomain subunit vaccine bacmam virus-based surface display of the infectious bronchitis virus (ibv) s1 glycoprotein confers strong protection against virulent ibv challenge in chickens progress and challenges toward the development of vaccines against avian infectious bronchitis molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china location of the amino acid differences in the s1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus the avian coronavirus spike protein the carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection a novel b-cell epitope of avian infectious bronchitis virus n protein generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus insect cell culture for industrial production of recombinant proteins optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins high-level protein expression following single and dual gene cloning of infectious bronchitis virus n and s genes using baculovirus systems induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus assembly and immunogenicity of baculovirus-derived infectious bronchitis virus-like particles carrying membrane, envelope and the recombinant spike proteins serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in guangxi identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus immune responses to structural proteins of avian infectious bronchitis virus immune responses to mucosal vaccination by the recombinant s1 and n proteins of infectious bronchitis virus infectious bronchitis virus: s1 gene characteristics of vaccines used in china and efficacy of vaccination against heterologous strains from china evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/97i strain of infectious bronchitis coronavirus evaluation of the protection conferred by several avian infectious bronchitis attenuated vaccines against the field strain ck/ch/ldl/97 i in china baculovirus induces an innate immune response and confers protection from lethal influenzavirus infection in mice immunogenicity of a recombinant pseudotype baculovirus expressing the s1 protein of infectious bronchitis virus in specific pathogen free (spf) chickens immunogenicity evaluation of s1 glycoproteins from infectious bronchitis virus isolates js/95/03 and sd/97/01 expressed by recombinant baculoviruses infectious bronchitis virus poly-epitope-based vaccine protects chickens from acute infection the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens involvement of the toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties andcrystallization expression and characterization of soluble human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein expression of recombinant proteinase 3, the autoantigen in wegener's granulomatosis, in insect cells key: cord-345088-krb1eidw authors: shen, s; law, y.c; liu, d.x title: a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature date: 2004-09-01 journal: virology doi: 10.1016/j.virol.2004.06.016 sha: doc_id: 345088 cord_uid: krb1eidw the spike (s) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. a number of variants with deletions and mutations in the s protein have been isolated from naturally and persistently infected animals and tissue cultures. here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (ibv) to a monkey kidney cell line. the complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. a single amino acid reversion (l(294)-to-q) in the s protein is sufficient to abrogate the ts phenotype. interestingly, unlike wt virus, the revertant grows well at and below 32 °c, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. if the two ts mutants were allowed to enter cells at 32 °c, the s protein was synthesized, core-glycosylated and at least partially modified at 40 °c. however, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 °c. evidence presented demonstrated that the q(294)-to-l(294) mutation, located at a highly conserved domain of the s1 subunit, might hamper processing of the s protein to a matured 180-kda, endo-glycosidase h-resistant glycoprotein and the translocation of the protein to the cell surface. consequently, some essential functions of the s protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished. the coronaviridae family contains causative agents of a wide spectrum of diseases affecting humans, mammals, and birds. it is the etiologic agent of severe acute respiratory syndrome (drosten et al., 2003; fouchier et al., 2003) . the molecular basis of host specificity and tissue or cell tropism of coronavirus partially resides in the specific interaction between the surface spike protein and cellular receptor(s). this interaction would determine the outcome of infection: whether it is acute or chronic and persistent (rowe et al., 1997a (rowe et al., , 1997b , which organs or tissues are targeted, what symptoms are shown (ballesteros et al., 1997; leparc-goffart et al., 1997; navas et al., 2001) , and even whether host specificity is changed (kuo et al., 2000) . coronaviruses are a group of enveloped rna viruses with positive-sense, single-stranded genomes of 27 to 32 kb that are packaged into helical structures by the nucleocapsid protein (n). the viruses acquire their envelopes by budding of the structural components into the intermediate compartment (ic) between the endoplasmic reticulum (er) and the golgi apparatus (krijnse-locker et al., 1994; lai and cavanagh, 1998; tooze et al., 1984) . the membrane glycoprotein (m) and the envelope protein (e) mediate the formation of the envelope, as they are involved in the induction of virion assembly and incorporation of other structural components into virus particles (vennema et al., 1996) . the spike (s) glycoproteins incorporate into the envelope (de haan et al., 1999) in the form of homooligomeric, 20 nm projections, giving the distinctive morphology of the coronavirus virion. some coronaviruses also contain another envelope protein, the hemagglutinin esterase (he). the largest structural s protein is a type i integral membrane glycoprotein and spans the viral envelope once. in some cases, the s protein is proteolytically cleaved into the n-terminal s1 and the c-terminal s2 subunits of equal size by a host proteinase . the s1 and s2 subunits are noncovalently associated to each other. a hydrophobic transmembrane domain (tm) of s2 anchors on the envelope with a short cytoplasmic-tail inside. one leucine-zipper (heptad repeats) domain overlaps with the tm domain and another extends outside the envelope, folding into a-helices and coiled-coil structures involved in oligomerization (de groot et al., 1987) . it was found that the tm domain and endodomain mediated the incorporation of s into virions but the details of the s/m interaction remain to be elucidated. although s protein is dispensable for the formation of virus-like-particles (vlps), its incorporation into virion is critical to the assembly of infectious virus particles. the s protein is cotranslationally n-glycosylated in the er and also forms stable complex with m in the pre-golgi membrane (holmes et al., 1981; opstelten et al., 1995; sturman et al., 1985) . it was then transported to the golgi apparatus where its high mannose side chains are subsequently trimmed and further modified. the matured s protein is an endo-glycosidase-h-resistant (endo-h), 180-kda form (luytjes et al., 1997) . at the trans-golgi, the matured virions are encapsulated into vesicles of the constitutive secretory pathway and released from infected cells. a portion of s protein might be transported to the plasma membrane and responsible for the cell-to-cell membrane fusion, resulting in the formation of typical syncycia and spread of virus infection to neighboring cells. the tm, cysrich and leucine-zipper domains of s2 and the disparate region of s1 are associated with the membrane fusion activity of the protein (chang et al., 2000; gallagher et al., 1991; krueger et al., 2001; luo et al., 1999; tsai et al., 1999) . cleavage of the s protein enhances the membrane fusion, though not necessarily required. the s protein contains receptor-binding domains in the s1 subunit (kubo et al., 1994; godet et al., 1994; saeki et al., 1997; suzuki and taguchi, 1996) . upon binding of the s protein to the receptor(s), conformation changes induce the virus -cell membrane fusion and subsequently unloading of the ribonucleocapsid inside the cell. it has been demonstrated that the cell-to-cell spread of mhv infection does not need the presence of the receptor, but the s protein is required to mediate the cell-to-cell fusion (gallagher et al., 1992; nash and buchmeier, 1996) . for coronaviruses, however, the detail of molecular mechanisms of membrane fusion, especially those linked to the s1 region, is yet to be fully elucidated. in this study, ts mutants were generated by growing the beaudette strain of ibv at progressively lower temperatures from 35 to 28 jc in vero cells. two ts mutants were isolated from passages grown at 29 jc (ts291602) and 28 jc (ts282902). a revertant was also obtained by growing the ts mutant at the nonpermissive temperature (40 jc). sequence comparison revealed that mutations in the s1 subunit were responsible for the ts phenotype. comparative studies of viral protein synthesis and growth properties of the wt, ts mutant, and revertant viruses demonstrated that the ts mutants were extremely unstable at the nonpermissive temperature. if they were allowed to enter cells by absorption for 1 h at the permissive temperature (32 jc), the s protein would be core-glycosylated and partially modified at the nonpermissive temperature, but was unable to assemble into virions. the membrane fusion activities of the ts mutants were totally abolished in virus-infected cells and in cells overexpressing the mutant s protein at the nonpermissive temperature. the beaudette strain of ibv, grown in vero cells at 37 jc, was plaque-purified and was initially adapted to grow at 35 jc. after 4 passages, the virus was subsequently adapted to grow at progressively lower temperatures by culturing at 34 jc for 5 passages, 33 jc for 7 passages, 32 jc for 44 passages, 30 jc for 12 passages, 29 jc for 21 passages, and 28 jc for 29 passages. when the virus was initially adapted to a lower temperature, no cpe was observed within 72 -96 h postinfection. apparent cpe appeared usually after 2 to 5 passages. the virus was continuously passaged at the same temperature until cpe became visible within 24 -48 h postinfection, and then shifted to a lower temperature. plaque assays of different passages were performed at both 32 and 40 jc. it was found that viruses from early passages grown at 32 jc could form plaques at both temperatures at similar levels. but passages grown at 29 jc produced much fewer plaques at 40 jc than at 32 jc, suggesting the emergence of ts mutants during cold-adaptation. these ts mutants dominated quickly at 28 jc. two ts mutants, designated ts291602 and ts282902, were plaque-purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. plaque assays showed that both ts291602 and ts282902 formed plaques only at the permissive temperature, but not at all at the nonpermissive temperature. by growing the ts mutant at 37 jc overnight and then at 40 jc for 2 days, a revertant, rev-1, was obtained which produced plaques at both 32 and 40 jc at a similar level ( table 1 ). the titers of rev-1 were much higher than that of the wild-type virus wt6501 (isolated from passage 65 at 37 jc) at 32 jc, as it was derived from the cold-adapted virus (table 1) . to map the defects responsible for the ts phenotype described above, the complete nucleotide sequences of wt6501, ts291602, ts282902, and rev-1 were determined using rt-pcr products and cdna clones from each virus. comparison of the sequence of wt6501 with ts291602 revealed 22 point mutations, a 5-base-deletion, a 1-base-, 3-base-and a 9-base-insertion (table 2 ). these alterations caused 15 amino acid substitutions in eight mature viral proteins, a single amino acid insertion in the 3a protein, a truncated 3b (compared with early passages of wt virus) caused by a single base insertion in the 3b of ts291602, and a 3-amino acid insertion in the n protein. in ts282902, similar point mutations, deletion and insertion were found ( table 2 ). the most different feature of the two ts mutant is the insertion in the 3b gene. in ts291602, a single a insertion was found at a poly-a stretch at positions 24075 to 24080, causing frameshift of the 3b gene and resulting in a c-terminally truncated 3b protein (shen and liu, 2003) . however, a three-a-insertion at the same poly-a stretch was found in ts282902, which does not affect the open reading frame of the 3b gene. comparison of the sequence of rev-1 with the two ts mutants showed only one amino acid change (q 294 -to-l 294 ) in the s protein and one amino acid deletion in the nonstructural protein 3a (i 28 ), which exists in both ts291602 and ts 282902 but not in rev-1 and wt virus. the genotype of the rev-1 at these two positions (q 294 and i 28 ) are therefore the same as that of wt6501, but different from the two ts mutants. as both wt virus and the revertant could form plaques at 32 and 40 jc, these two mutations might be responsible for the ts phenotype. a blast search of genbank clearly showed that the amino acid at position 294 of the s protein was a conserved glutamine. the leu 294 residue in the two ts mutants is located immediately downstream of a hypervariable region and the upstream of a conserved region of the s1 subunit. one additional mutation, d 709 -n 709 , was found in ts291602 only, and an i 769 -m 769 mutation in the two ts mutants and rev-1. after mapping the potential determinants responsible for the ts phenotype, we would like to explore the mechanisms by which these mutations affect the propagation of the ts mutants at the nonpermissive temperature. synthesis of the s protein in cells infected with wt virus, ts mutants, and revertant was analyzed. two sets of experiments were carried out. first, vero cells were infected with wt6501, ts291602, ts282902, and rev-1 at 32 and 40 jc, respectively, and lysates were immunoprecipitated with anti-ibv antibodies. it was obvious that synthesis of the ts mutant s intergenic region between m and 5a 25,346 table 1 titers of wild type, mutant and revertant viruses at 32 jc and 40 jc (pfu/ml) titer 32 jc 4 0 jc wt6501 8.5 â 10 3 5.5 â 10 6 ts291602 2.0 â 10 6 0 ts282902 3.2 â 10 6 0 rev-1 7.0 â 10 6 5.5 â 10 6 protein was detectable at 32 jc but not at 40 jc (fig. 1a , lanes 3, 4, 5, and 6) under these conditions. in contrast, expression of the s protein was observed in cells infected with wt6501 and rev-1 at both temperatures ( fig. 1a , lanes 1, 2, 7, and 8), though the level of the proteins detected in cells infected with wt6501 was much lower at 32 jc. this was consistent with the lower titers of wt virus at 32 jc. in the second set of experiments shown in fig. 1b , cells were absorbed with each virus at 32 jc for 1 h and one of the duplicates was shifted to 40 jc. under these conditions, the two ts mutant s proteins were synthesized at both 32 and 40 jc (fig. 1b, lanes 3 , 4, 5, and 6), like those of the wt virus and revertant (fig. 1b , lanes 1, 2, 7 and 8). these results indicated that the viral structural protein could be synthesized at nonpermissive temperature if the mutants were allowed to enter cells. the viral subgenomic rna and one of the processed proteins, the 3c-like proteinase, were also detected in the same ways (data not shown), and the results were consistent with those described above. comparison of the nucleotide sequences and the s protein synthesis of wt, ts mutants and revertant indicate that the single amino acid mutation (q 294 -l 294 ) in the s protein may be responsible for the temperature sensitivity of the mutant virus. this possibility was studied by biochemical and functional characterization of the s protein from the revertant and a ts mutant. the s gene of rev-1 and ts291602 was cloned under the control of a t7 promoter to investigate whether membranefusion activity was affected by the mutations in the s protein. cells were infected with recombinant vaccinia/t7 virus and were transfected with plasmids containing the s gene from either ts291602 or rev-1. the expression of the s proteins was analyzed by western blotting using anti-ibv antibodies. as shown in fig. 2a , the s protein of both revertant and ts mutant was expressed at 32 and 40 jc (lanes 3, 4, 5, and 6), though the s protein of ts291602 was expressed at a relatively lower level than the rev-1 at 40 jc (compare lanes 4 and 6). the fusion activity of the s protein derived from the revertant and ts291602, respectively, was then examined. as shown in fig. 2b , membrane fusion was observed in cells expressing the s gene of ts291602 at 32 jc (panel c) but not at 40 jc (panel f), at 48 h posttransfection. in contrast, membrane fusion was observed in cells expressing the s gene of revertant at both 32 and 40 jc (panels b and e). interestingly, membrane fusion appeared at 72 h posttransfection, after the cells expressing the s gene of ts291602 were shifted from 40 to 32 jc at 48 h posttransfection (panel i). the recombinant s genes of the ts mutant and revertant were transfected into cells in 35 mm dishes at 32 jc and one of the duplicates was shifted to 40 jc at 4 h posttransfection. radiolabeled proteins were immunoprecipitated and treated with endo-h at 37 jc. as shown in fig. 3a , the ts mutant s protein, synthesized at 40 jc, was sensitive to endo-h digestion, as no 180-kda form was observed (lane 12). in contrast, a portion of the ts mutant s protein, synthesized at 32 jc (lanes 10), was resistant to endo-h digestion. the revertant s protein, synthesized at both 32 and 40 jc, was also resistant to endo-h digestion (lanes 6 and 8). these a b fig. 1 . analysis of the expression of the s protein from wt (wt6501), ts mutants (ts291602 and 282902) and revertant (rev-1) viruses. (a) cells were infected with each virus at 32 jc and 40 jc as indicated on the top for 1 h and were maintained at the same temperatures. (b) two dishes of cells were infected with each virus at 32 jc for 1 h. one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7) and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). radiolabeled cell lysates were immunoprecipitated with anti-ibv antibodies. the proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular mass in kilodalton and the position of the s protein is indicated on the right. results indicate that the ts mutant s protein synthesized at 40 jc was not a mature form of the glycoprotein. in addition, a novel glycosylated form of the s protein, migrating between the endo-h treated 130-kda and the matured 180-kda forms, was observed. it might represent trimming of an initial, core-glycosylated form of the s protein in the er and the cis-golgi. interestingly, this band was also observed in ts291602-infected cells (fig. 3b, lane 2) , when the incubation time at 32 jc was extended before the culture was shifted to 40 jc. this band is much stronger than the 180-kda form (lane 2), giving direct evidence that the mutations in the s protein of ts291602 dramatically hampered the maturation process of the s glycoprotein. compared with ts282902, the s protein of ts291602 contains another amino acid substitution, d 709 -n 709 (table 1) , which is not present in the s protein of rev-1 and wt6501. as this band was not observed in ts281602-infected cells (data not shown), it is not clear at this point if this additional mutation may also play a role in the maturation of the s protein. it was also noted that, at 40 jc for all viruses, the s1 and s2 subunits were hardly detected in cell lysates but abundant in the supernatants (fig. 3b) . at the nonpermissive temperature, the s protein was not cleaved at all (lanes 2 and 6). these results may suggest that only the mature form of the s glycoprotein could be cleaved into s1 and s2 and fig. 2 . membrane fusion activity of the s protein expressed from wt and ts291602. (a) vero cells were transfected with plasmids without insert (lanes 1 and 2) or with the s gene from the wt (lanes 3 and 4) and ts291602 (lanes 5 and 6), and were cultured at 32 jc (lanes 1, 3, and 5) or 40 jc (lanes 2, 4, and 6). the expression of the s protein was examined in western blot using anti-ibv antibodies, and h-tubulin was immunostained as loading controls. (b) the membranefusion activity of the s protein from the wt (panels b, e, and h) and ts mutant (panels c, f, and i) at 32 jc (panels b and c) and 40 jc (panels c and f) 2 days post-transfection was compared. panels h and i show induction of membrane fusion on cells transfected with the wt and ts291602 by shifting cells shown in panels e and f to 32 jc for 1 day. panels a, d, and g show cells transfected with empty plasmids. reinforce the conclusion that the majority of the s protein of the ts mutants synthesized at the nonpermissive temperature may represent a pre-mature form of the s glycoprotein. to detect whether the ts mutant s protein would be transported to the plasma membrane at the nonpermissive temperature, the s protein was transiently expressed in vero cells using the vaccinia virus-t7 expression system. the transfected cells were treated with cycloheximide at 3.5 h posttransfection for 30 min to inhibit further protein synthesis. the cells were then incubated at 32 and 40 jc, respectively. the subcellular localization of the s protein was analyzed by indirect immunofluorescent staining at 4, 8, 16, and 24 h postinfection, respectively. the confocal microscopy images of transfected cells are shown in fig. 4 . plasma membrane staining of the ts mutant s protein was not observed at any time points posttransfection at 40 jc, but was clearly seen at 32 jc at 16 and 24 h postinfection (fig. 4) . the revertant s protein was expressed on the cell surface at both temperatures at 16 and 24 h posttransfection. these results may explain the lack of membrane fusion activity of the ts mutant s protein in both infected and transfected cells. endo-h treatment of the s protein from rev-1 and ts291602. the s protein derived from rev-1 (lanes 5, 6, 7, and 8) and ts291602 (lanes 9, 10, 11, and 12) were expressed in vero cells at 32 jc (lanes 5, 6, 9, and 10) and 40 jc (lanes 7, 8, 11, and 12), using a t7-vaccinia expression system. the radiolabeled proteins were immunoprecipitated with anti-ibv antibodies, the eluted proteins were endo-h-(lanes 6, 8, 10, and 12) or mock-treated (lanes 5, 7, 9, and 11) and analyzed by sds-page. (b) detection of the less matured s protein from ts291602 at 40 jc and the defect in cleavage of the mutant s protein. cells were infected with ts291602 and rev-1 for 5 h at 32 jc, one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7), and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). cells were radiolabeled and viral proteins were immunoprecipitated with anti-ibv antibodies and analyzed by sds-page. lanes 1, 2, 3, and 4 refer to viral products detected from cell lysates and lanes 5, 6, 7, and 8 refer to viral proteins detected from virus particles released to the cultured media. quantitative analysis of surface expression of the wild type and ts mutant s protein was carried out by immunofluorescent staining with anti-s protein antiserum and the positive staining cells were sorted by flow cytometry. as shown in fig. 5 , 5.5% of nonpermeabilizing (panel a) and 0.7% of permeabilizing (panel f) cells expressing the empty plasmid showed background staining. when the cells were incubated at the permissive temperature, 3.8% (9.2 -5.5) of hela cells expressing the wt s protein (panel b) and 4.1% (9.6 -5.5) of cells expressing the ts mutant s protein (panel c) displayed surface staining. after permeabilizing with 0.1% saponin, 11.8% (12.5 -0.7) of cells expressing the wt s protein (panel g) and 10.9% (11.6-0.7) of cells expressing the ts mutant s protein (panel h) showed positive staining. when the cells were incubated at the nonpermissive temperature, 10.4% (15.9 -5.5) of cells expressing the wt s protein (panel d) and 2.3% (7.8 -5.5) of cells expressing the ts mutant s protein (panel e) exhibited surface staining. after permeabilizing with 0.1% saponin, 9.3% (10 -0.7) of cells expressing the wt s protein (panel i) and 9.7% (10.4 -0.7) of cells expressing the ts mutant s protein (panel j) showed positive staining. these results confirm that the ts mutant s protein could not be efficiently translocated to the cell surface at the nonpermissive temperature. to investigate whether the s protein of ts mutant was assembled into virion or not, ts291602 and rev-1-infected cells were radiolabeled and the virus particles were purified through sucrose gradients twice. immunoprecipitation of the purified virions using anti-ibv antibodies showed that the s protein of ts291602 was not detected at 40 jc (fig. 6 , lane 2), but was observed at 32 jc (fig. 6, lane 1) . the s protein was detected at both temperatures for rev-1 (fig. 6, lanes 3 and 4) . these results render support that the spike protein of the ts mutant may not be assembled into virus particles at 40 jc. coronavirus s protein is responsible for receptor binding and membrane fusion. it also induces neutralizing antibodies and bears determinants for virulence. a considerable diversity in s protein among coronaviruses exists, which contributes to host specificity, cell and organ tropisms, and pathogenesis. characterization of mutants with point or deletion mutations in the s protein has helped to establish links between the variations and the functions or altered antigenicity and virulence of viruses. through sequence analysis of wt virus, two ts mutants, a revertant and different passages of a cold-adapted ibv, data presented in this study not only mapped the defects of the ts mutants to the s gene, but also revealed the molecular events occurred during evolution of the ibv s gene. compared with the parental wt virus, mutations that are identical in the two ts mutants and the revertant might be associated with the cold-adaptation phenotype, while those identical only in the two mutants but different from the revertant and wt virus were considered to be linked to the ts phenotype. according to this criterion, the q 294 -to-l 294 mutation in the s protein that exists in the two ts mutants, and interestingly, occurred at a highly conserved domain among ibv viruses immediately downstream a variable domain in the s1 subunit may be responsible for the ts phenotype. the mutants with this mutation accumulated and became dominant under selective pressures, in this case, changes in the hosts and temperatures. the i 769 -m 769 mutation emerged earlier than the q 294 -l 294 mutation, which among variations in other gene products may be associated with cold-adaptation, suggesting potential segregation of mutations responsible for either cold-adaptation or ts phenotypes. further confirmation of the possibility that the q 294 -to-l 294 mutation may cause the ts phenotypic changes is currently being carried out by introducing the mutation to an infectious ibv clones and isolation of a recombinant virus containing this mutation only. the s protein is co-translationally n-glycosylated in the er, oligomerized (luytjes et al., 1997) if folded properly, and associated with the m protein in pre-golgi membrane (opstelten et al., 1995) . one of the essential steps in nlinked glycosylation is transfer of a preformed, 14-coreunit-oligosaccharide to a specific asn residue in the sequence asn-x-ser/thr (where x is any residue except pro, asp, or glu). the oligosaccharide chain is donated by dolichol-pyrophosphate-oligosaccharide to the protein chain in the er. it is trimmed down in the er and cis-golgi, and different external sugars are then added to the trimmed chain in the medial-and trans-golgi. glycoproteins with high mannose oligosaccharides in the er and cis-golgi remain sensitive to endo-h. they become endo-h resistant after being processed by the medial-and trans-golgi resident enzymes to glycoproteins with complex oligosaccharides. the acquisition of endo-h resistance is therefore an indication that viral n-linked glycoproteins are properly processed and transported to the golgi apparatus (luo and weiss, 1998; luo et al., 1999; luytjes et al., 1997) . examination of the amino acid sequence of ibv s protein showed that there are 13 and 9 potential glycosylation sites in the s1 and s2 subunits, respectively. the calculated molecular weight of the s protein is 128 kda. if all 21 sites are used, the initially transferred oligosaccharides account for 50 kda. in cells overexpressing s protein, core-glycosylation of the s protein in the er resulted in a 180-kda product as expected. trimming of the s glycoprotein in the er and cis-golgi led to a partially processed 155-kda form, larger than the endo-h-treated 130-kda form which only has one sugar residue left at each site. both the trimmed 155-and core-glycosylated 180-kda forms were sensitive to endo-h digestion. at the permissive temperature, further modification of the mutant s protein in the medial-and trans-golgi by addition of sugars to the trimmed chains resulted in the maturation of s, which co-migrates with the core-glycosylated 180-kda form but is resistant to endo-h digestion. at the nonpermissive temperature, however, no mature form of the fig. 6 . analysis of the structural proteins on purified ts291602 and rev-1 virions. immunoprecipitations were performed using virions, purified from ts291602-(lanes 1 and 2) and rev-1-infected (lanes 3 and 4) vero cells at 32 jc (lanes 1 and 3) and 40 jc (lanes 2 and 4) . the viral proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right. mutant s protein was produced. in virus-infected cells, the endo-h sensitive, 155-kda form of ts291602 was abundant at the nonpermissive temperature, clearly indicating that the mutant s protein was trimmed in the er and cis-golgi but was not transported to the trans-golgi. furthermore, the mutant s protein was not cleaved into s1 and s2 at the nonpermissive temperature, which usually occurs in the trans-golgi shortly before the release of virions from cells (frana et al., 1985) . as only spikeless virions were produced, these results demonstrated that the core-glycosylated or the trimmed forms of s were unable to incorporate into virions. in addition, the mutant s protein fails to induce cell -cell fusion at the nonpermissive temperature. as the q 294 -to-l 294 mutation is not in the fusogenic domain of s, it renders support to the conclusion that the mutation causes the defect in its modification and intracellular transport, instead of having direct impact on the fusion process. taken together, these studies clearly indicate that the mutant s protein could not reach the site where the s protein is incorporated into virions. the reasons for the retention of the mutant s protein in the er and cis-golgi are yet to be explored. previous studies show that the retention of monomeric s protein in the er is due to misfolding induced by disruption of disulfide bonds (opstelten et al., 1993) , and also caused by lack of oligomerization of endo-h-sensitive s protein of ts mutants though the locations were not determined (luytjes et al., 1997) . it is not clear whether mutations in ibv s protein have an effect on its folding or oligomerization. nevertheless, in both circumstances, the intracellular transport, modification of the s protein and its incorporation into virion would have been affected. studies with some of the other enveloped viruses demonstrated that molecular chaperones (like calnexin and calreticulin) in the er transiently and specifically interact with partially trimmed, monoglycosylated form of viral n-linked glycoproteins (tatu and helenius, 1997) . for instance, calnexin interacts with the influenza hemagglutinin and vesicular stomatitis virus g protein (hebert et al., 1996; hammond and helenius, 1994) . these and other proteins could facilitate proper folding and ensure quality control for viral glycoproteins. proteins that fail to fold and oligomerize are prevented from transport and ultimately degraded. with the ts mutants, it is possible to explore the interaction of the s protein with molecular chaperones and folding enzymes. this approach would provide new insights into the maturation and assembly of the coronavirus s protein. the ts mutant quickly lost its infectivity at the nonpermissive temperature. this low thermostability indicated that even if the mutant s protein was synthesized, fully modified and assembled into virions at the permissive temperature, the resulting particles were relatively unstable and lost infectivity at the nonpermissive temperature. it suggests that the q 294 residue in the conserved s1 domain of ibv may also play an essential role in maintaining the thermal stability of the spike in the virion, resembling the s 287 residue in the s1 region of an mhv ts mutant (ricard et al., 1995) . in addition, if cells were incubated with the ts mutants at the nonpermissive temperature, viral proteins were hardly detected, indicating that at the nonpermissive temperature, the ts mutant s protein may be unable to either bind efficiently to the receptor or induce virus -cell membrane fusion in contrast to the s protein of the revertant and wt virus. this observation suggests that the mutation in the s protein might induce conformation changes of the spikes on virions at the nonpermissive temperature and hamper either process. vero cells were maintained in complete dmem medium (gibco brl), supplemented with newborn calf serum (10%), streptomycin (1000 ag) and penicillin (1000 units/ml). the beaudette stain of ibv was purchased from atcc and propagated in chicken embryonated eggs for three passages. the virus was then adapted to grow and passage on vero cells for 65 times at 37 jc. in this study, the viruses were further passaged on vero cells at progressively lower temperatures for 121 passages (at 35, 34, 33, 32, 30, 29, and 28 jc for 4, 5, 7, 44, 12, 21, and 29 passages) . virus stocks were used for plaque assays, plaque-to-plaque purification and viral rna extraction as described previously (shen et al., 1994) . the wild-type virus, wt6501, was plaque-purified from passage 65 grown at 37 jc, while ts mutants, ts291602 and ts282902, were purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. the revertant, rev-1, was plaque-purified from cells infected with ts 291602 for 12 h at 37 jc and then shifted to 40 jc for 2 days. recombinant vaccinia/t7 (v/t3) virus was propagated and was tittered on vero cells. virus stocks were kept at à80 jc until use. confluent monolayers of vero cells were infected with viruses at a multiplicity of infection (moi) of 0.1. after 2h absorption, the viruses were radiolabeled by replacing medium with methionine-free dmem supplemented with 30 aci/ml [ 35 s]-methionine. after 16-h incubation at temperatures appropriate for each virus, cells were harvested and virus stock was prepared by freezing and thawing three times. cell debris was removed by centrifugation at 5000 rpm for 15 min (beckmen, 25.50). the supernatant was centrifuged through a 20% sucrose cushion and the resulting pellet was resuspended in tne buffer (50 mm tris -hcl, ph 7.4, 100 mm nacl, 1 mm edta). the viruses were further purified by centrifugation through a 20-55% su-crose gradient in tne buffer at 45,000 rpm (beckmen, sw50) twice. fractions containing the virus were pooled together and used for protein analysis. viral structural proteins were labeled with [ 35 s]-methionine, immunoprecipitated and separated on 12.5% polyacrylamide gels as described previously (liu et al., 1998) . the anti-ibv serum was raised in rabbit against purified ibv virions and was used in immunoprecipitation and western blot as previously described (liu and inglis, 1991) . a portion of cell lysates was immunoprecipitated with anti-ibv serum. the pellets were washed three times with standard ripa buffer, dissolved in 20 al of digestion buffer (50 mm tris, ph 6.8, 0.25% sds) and incubated at 95 jc for 5 min. ten microliters of the supernatants were mixed with 10 al of digestion buffer with or without endo-h (0.2 mu, boehringer mannheim) and incubated for 4 h at 37 jc. confluent monolayers of vero cells were infected with recombinant vaccinia/t7 viruses at a moi of 0.1. after 1h absorption, cells (2 â 10 7 ) were trypsinized, centrifuged at 250 g and resuspended in 2 ml of pbs. the cells (0.3 ml) were mixed with 5 ag plasmid and electroporated (easyject, equibio) at 600 v in a 0.4-cm cuvette (biorad). the cells were then plated in a 35-mm dish containing 3 ml of dmem with 2% of newborn calf serum and incubated at temperatures indicated. viral rna was extracted from the purified viruses using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription and polymerase chain reaction (rt-pcr) were performed using the expand reverse transcription and high fidelity pcr kits (boehringer mannheim). annealing and extension times of pcr were optimized for amplification of pcr products with different sizes using different primers. more than 100 specific primers were used for amplification, sequencing and cloning. automated sequencing was carried out using pcr products or cdna clones and specific primers as previously described (shen et al., 1999) . sequence analysis was carried out using the gcg and blast suite of programs as described previously (shen and liu, 2000) . hela cells were infected with vaccinia/t7 virus, transfected with constructs encoding wt and ts mutant s protein using qiagen effectene transfection reagent, and incubated at the permissive and nonpermissive temperatures, respectively, for 18 h. cells were harvested, washed once with pbs, resuspended in blocking buffer (20% fbs and 1% bsa in pbs), incubated on ice for 30 min. a half of the cells were permeabilized with 0.1% saponin in facs washing buffer (2.5% fbs and 0.05% sodium azide in pbs) incubated for 10 min at room temperature, and stained with 1:100 diluted primary antiserum, the rabbit anti-ibv s protein. the other half was stained directly with the same primary antibody. cells were then washed two times with the facs washing buffer, stained with 1:20 diluted fitc conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were then fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein evidence for a coiled-coil structure in the spike of coronaviruses mapping of the coronavirus membrane protein domains involved in interaction with the spike protein identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein folding of vsv g protein: sequential interaction with bip and calnexin calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses altered pathogenesis of a mutant of the murine coronavirus mhv-a59 is associated with a q159l amino acid substitution in the spike protein association of the infectious bronchitis virus 3c protein with the virion envelope proteolytic mapping of the coronavirus infectious virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-tocell fusion characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a59 with maturation defects in the spike protein spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly a conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp2 gene with an unique insertion emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication rearrangement of the vp6 gene of a group a rotavirus in combination with a point mutation affecting trimer stability sequence analysis and in vitro expression of genes 6 and 11 of an ovine group b rotavirus isolate, kb63: evidence for a non-defective, c-terminally truncated nsp1 and a phosphorylated nsp5 proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments analysis of the receptor-binding site of murine coronavirus spike protein interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum replication of coronavirus mhv-a59 in sac-cells: determination of the first site of budding of progeny virions a 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes this work was supported by the biomedical research council, agency for science technology and research, singapore. key: cord-342176-tewfm8it authors: kjærup, rikke m.; dalgaard, tina s.; norup, liselotte r.; bergman, ingrid-maria; sørensen, poul; juul-madsen, helle r. title: adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations date: 2013-11-08 journal: immunobiology doi: 10.1016/j.imbio.2013.10.013 sha: doc_id: 342176 cord_uid: tewfm8it mannose-binding lectin (mbl) plays a major role in the immune response as a soluble pattern-recognition receptor. mbl deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. in humans and chickens, several studies have shown that mbl participates in the protection of hosts against virus infections. infectious bronchitis (ib) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (ibv). mbl has earlier been described to play a potential role in the pathogenesis of ibv infection and the production of ibv-specific antibodies, which may be exploited in optimising ibv vaccine strategies. the present study shows that mbl has the capability to bind to ibv in vitro. chickens from two inbred lines (l10h and l10l) selected for high or low mbl serum concentrations, respectively, were vaccinated against ibv with or without the addition of the mbl ligands mannan, chitosan and fructooligosaccharide (fos). the addition of mbl ligands to the ibv vaccine, especially fos, enhanced the production of ibv-specific igg antibody production in l10h chickens, but not l10l chickens after the second vaccination. the addition of fos to the vaccine also increased the number of circulating cd4+ cells in l10h chickens compared to l10l chickens. the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8α−γδ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination. as mbl ligands co-administered with ibv vaccine induced differences between the two chicken lines, these results indirectly suggest that mbl is involved in the immune response to ibv vaccination. furthermore, the higher antibody response in l10h chickens receiving vaccine and fos makes fos a potential adjuvant candidate in an ibv vaccine. the first line of host defence against pathogens involves the innate immune system. pathogens have specific microorganismassociated molecular patterns (mamps) that are recognised by pattern-recognition receptors (prrs). two kinds of prrs exist: surface prrs and soluble prrs. prrs trigger intracellular signalling cascades upon mamp recognition culminating in activation of antigen presenting cells and production of co-stimulatory molecules as well as pro-inflammatory cytokines. the production of costimulatory molecules and pro-inflammatory cytokines initiates the early host response to infection and also partakes in the activation and shaping of the adaptive immune response (crozat et al. 2009; de visser and coussens 2005; hoffmann et al. 1999) . several soluble prrs have been described, and an example of such is mannose-binding lectin (mbl). mbl has various functions in, for example, complement activation, promotion of complementindependent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance (dommett et al. 2006) . mbl is a collectin consisting of multiple identical polypeptide chains oligomerised into different sizes. the chains are made up of four distinct domains. these are a cysteinerich n-terminal domain, a collagenous domain, a neck domain, and a calcium-dependent carbohydrate-recognition domain (crd) at the c-terminal (takahashi 2011) . it is the crd that permits mbl to bind in a ca 2+ -dependent manner to mamps such as polysaccharides. in fact, terminal sugars, such as d-mannose, l-fucose and n-acetyl-d-glucosamine, found on the surface of many microorganisms, contain equatorial 3-and 4-hydroxyl groups to which mbl binds. mbl does not bind to d-galactose and sialic acid found on the surface of many animal cells. beside sugars, mbl has also been found to bind to phospholipids, nucleic acids, and non-glycosylated proteins (ip et al. 2009 ). the mbl genes in human (madsen et al. 1994 (madsen et al. , 1995 steffensen et al. 2000; sumiya et al. 1991) , porcine (juul-madsen et al. 2011a; lillie et al. 2007) , bovine (liu et al. 2011) and chickens (kjaerup et al. 2013) have been found to have polymorphisms resulting in wide variations in mbl serum levels in the organisms. over the last decades mbl deficiency and the influence on the susceptibility to different types of infections have been subject to extensive studies as reviewed (heitzeneder et al. 2012; mayilyan 2012; takahashi 2011) . some results indicate that mbl deficiency may actually be beneficial with regard to disease, for example visceral leishmaniasis (santos et al. 2001 ). however, most results suggest that mbl deficiency leads to a weaker immune response. in humans, several studies have shown that mbl participates in the protection of hosts against virus infections, such as infections with influenza a virus (chang et al. 2010 ), hepatitis c virus (brown et al. 2010) , ebola virus (michelow et al. 2011) , and severe acute respiratory syndrome (sars) coronavirus (ip et al. 2005; zhou et al. 2010) . thus, mbl in chickens may also play a role in the pathogenesis of chicken virus infections and the production of antibodies as suggested by juulmadsen et al. (2007) . selective breeding of chickens for low or high serum mbl concentrations has been performed for several generations at our department as published by juulmadsen et al. (2007) . this has resulted in two distinct chicken lines designated high (l10h) or low (l10l) with mean serum mbl concentrations of 33.4 g/ml serum (l10h) and 7.6 g/ml serum (l10l) (f14 generation, unpublished) . studies using these chicken sublines as well as outbred chickens have shown an inverse relationship between the mbl concentrations and the pathogen-specific antibody response (juul-madsen et al. 2007; schou et al. 2008 ). studies in mice have shown that mbl deficiency may result in a higher igg antibody response after infections (carter et al. 2007 ) and vaccinations (guttormsen et al. 2009 ). from these results it can be hypothesised that basal mbl plasma levels may influence specific humoral immune responses. this explanation for this may be that either: (1) mbl pushes the immune response into a more cellular response (th1 vs. th2); (2) mbl efficiently neutralises the pathogen via the complement membrane-attack complex and no adaptive immune response is needed; or (3) mbl influences the pro-inflammatory cytokine production via interaction with surface receptors, such as toll-like receptors (ip et al. 2008) . infectious bronchitis (ib) is a highly contagious disease of economic importance in the poultry industry with symptoms such as sneezing, tracheal rales, and coughing. furthermore, ib may cause a decline in egg quality and production in layers (raj and jones 1997) . ib is caused by the coronavirus infectious bronchitis virus (ibv) which is highly able to genetically mutate and recombine. as a result, there is a continuous development of new strains throughout the world. different strains can co-circulate within a region, and the severity of the disease varies from strain to strain and from flock to flock (capua et al. 1999; cavanagh 2007; cook et al. 2012) . consequently, applied vaccines sometimes provide insufficient protection, as vaccination with one strain of ibv may not be protective against other strains. vaccine efficacy may be improved by the use of adjuvants. good candidates for vaccine adjuvants are carbohydrates since they are mostly of low toxicity and high biocompatibility and furthermore play major roles within the immune system (petrovsky and cooper 2011) . carbohydrates such as mannan (liu et al. 2012) , chitosan (rauw et al. 2010) , and fructooligosaccharide (fos) (benyacoub et al. 2008 ) have previously been used in vaccines or diets as modulators of the immune response. these three carbohydrates are potential mbl ligands owing to their content of sugar units. the hypothesis of this study was that immunity after ibv vaccination may be improved after temporarily inhibition of the mbl function. this was achieved by adding an mbl ligand (mannan, chitosan, or fos) to the vaccine given to chickens and thereby creating an artificial mbl deficiency during vaccination. innate as well as adaptive immunological parameters were measured throughout the experimental period. all chemicals were obtained from sigma-aldrich, ballerup, denmark, except when noted. dreamtaq tm master mix was obtained from qiagen, and oligonucleotide primers and probes were obtained from eurofins mwg operon, ebersberg, germany. purified chicken mbl was bought from the department of cancer and inflammation research, university of southern denmark. it was purified from chicken serum as previously described (laursen et al. 1998a ). the binding capacity of mbl to ibv was measured using elisa. dilutions of purified mbl and serum samples from l10l or l10h chickens were made with or without addition of saccharides. biowhittaker ® veronal buffer (lonza, walkersille, md, usa; cat. no. 12-624e) adjusted to a final concentration of 5 mm mgcl 2 and 10 mm cacl 2 was used for diluting the samples. for the titration of mbl binding, concentrations of 0, 1, 2, 4, 8, 16, 24 and 32 g/ml purified mbl were used, and 5 l serum samples were diluted 1:50. the concentrations of the saccharides added were as follows: mannan 100 mg/ml; fos 100 mg/ml; chitosan 10 mg/ml; galactose 9 mg/ml; and edta 20 mm. one-hundred microlitres of the dilutions and saccharides were mixed in a nunc 96-well polypropylen microwell plate (thermo fisher scientific, slangerup, denmark; cat. no. 442587) and incubated for 5 min before the samples were transferred to a 96-well microtitre plate coated with ibv antigen (from the proflok ® ibv antibody test kit from synbiotics corporation, san diego, ca, usa; cat. no. 96-6506). wells receiving only buffer were used as negative controls. all dilutions were added in triplicates. the plate was then incubated at room temperature for 1 h. after a washing step with gibco ® dpbs supplemented with calcium and magnesium (life technologies europe bv, naerum, denmark; cat. no. 14080-048) and ph adjusted to 7.4 followed by supplementation with 0.1% bsa (hereafter called pbs+), the wells were incubated for 45 min at room temperature with 1 g/ml of biotinylated monoclonal mouse anti-cmbl (bioporto diagnostics a/s, gentofte, denmark; cat. no. hyb 182-01) in pbs+. after another washing step streptavidin horseradish peroxidase (sav-hrp) (bd bioscienses, albertslund, denmark; cat. no. 554066) diluted 20,000-fold in pbs+ was added. after 30 min of incubation and washing with pbs+, the presence of sav-hrp was detected by adding 100 ml of substrate solution (<0.05%, wt/wt; 3,3 ,5,5 tetramethylbenzidin). colour development was stopped with a 1 m h 2 so 4 solution. the colour development was determined by reading the absorbance at 405 nm with absorbance at 650 nm as reference. generation 13 from the two au inbred sub-lines l10l and l10h (juulmadsen et al. 2007 ) was used in this study (n = 72). chickens from l10 consist of 67.5% um-b19 and 33.5% white cornish (laursen et al. 1998b ). the offspring were reared together in a biosecured ibv-free environment until they were 3 weeks of age and then allocated into 4 different treatments groups with 9 birds from each subline in each group. the first treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ® ib ma5 (msd animal health, ballerup, denmark; danish product license no. 8674) per animal. the second treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 200 mg/ml purified mannan from saccharomyses cerevisiae per animal aiming at 100 g mannan per gram body weight (juulmadsen et al. 2011b ). the third treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 50 mg/ml deactylated chitosan from shrimp shells per animal. the fourth treatment group was treated with 100 l deionized water containing one dose of live attenuated nobilis ib ma5 vet and 200 mg/ml fos (orafti ® p95 from alsiano a/s, birkerød, denmark) per animal aiming at 100 g fos per gram body weight. all solutions were shaken before nasally applied to the chickens. after three weeks, the four treatment groups were vaccinated again with the same amount of vaccine and saccharides as for the first vaccination. the chickens were fed diets that met or exceeded nrc requirements. food and water were provided ad libitum. the experimental procedures were conducted under the protocols approved by the danish animal experiments inspectorate and complied with the danish ministry of justice law no. 382 (10 june 1987) and acts 739 (6 december 1988) and 333 (19 may 1990) concerning animal experimentation and care of experimental animals. serum was collected from blood samples (0.5-0.7 ml) taken from the jugular vein or the wing vein from the experimental chickens on days 0, 1, 2, 3, 4, 5, 7, 14, 21, 22, 23, 24, 25, 26, 28, 35, 42, 49 and 56 post vaccination 1 (pv1). heparin-stabilised blood for immunophenotyping was collected once a week on days 0, 7, 14, 21, 28, 35, 42, 49 and 56 pv1 and the collected blood (0.5-0.7 ml) was divided into one serum tube and one heparin tube. oropharyngeal airway (opa) swab samples were collected on days 0, 2, 3, 4, 5, 7, and 23 pv1. swab samples were kept in 0.5 ml virus media (20.4% gibco ® penicillin-streptomycin (life technologies europe bv, naerum, denmark; cat. no. 15140-122); 74% biowhittaker ® pbs (lonza, verviers, belgium; cat. no. be17-517q); 5% biowittaker ® foetal bovine serum (lonza, verviers, belgium; cat. no. de14-801f); and 0.01‰ phenol red (merck, darmstadt, germany, cat. no. 1072410005) at −20 • c until testing by real-time quantitative reverse transcription pcr (qrt-pcr) after the termination of the experiment. mbl haplotypes were determined by means of the taqman ® snp genotyping technique (applied biosystems, foster city, ca, usa). two assays were designed, using the custom taqman ® assay design tool according to the instructions on the website (https://www5.invitrogen.com/custom-genomic-products/tools/ genotyping/). these assays distinguish between the cg and ta alleles of snp1 and the gggg and agga alleles of snp2 (kjaerup et al. 2013) . the assays were validated and run on 384-well microtitre plates in an abi prism 7900ht sequence detection system instrument, using version 2.2 of the sds software. a total reaction volume of 10 l was applied, each sample containing 5 l taqman ® universal pcr mastermix (applied biosystems, life technologies europe bv, naerum, denmark; cat. no.4364338), 0.25 l assay mix, 3.75 l h 2 o, and 1 l dna at a concentration of 5 ng/l. to ensure optimal clustering, 24 positive controls, representing animals of known genotypes, were included in each run, together with four non-template (negative) controls. amplification was obtained through an initial incubation period of 10 min at 95 • c followed by 40 cycles of denaturation for 15 s at 92 • c and annealing/extension for 1 min at 60 • c. after amplification, end-point reads were carried out, and analysis was performed with 2-cluster calling enabled in the sds software version 2.2. forty-seven microlitres of three swab samples from each subline, treatment group and day were pooled; thus, 3 pools per subline were analysed for each time point. rna purification was done using the qiaamp ® viral rna mini kit (qiagen, copenhagen, denmark; cat. no. 52904) according to the manufacturer's instructions. the reverse transcription pcr was carried out using the high-capacity cdna archive kit (applied biosystems, life technologies europe bv, naerum, denmark; cat. no. 436881) according to the manufacturer's instructions with 25 l of rna. the real time quantitative reverse transcription pcr (qrt-pcr) reaction was performed with the forward primer ibv5 gu391 (5 -gcttttgagcctagcgtt-3 ), the reverse primer ibv5 gl533 (5 -gccatgttgtcactgtctattg-3 ) and the dual-labelled probe ibv5 g probe (5 -fam-caccaccagaacctgtcacctc-bhq1-3 ) previously described to amplify at least 15 different strains of ibv (callison et al. 2006 ). the total reaction volume was 20 l containing 11 l taqman ® universal pcr master mix (applied biosystems, life technologies europe bv, naerum, denmark; cat. no. 4304437), 0.9 m primers, 0.125 m probe and 0.9 l cdna as template. the reaction was performed in an abi prism 7900ht sequence detection system at 50 • c for 2 min, 95 • c for 10 min with optic off; 40 cycles of 95 • c for 15 s followed by 60 • c for 1 min with optics on. standard curves were included in each qrt-pcr run and were generated from dilutions of cdna originating from purified rna from the live attenuated nobilis ib ma5 vet. the standard curves were made as 5-fold dilutions starting at one dose, corresponding to minimum 10 3 eid 50 according to the manufacturer's instructions. results are expressed as viral load according to the standard curve. however, the viral load is only a measure of viral rna, not true viral replicates. each qrt-pcr experiment contained triplicate no-template controls, test samples and dilution series of nobilis ib ma5 vet cdna. the igg-specific antibody titres against ibv in serum were measured as previously described (juul-madsen et al. 2011b ) using the proflok ® ibv antibody test kit from synbiotics corporation (san diego, ca, usa; cat. no. 96-6506) according to the manufacturer's instructions. the absolute numbers of different t-cell subsets in peripheral blood were measured once a week using a no-lyse no-wash flow cytometric method. fifty microlitres of 25 times diluted heparinstabilised blood was mixed with 50 l antibody solution containing and 0.25 l anti-cd8␣-cy5 (clone 3-298) in facs buffer (0.2% bsa, 0.2% sodium azide and 0.05% normal horse serum in pbs). all monoclonal antibodies were obtained from southern biotech (birmingham, al, usa). the samples were incubated at room temperature for 20 min in a 4 ml tube in darkness, and immediately before acquisition 400 l facs-buffer and 25 l flow-count tm fluorospheres (beckman coulter ireland, mervue, galway, ireland; cat. no. 7547053) were added. b-cell numbers were only measured at week 9 pv1. the protocol was as described above but using 1 l anti-bu-1-rpe (clone av20) in facs buffer. bu-1 positive macrophage and monocyte subsets were avoided by fsc/ssc gating on small lymphocytes. all flow cytometric analyses were performed on a bd facscanto tm (bd biosciences, san jose, ca, usa) equipped with a 488 nm blue laser and a 633 nm red laser. using the facsdiva software each sample was acquired and recorded for 1 min at medium flow rate. single-stained compensation controls as well as negative fluorescence minus one (fmo) controls were included and titration of all antibodies was performed prior to the experiment in order to determine the optimal staining concentrations. the count of cells in the samples was calculated according to the manufacturer's instructions as: cells/l = (total number of cells counted × dilution factor × l fluorospheres added × assayed concentration of fluorospheres)/(total number of fluorospheres counted × total volume). license to conduct the animal experiment was obtained from the danish ministry of justice, animal experimentation inspectorate by helle r. juul-madsen. the experiment was conducted according to the ethical guidelines. all data, except for the viral load measured by qrt-pcr, were found to be normally distributed. to analyse the data from the mbl-ibv binding assay the values from no addition of mbl ligand were compared with the values from each of the specific ligands added using the analysis of variance principle. in cases of significant effect (p < 0.05) the given mbl ligand was considered to affect the mbl-ibv binding. the viral loads were, due to pooled samples (only 3 pools per group), only tested for subline differences. the viral loads were far from being normally distributed and so an 2 -test was used to test if the sublines had an effect on the viral load. the test was performed on data from days 1 to 7 pv1. for test of the subline effect the observation was distributed into three viral load categories: viral load = 0, 0 < viral load < 0.12 × 10 −5 , and viral load > 0.12 × 10 −5 . the 2 -test tested the null hypothesis: that the observations were distributed equally over the sublines for all categories. the model used for the ibv-specific igg antibody titre measured by elisa was: y ijk = + t i + w j + l k + twl ikj + e ijk , where = overall means, t i = fixed effect of treatment i, w j = fixed effect of week j pv1, l k = fixed effect of line k. the various interaction effects and y ijk and e ijk were expected to be normally distributed. to avoid weeks with little or no antibody titre, the antibody titre was only statistically analysed from week 2 to 9. the model used for the absolute counts of tcell subsets measured by flow cytometry was: y ijk = + t i + w j + l k + lw jk + tl ik + twl ikj + e ijk , where = overall means, t i = fixed effect of treatment i, w j = fixed effect of week j pv1, and l k = fixed effect of line k. the various interaction effects and y ijk and e ijk were expected to be normally distributed. the model used for the absolute counts of b-cells measured by flow cytometry was: y ij = + t i + l j + tl ij + e ij , where = overall means, t i = fixed effect of treatment i, l j = fixed effect of line j. the various interaction effects and y ij and e ij were expected to be normally distributed. the analysis of the variance was performed by the glm procedure of the sas software (sas institute inc. 2009). an elisa protocol was used for measuring the binding of mbl to ibv. the results (fig. 1) showed that purified mbl binds to ibvcoated plates in a dose-dependent manner (fig. 1a) . mbl in serum samples was also shown to bind to ibv ( fig. 1b and c) . this binding was inhibited by adding mbl ligands. thus, the addition of mannan and fos reduced the mbl-ibv binding significantly (p < 0.01) for l10h serum samples. addition of chitosan only showed a tendency for inhibition of mbl-ibv binding (p = 0.0513). for the mbl in l10l serum the binding was significantly inhibited by mannan and fos (p < 0.01), but not by chitosan. addition of edta to the serum samples inhibited the mbl-ibv binding significantly (p < 0.01) in both sublines, indicating that the binding is calcium dependent. on the other hand the negative control, galactose, had no influence on the mbl-ibv binding. chickens were tested for their mbl haplotype as described by kjaerup et al. (2013) and were determined through the taqman ® snp genotyping technique (data not shown). all l10l chickens were homozygous for the a1 haplotype. all l10h chickens were homozygous for the a3 haplotype, except for five chickens which were heterozygous a1/a3 and were found in groups 1 (n = 2), 3 (n = 2) and 4 (n = 1). ibv-specific qrt-pcr was used for monitoring the presence of ibv genomes in the opa swabs sampled from days 0 to 21 pv1 (fig. 2) . three swab samples from each subline were pooled based on treatment group and day. the results were statistically analysed regardless of treatments (table 1) , since three measurements per subline, treatment group and day were considered insufficient for statistics. in general, the l10l chickens had a higher viral load than the l10h chickens. the viral load in l10h peaked at day 2 pv1, whereas in l10l it peaked at days 2-3 pv1. the viral load in both (table 1) . l10h had a significantly larger number of chickens with low viral load (<0.12 × 10 −5 ) than l10l. contrary to this, l10l had a significantly larger number of chickens with high viral load (>0.12 × 10 −5 ) than l10h (p < 0.01). hence, the viral loads indicated that l10h chickens were less severely affected by the infection than l10l chickens. the ibv-specific igg antibody titres were measured using elisa (fig. 3) . a few chickens were non-responders and remained seronegative. these chickens were two l10h from treatment groups 1 (n = 1) and 4 (n = 1), and four l10l from treatment groups 1 (n = 1), 3 (n = 1) and 4 (n = 2). they were excluded from the statistical analysis of the ibv-specific igg titre. these chickens responded as the other chickens in all other parameters. therefore, the measurements for these chickens were maintained in the statistical analysis of all other parameters. when comparing the sublines, group 4 was the only group where l10h and l10l differed significantly from each other in the ibv-specific igg antibody titre (p < 0.01). in this group the antibody titres in l10h chickens were higher than the antibody titres in l10l chickens after the second vaccination (week 3) and for the rest of the experimental period. the antibody titres showed no difference between the l10l chickens in the four treatment groups. on the other hand, the antibody titre in l10h showed differences between the treatment groups at week 4 (group 1 / = groups 3 and 4; and group 4 / = groups 2 and 3), week 5 (group 4 / = groups 1, 2 and 3), week 6 (group 4 / = groups 1, 2 and 3) week 7 (group 4 / = group 1), week 8 (group 4 / = group 1) and week 9 pv1 (group 4 / = group 1), where p < 0.04. in summary, the ibv-specific igg antibody titres were the same for l10l chickens between different treatments, whereas the addition of an mbl ligand to the vaccine, especially fos, seemed to enhance the production of ibv-specific igg antibody in l10h chickens, but not in l10l chickens. an absolute count flow cytometric protocol using a no-lyse no-wash method, as described by seliger et al. (2012) was used for quantifying t-cell subsets in whole blood. the t-cell subsets were identified by fsc/ssc gating on small lymphocytes, followed by identification of cd3+ cells (t-cells) and finally by subdividing the cd3+ cells into total cd4+ cells (i.e. cd4+cd8␣+ and cd4+cd8␣−), cd4−cd8␣+ cells, and cd4−cd8␣− cells (fig. 4a and b). cd4+cd8␣+ and cd4+cd8␣− cells were combined as total cd4+ cells, due to individual differences in the counts of cd4+cd8␣+ cells between animals which have an heritable origin (hala et al. 1992) . the results are presented in figs. 5 and 6. comparing the counts between the two sublines (l10h and l10l) within each treatment group (fig. 5) , the numbers of total cd4+ cells showed no difference between l10h and l10l in groups 1 and 3. in group 2 the counts of total cd4+ cells in l10h were significantly higher than in l10l at week 6 pv1 (p = 0.03). a more pronounced difference between l10h and l10l was seen in group 4, where the counts of total cd4+ cells were significantly higher for l10h than l10l at weeks 2, 5, 6, 7, 8 and 9 pv1 (p ≤ 0.03). counts of cd4−cd8␣− cells for l10h were higher than l10l in groups 3 and 4 at week 1 pv1 (p < 0.05), whereas counts of cd4−cd8␣− cells were higher for l10h than l10l in group 2 at weeks 0, 1, and 2 pv1 (p ≤ 0.02). cd4−cd8␣+ cells only differed between l10h and l10l within groups in group 1 at week 5 (p = 0.02). briefly, the addition of fos to the vaccine increased the number of total cd4+ cells in l10h chickens compared to l10l chickens, and the l10h chickens showed a significant increased number of cd4−cd8␣− cells after the first vaccination when an mbl ligand was added to the vaccine compared to l10l chickens. the comparison of counts between treatment groups within each subline (l10h and l10l) is presented in fig. 6 . the counts of total cd4+ cells showed no difference between treatment groups neither for the l10h nor l10l sublines, except for the l10l subline at week 6 pv1 (group 3 / = group 4) where the l10l chickens in treatment group 3 showed a significantly higher count of total cd4+ cells than l10l chickens in treatment group 4 (p = 0.03). the numbers of cd4−cd8␣+ cells for the l10h sublines differed significantly between treatment groups at weeks 6-9 pv1 (group 1 / = group 3), where the l10h chickens in treatment group 3 had significantly lower numbers than the l10h chickens in treatment group 1 (p < 0.05). besides this, the numbers of cd4−cd8␣+ cells differed significantly for l10h between treatment groups at week 6 pv1 (group 2 / = group 4), where p = 0.01. the numbers of cd4−cd8␣+ cells for l10l only differed significantly between treatment groups at week 3 pv1 (group 1 / = groups 2 and 4), where p = 0.04. the number of cd4−cd8␣− cells differed significantly for the l10h sublines between the treatment groups at week 1 (group 1 / = groups 2 and 4), week 2 (groups 1 / = 2 and 5), week 3 (group 1 / = 2), week 5 (group 1 / = 3), and week 6 pv1 (group 1 / = 3), where p ≤ 0.04. the number of cd4−cd8␣− cells for l10h in treatment group 1 was lower than the other treatment groups at weeks 1, 2, 3, 5 and 6 pv1. besides this, the number of cd4−cd8␣− cells differed significantly for l10l between the treatment groups at week 1 (group 1 / = 3), week 2 (group 1 / = 4), and week 5 pv1 (group 1 / = 3) where p ≤ 0.05. the l10l chickens in treatment group 1 were lower in the number of cd4−cd8␣− cells than the other treatment groups at weeks 1, 2, and 5 pv1. briefly, the addition of an mbl ligand had no influence of total cd4+ cells within a l10h or l10l subline. however, the addition of chitosan to the vaccine decreased the number of cd4−cd8␣− cells in the l10h chickens at week 6 to 9 pv1. also, the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8␣−␥␦ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination. the b-cells were identified by fsc/ssc gating on small lymphocytes, followed by identification of bu-1 positive cells ( fig. 4a and c) . results are shown in table 2 . a significant difference between fig. 3 . ibv-specific igg antibody titres as measured by elisa. results are shown as mean values ± sem. the asterisks indicate statistically significant differences (p < 0.05) between groups or sublines. the double asterisks indicate statistically significant differences (p < 0.01) between groups or sublines. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). l10h and l10l chickens were observed in treatment group 1 where the absolute counts of b-cells were significantly lower for l10h chickens than for l10l chickens (p = 0.001). no significant differences were observed between l10h and l10l chickens in the other treatment groups. the main purpose of the present study was to investigate innate and adaptive immune responses following ibv vaccination when temporarily inhibiting mbl function by co-administering mbl ligands. the binding of pathogens by prrs, such as mbl, is an important step to initiate the early host response to infection. if mbl efficiently neutralises the pathogen via the complement membrane-attack complex, no adaptive immune response is needed. thus, poor adaptive memory is obtained and the purpose of the vaccine is unachieved. to our knowledge the current study is the first to show that mbl binds to ibv. binding was shown to occur through the crd of mbl since binding was inhibited by edta as well as mannan. previous studies have shown that human mbl binds to several viruses, such as hiv (saifuddin et al. 2000) and sars (ip et al. 2005) . these bindings have also been shown to occur through the crd of mbl. further, the current study shows that mbl binds to ibv in a dosedependent manner. besides this, it also provides evidence that fos is an mbl ligand with the potential to inhibit mbl-ibv binding. molecules (epstein et al. 1996; takahashi et al. 2011 ). in the serum samples from l10h chickens chitosan showed a tendency to inhibit the mbl-ibv binding (p < 0.0513) indicating that chitosan may also be an mbl ligand. chitosan is a highly basic polysaccharide obtained by deacetylation of chitin which is a linear polymer of n-acetyld-glucosamine (ravi kumar 1999) , which is known to be an mbl ligand (epstein et al. 1996) . chitosan is insoluble in water, which is why there is an uncertainty concerning the precise amount of chitosan added in the binding assay and to the vaccine given to the chickens. in a previous study both chitosan and its derivative n,n,ntrimethylated chitosan (tmc) were used as adjuvants in vaccines in raccoons (fry et al. 2012) . this study showed a higher number of responders to the vaccine when tmc was added instead of chitosan. tmc is water-soluble and have the same immunogenic and adhesive properties as chitosan (kotze et al. 1997) , which is why, with hindsight, that tmc instead of chitosan probably would have been a better choice as a possible mbl ligand added to the ibv vaccine to avoid the uncertainty about the concentrations used. following the first ibv vaccination the presence of viral genomes was observed in the opa of all the birds. however, the viral loads were lower for l10h chickens than l10l chickens (fig. 2) , indicating that l10h chickens were less severely affected by the infection than l10l chickens. previously, juulmadsen et al. (2011b) did not find any statistical difference in virus load between l10l and l10h chickens after vaccination. some of the incongruence between the current study and the vaccination part of the previous study could be explained by the larger animal groups in the current study, giving a more accurate outcome. besides, the estimation of viral load using qrt-pcr is more accurate than estimating the viral load by gel. the higher viral load in the l10l does, however, support the previous suggestions that mbl is associated with susceptibility to ibv infection (juul-madsen et al. 2007; juul-madsen et al. 2011b) . the ibv-specific igg antibody titres after the first vaccination did not differ between sublines (l10h and l10l) or treatment. after the second vaccination (week 3) of chickens receiving vaccine alone no boosting effect of the vaccine was found in the two sublines. on the other hand, the ibv-specific igg antibody titres after the second vaccination for l10h chickens receiving an mbl ligand together with the vaccine increased more than the antibody titre for l10h receiving vaccine alone (fig. 3) . this was most pronounced for the l10h chickens receiving vaccine and fos. this is also in contrast to the challenge study by juulmadsen et al. (2011b) , where the titre was lower for l10h chickens receiving mannan together with the vaccine compared with chickens receiving vaccine alone -we have no explanation for that. the antibody titres were the same for the l10l chickens between treatment groups. this, in combination with the results of the mbl-ibv binding assay, where the mbl-ibv binding was clearly inhibited by the addition of ligands in serum from l10h but not l10l, implies that the combination of high mbl serum concentrations and the addition of an mbl ligand have an impact on the development of ibv-specific igg antibodies. an increased antibody titre has earlier been reported in chickens vaccinated against eimeria (janardhana et al. 2009 ) and in mice vaccinated and infected with salmonella (benyacoub et al. 2008 ) when feeding a diet containing fos. the dramatic increase in the antibody titre after vaccination with fos may be beneficial in breeding, since the transfer of maternal antibodies to offspring is of importance in protecting the newly hatched chicks. this protection would be increased by an increase in the amount of antibodies in the dams, since there is a direct relationship between ibv-specific igg levels in the dam and those in her offspring (hamal et al. 2006) . flow cytometry-based methods for counting absolute numbers of peripheral blood cells have previously been described (burgess and davison 1999; seliger et al. 2012 ). in the current study flow cytometry was used to identify phenotypic differences between the chicken sublines vaccinated with or without the addition of mbl-ligand. no general differences were observed in the amount of total cd4+ cells for both sublines and between treatment groups. nor was any difference between the two sublines within each treatment group observed in groups 1, 2 and 3. however, the addition of fos to the vaccine gave a higher number of total cd4+ cells in the l10h chickens than in the l10l chickens (fig. 5) . despite of this, no difference in the amount of b-cells at week 9 pv1 (table 2) was observed in this group, indicating that the increase may have been caused by th1-cells instead of th2-cells. this is supported by a study by guo et al. (2008) which indicates that th1-cells are activated immediately after ibv infection. the response of cd4−cd8␣+ cells, mostly cytotoxic t-lymphocytes (ctl), is crucial for the elimination of the virus from local infection sites (collisson et al. 2000; guo et al. 2008; juul-madsen et al. 2011b ). an increased number of cd4−cd8␣+ cells were indeed observed in the current study both pv1 and pv2 which may support the suggestions by collisson et al. (2000) that ctls are important in the protection against ibv. however, this increase was repressed in l10h chickens treated with fig. 4a and b. the absolute counts of total cd4+ cells are shown in the left column, the absolute counts of cd4−cd8␣+ cells are shown in the middle column, and the absolute counts of cd4−cd8␣− cells are shown in the right column. the upper panels show the comparison of sublines in group 1, the second upper panels the comparison of sublines in group 2, the second lower panels the comparison of sublines in group 3, and the lower panels the comparison of sublines in group 4. results are shown as mean values ± sem. the asterisks indicate statistically significant differences (p < 0.05) between groups. the double asterisks indicate statistically significant differences (p < 0.01) between groups. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). vaccine and an mbl ligand at weeks 6-9 pv1 (fig. 6) . as this study does not contain a mock vaccine group, it cannot be ruled out that the increase is age-dependent. involvement of other receptors specific for the used mbl ligands may have influenced some of the differences observed in the current study. however, mbl has an influence as the difference was more pronounced for the l10h chickens than l10l chickens for both the ibv-specific igg antibody titres and the numbers of cd4−cd8␣+ and cd4−cd8␣− cells. the chicken cd3+cd4−cd8␣− cells in circulation are mostly ␥␦ t-cells which may contribute with up to 60% of the circulating tcells in a healthy adult chicken (dalgaard et al. 2010 ) indicating that they may play an important role in the chicken immune system. for the three groups receiving vaccine and an mbl ligand, the ␥␦ t-cell response was more pronounced in the l10h chickens than in the l10l chickens pv1 (fig. 5) . there was also a tendency (p = 0.0517) for this in the group receiving vaccine alone. this increase was also observed in the study by juulmadsen et al. (2011b) . this study showed a higher increase in the percentage of ␥␦ t-cells after ibv vaccination in the group receiving vaccine and mannan. in the current study no difference was observed in the ␥␦ t-cell response pv2 between l10h and l10l chickens within treatment groups (fig. 5) . these inconsistencies may be explained by the use of live virus in the previous study versus the use of attenuated live virus in the current study as already suggested. however, at least a tendency for a higher number of ␥␦ t-cells was observed in the current study until week 6 pv1 for l10h chickens receiving vaccine and mbl ligand compared to l10h chickens receiving vaccine alone (fig. 6) . the same was observed for l10l chickens receiving vaccine and chitosan or fos until week 3 pv1 and at week 5 the asterisks indicate statistically significant differences (p < 0.05) between groups. the double asterisks indicate statistically significant differences (p < 0.01) between groups. h or l indicates the sublines with high or low mbl serum concentration, respectively; 1, 2, 3 or 4 indicates the treatment groups "vaccine" (1), "vaccine + mannan" (2), "vaccine + chitosan" (3) or "vaccine + fos" (4). pv1 for l10l treatment groups receiving vaccine and mbl ligand (fig. 6) . the function of chicken ␥␦ t-cells is still under debate, but studies have indicated that ␥␦ t-cells respond to pathogens and provide a protective immune response after immunisation with both live attenuated and non-attenuated salmonella strains (berndt et al. 2006) . the results of the current study may support these indications since our results showed that cd4−cd8␣−␥␦ t-cells are increased after ibv vaccination when mbl is inhibited. a difference in the amount of b-cells at week 9 pv1 (table 2) was observed between the l10h and l10l chickens receiving only vaccine, but no difference was observed for ibv-specific antibody titres in this group. in the treatment group receiving vaccine and fos a major difference between l10h and l10l chickens was observed for the ibv-specific antibody titres, even though only a tendency (p = 0.078) for a difference in the amount of b-cells at week 9 pv1 was observed between these chickens. these findings support the suggestions made by guttormsen et al. (2009) that an increased igg response in mbl-deficient mice is not only attributed to an increase in the number of b-cells. the increase in igg may also have been caused by ig class switching, as previously observed in mice for dietary fos by nakamura et al. (2004) . previous challenge studies have shown an up regulation of igg in species with low amount of mbl in both mice (guttormsen et al. 2009 ) and chickens (juul-madsen et al. 2007 ). likewise, ruseva et al. (2009) argue that genetic environment influences the modifying effect of mbl. we cannot conclude whether the different amounts of bcells at week 9 pv1 observed in chickens receiving vaccine alone in the current study was caused by vaccine or age. if the difference was caused by the vaccine, the mbl ligands eliminate this difference. in conclusion, the ibv-specific igg antibody titres were the same for l10l chickens between different treatments, whereas the addition of an mbl ligand to the vaccine, especially fos, seemed to enhance the production of ibv-specific igg antibody in l10h chickens, but not in l10l chickens. the addition of fos to the vaccine also increased the number of total cd4+ cells in l10h chickens, in combination with an unchanged amount of b-cells at week 9 pv1 compared to l10l chickens. the l10h chickens as well as the l10l chickens also showed an increased number of cd4−cd8␣−␥␦ t-cells when an mbl ligand was added to the vaccine, most pronouncedly after the first vaccination, suggesting that cd4−cd8␣−␥␦ t-cells may also play a role in the immune response against ibv. these results indicate, as previously suggested by juulmadsen et al. (2011b) that mbl is involved in the adaptive immune response to ibv vaccination. mbl inhibition may therefore be beneficial to achieve high antibody response during vaccinations. further studies are needed to elucidate whether the addition of mbl ligands to ibv vaccine gives a better immune protection against ibv infection. 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mannan-binding lectin (mbl) in two chicken breeds and the correlation with experimental pasteurella multocida infection a rapid high-precision flow cytometry based technique for total white blood cell counting in chickens detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (mbl) gene by polymerase chain reaction with sequence-specific primers molecular basis of opsonic defect in immunodeficient children mannose-binding lectin and the balance between immune protection and complication dietary sugars inhibit biologic functions of the pattern recognition molecule, mannose-binding lectin a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms the authors wish to thank l.r. dal and h. svenstrup for technical assistance and k.v. østergaard for critical reading of the manuscript. this work was supported by the project "development of genetic selection technology for polyvalent resistance to infectious diseases" (poly-reid) (grant number 10-093534) granted by danish council for strategic research, danish poultry council, the hatchery hellevad, and cobb-vantress, and grants from the graduate school of science and technology at aarhus university and european community's seventh framework programme the authors declare that they have no conflicts of interest. key: cord-324324-8ybfiz8f authors: decaro, nicola; lorusso, alessio title: novel human coronavirus (sars-cov-2): a lesson from animal coronaviruses date: 2020-04-14 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108693 sha: doc_id: 324324 cord_uid: 8ybfiz8f the recent pandemic caused by the novel human coronavirus, referrred to as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), not only is having a great impact on the health care systems and economies in all continents but it is also causing radical changes of common habits and life styles. the novel coronavirus (cov) recognises, with high probability, a zoonotic origin but the role of animals in the sars-cov-2 epidemiology is still largely unknown. however, covs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face cov infections in animals, which could represent a model for sars-cov-2 infection in humans. in the present paper, we provide an up-to-date review of the literature currently available on animal covs, focusing on the molecular mechanisms that are responsible for the emergence of novel cov strains with different antigenic, biologic and/or pathogenetic features. a full comprehension of the mechanisms driving the evolution of animal covs will help better understand the emergence, spreading, and evolution of sars-cov-2. eighteen years after the emergence of severe acute respiratory syndrome (sars) in china and 8 years after the emergence of middle east respiratory syndrome (mers) in saudi arabia, a novel coronavirus (cov) epidemic, recently classified as pandemic by the who, is threatening the human population worldwide (zhou et al., 2020) . the disease, now referred to as coronavirus disease 2019 , is caused by a novel human cov, which was initially denominated 2019 novel coronavirus (2019-ncov) and later renamed as sars coronavirus 2 (sars-cov-2) the by coronavirus study group of the international committee on taxonomy of viruses (gorbalenya et al., 2020) . covid-19 emerged in december 2019 in wuhan city, hubei province, china, in humans exposed to wildlife at the huanan seafood wholesale market, which is the largest seafood market in central china, and where different species of farm and wild animals are commonly sold (lorusso et al., 2020) . the epidemic has then expanded not only to neighbouring asian countries, but also to other continents (https://www.who.int/ docs/default-source/coronaviruse/situation-reports/20200415-sitrep-86-covid-19.pdf?sfvrsn=c615ea20_2). . a list of human covs is showed in table 1 . historically, only two human covs (hcovs) had been known before the sars emergence, namely hcov-229e, an alphacoronavirus originated in bats and transmitted to humans through alpacas, and hcov-oc43, a betacoronavirus which had passed from rodents to humans through cattle (corman et al., 2015 (corman et al., , 2018 . after 2002-2003 sars epidemic, the renovated interest in hcovs allowed the discovery of two additional viruses, the alphacoronavirus hcov-nl63 and the betacoronavirus hcov-hku1, derived from bats and rodents, respectively (tao et al., 2017) . all these four viruses are usually responsible for mild respiratory symptoms in immunocompetent patients. sars-cov and mers-cov are two unrelated betacoronaviruses originated in bats and transmitted to humans by wild carnivores and dromedary camels, respectively. in contrast to other hcovs, these two viruses displayed an increased virulence, causing severe pneumonia and even the death of affected people, with mortality rates of about 10 % and 30 %, respectively (guarner, 2020) . the occurrence of three highly pathogenic covs with a zoonotic origin in less than two decades, highlights the role of animals in generating covs with increased virulence that can adapt to humans, causing epidemics (and eventually pandemics) with high impact on human health. indeed, cov infections of veterinary interest have been known since almost a century (cavanagh, 2007; pedersen, 2014; decaro et al., 2020) , so that animal covs are paradigmatic of how this large family of viruses evolves, generating strains with different biological properties. in addition, the efforts done in veterinary medicine https://doi.org/10.1016/j.vetmic.2020.108693 received 10 march 2020; received in revised form 10 april 2020; accepted 10 april 2020 evolve rapidly, changing their antigenic profile, tissue tropism or host range by means of two distinct mechanisms. the viral replicase (an rna dependent-rna polymerase) does not possess a good proof reading activity, therefore the incorporation of wrong nucleotides at each replication cycle and the consequent accumulation of mutations in the viral genome lead to a progressive differentiation of the viral progeny from the parental strain. this mechanism, which is well known for influenza viruses being responsible for the so called antigenic drift, may cause the progressive adaptation of the viral surface proteins to the cell receptors of new animal species, increasing the viral fitness. in addition, the particular replicating machinery of covs facilitates recombination events due to the presence of consensus sequences upstream each gene. therefore, in the case of coinfection by more than one cov strain, the rna polymerase can jump from the rna of a strain to that of the other one, synthetizing a hybrid rna containing sequences from both viruses. recombination can occur not only with genomic sequences of other covs (homologous recombination), but also with rnas of different viruses and other organisms (heterologous recombination) (luytjes et al., 1988; banner and lai, 1991; lai, 1996; zeng et al., 2008; huang et al., 2016) . recombination is an alternative mechanism that let covs acquire novel biological properties in terms of virulence, host range and tissue tropism, so that cov strains, which are non-pathogenic or lowpathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species spreading in the new host with exceptional rapidity (banner and lai, 1991) . the occurrence of three human cov epidemics in less than 20 years, along with the emergence of less pathogenic human covs, arises some questions on how these viruses that have their reservoirs in bats and rodents may overcome the species barriers jumping to humans. the animal-to-human transmission of viruses has been already occurred in the past, but it seems that its frequency has been increased in the last decades, involving in a short time span not only covs, but also a plethora of genetically and biologically different viruses with zoonotic potential, such as ebola virus, influenza viruses, flaviviruses, hendra and nipah viruses (mcmahon et al., 2018) . climate changes that are intensifying in this first quarter of the 21st century are favouring the spread of vector-borne diseases through increasing the proliferation of vectors and predisposing to their occupation of new ecological niches. the emergence in temperate climate areas such as europe of vectorborne diseases caused by viruses considered exotic until few years ago (west nile virus, usutu virus, chikungunya virus) accounts for a progressive geographic expansion of tropical diseases thanks to the ongoing phenomenon of tropicalisation (mcmahon et al., 2018) . deforestation and urbanization are other major factors that facilitate the spill-over of zoonotic agents to humans by reducing the habitat of wildlife and increasing the chances of contacts between wild animals (like bats, rodents and birds) and human beings (beena and saikumar, 2019; lorusso et al., 2020) . this could be the case of ebola virus, hendra and nipah viruses, hantavirus and coronavirus infections. in addition, the close contact between human beings and different animal species sold at the wet markets of east asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like covs. it is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic h5n1 avian influenza and sars) have emerged in the same chinese province of guangdong where the contact between humans and animals is closer (lorusso et al., 2020) . table 2 reports the most important avian cov species recognised so far and their associated diseases. the number of avian species in which covs have been detected in the last years is humongous. since the emergence of sars-cov in 2002, there has been increased interest in table 2 main coronaviruses in domestic and domesticated avian species. schalk and hawn (1931); beach and schalm (1936) ; beaudette and hudson (1937) turkey ( respiratory and kidney disease spackman et al. (1983) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 covs in other species, including birds. prior to that time, our knowledge of covs in avian species was limited largely to three birds of the order galliformes, i.e., domestic fowl (gallus gallus), turkeys (genus meleagris) and pheasants (phasianidae), with their infectious bronchitis virus (ibv), turkey coronavirus (tcov), and pheasant coronavirus (phcov), respectively. these three viruses were considered for a long-time different species for several reasons such as the diverse pathotype (enterotropic or respirotropic), host range and genetic relatedness of the s protein (cavanagh, 2007) . this scenario radically changed after the discovery of several novel covs with high genetic diversity from different avian species and the novel rules for species designation of the coronavirus study group (csg https://talk.ictvonline.org/ictv-reports/ ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/ 222/coronaviridae). all these viruses as well as analogous ibv-like covs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (cavanagh et al., 2002; circella et al., 2007; domanska-blicharz et al., 2014; torres et al., 2013; hughes et al., 2009; liu et al., 2005; wille et al., 2016; jordan et al., 2015; bande et al., 2016; suryaman et al., 2019) have been assigned to the same viral species known as avian coronavirus (acov) within the subgenus igacovirus of genus gammacoronavirus. ibv and ibv-like strains are commonly detected in both gallinaceous and non-gallinaceous birds, also asymptomatically (cavanagh, 2005) . this might suggest that these species would act as wild reservoirs, spreading ibv strains over the world (de wit et al., 2011) . as for the huge economic impact of the disease it causes, ibv is one of the most studied covs over the last decades. ibv causes the infectious bronchitis (ib), a term adopted in 1931 for describing the main clinical characteristics of a transmissible respiratory disease of poultry detected for the first time in north dakota (usa). ib has been now diagnosed worldwide and is one of the most important viral diseases of poultry characterised by respiratory signs, but it can also affect the kidneys and reproductive tract following viremia with a severity that differs depending on the involved viral strain (cavanagh and gelb, 2008) . the disease also affects wild and ornamental birds chen et al., 2013) . ib control has been hampered by the intricate ibv evolution, which has been entailed, over the years, by the emergence of many different antigenic or genotypic types, commonly referred to as variants, with divergent molecular, biological, and antigenic properties. being a cov, ibv has, indeed, a considerable ability to change both by mutation and by homologous recombination events, which may cause, along with replicase stuttering or slippage, also insertions and deletions in the genome (cavanagh and gelb, 2008) . if these mechanisms involve the hypervariable region s1, they frequently result in the emergence of new ibv variants. although many new variants are not successful, a few may emerge, spread, causing devastating disease either worldwide or in limited geographic areas. currently 32 lineages have been recognized, categorized into six genotypes (gi to gvi) (valastro et al., 2016) . through their s protein, ibv and ibv-like viruses recognise as cellular host receptor the α2,3-linked sialic acid glycan, widely distributed in the respiratory tract and in several other host tissues, factor which may explain the tropism also for several organs of the infected host (winter et al., 2006 (winter et al., , 2008 shahwan et al., 2013; ambepitiya wickramasinghe et al., 2011) . extensive use of vaccines has greatly contributed to the high variability of ibv strains thorough recombination between vaccine and field viruses and viral selection pressure resulting from vaccination and presence of partially immune birds (gandon and day, 2008; gandon et al., 2001; bande et al., 2017) . important ibv-like strains are tcov, responsible for enteritis in turkey (also known as bluecomb disease), guinea fowl coronavirus (gfcov) and quail coronavirus (qcov) responsible for fulminating enteric disease in guinea fowl and quail, respectively cavanagh, 2005; liais et al., 2014) . tcov, gfcov and qcov are evolutionarily distant from acov based on the s protein. while ibv is a primarily respiratory pathogen, tcov causes gastrointestinal disease (liais et al., 2014; guy, 2008) . enterotropism has also been observed for some ibv serotypes; however, all ibv strains infect primarily the respiratory tract, resulting in mild to severe inflammation of the nasal and tracheal epithelia (cavanagh, 2005 (cavanagh, , 2007 . the s1 domain of the s protein is highly variable, with the amino acid sequences of ibv and tcov from the usa sharing < 25 % sequence identity. phylogenetic analysis of the s1 gene shows, indeed, grouping of ibv and ibv-like viruses on the one hand and tcov-us, gfcov and qcov on the other hand (ambepitiya wickramasinghe et al., 2015a) . accordingly, the emergence of covs in turkeys in the usa was proposed to have resulted from recombination events involving ibvs and an as-yet-unidentified cov donating a novel s gene. this switch contributed largely to determine the in vivo tissue tropism of tcov and related viruses. intriguingly, the s protein of these covs requires nonsialylated type 2 poly-lacnac structures on n-glycan cores for binding. this is in marked contrast to the α2,3-linked sialic acid glycan binding of ibv and ibv-like viruses (ambepitiya wickramasinghe et al., 2015b) . the s1 subdomain of a tcov isolate from france in 2008 (tcov-fr) had only 42 % sequence identity to that of the tcov-us strain (maurel et al., 2011) . this diversity was biologically evident by the prominent tropism for the epithelium of the bursa of fabricius and only mild tropism for the small intestine of turkey. tcov-fr s1 protein did not show, indeed, affinity for nonsialylated type 2 poly-lacnac (ambepitiya wickramasinghe et al., 2015a) . this genetic diversity between tcovs is in accordance with several recombination events involving ibvs on different continents with several unknown covs. on the one hand, the s genes of gfcov/fr/2011 (isolated in france in 2011) and tcov-us share significant genetic relationships, and thus these viruses must have acquired their s gene from a common ancestor. on the other hand, gfcov/fr/2011 and fr tcov have a very similar genetic background in other genes. two recombination events may be responsible for the genesis of tcov-us and fr tcov. a first event occurred between an ibv eu recipient strain and an unknown acov donor, resulting in a virus with a new s gene, whose evolution would have resulted in fr tcov and gfcov/fr/2011. a second recombination event involving a us ibv recipient and gfcov/fr/2011 would have generated us tcov viruses, which share a stronger s gene similarity with gfcov/fr/2011 than with fr tcov . additional covs distinct from acovs and mainly circulating in ducks (duck coronavirus, dcov), pigeons (pigeon coronavirus, pcov), or geese (goose coronavirus, gcov) have been identified (cheng et al., 2013; jonassen et al., 2005; muradrasoli et al., 2010; kim and oem, 2014; zhuang et al., 2015; papineau et al., 2019) . although their genome seems to fulfill the official ictv criteria required to distinguish a new species within the gammacoronavirus genus, ictv approval is still pending. historically, covs of birds were all included in the gammacoronavirus genus and, in turn, all covs belonging to this genus were identified only in birds. however, this suggestion was rebutted by the evidence of a cov belonging to the gammacoronavirus genus in a beluga whale first discovered in 2008 (viral species beluga whale coronavirus sw1 species, subgenus cegacovirus, genus gammacoronavirus) (mihindukulasuriya et al., 2008) , and of three novel covs, bucov hku1, thcov hku12, and mucov hku13 in birds of the order passeriformes, namely bulbuls (pycnonotus jocosus), thrushes (turdidae) and munias (lonchura punctulate), respectively, which did not cluster phylogenetically with extant covs identified in birds. these latter three viruses were distinct from known covs forming a unique cluster in the phylogenetic tree, which was the basis for generation of the deltacoronavirus genus (woo et al., 2009) . importantly, additional novel viruses belonging to this novel genus were detected in wild birds chu et al., 2011; durães-carvalho et al., 2015; torres et al., 2016) . these viruses cluster with previously unclassified covs detected in various asian carnivores, i.e., the asian leopard cat (prionailurus bengalensis) and chines ferret badger (nyctereutes procyonoides) (dong n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 et al., 2007) . covs belonging to the betacoronavirus genus, which are strictly related to mouse hepatitis virus (mhv), were also described in wild birds, including parrots, in brazil (durães-carvalho et al., 2015) . interestingly, this was not the first detection of viruses belonging to the betacoronavirus genus in birds. often overlooked is the discovery over 38 years ago of a cov from the manx shearwater (puffinus puffinus), a bird that visits the shores of britain in summer (nuttall and harrap, 1982; cavanagh et al., 2007) . this virus was also related to mhv. however, at that time, considering the unusual finding and that the virus was isolated by passage of shearwater material in the brains of mice, it was speculated that the detected virus was an mhv strain already present in the mice before inoculation (cavanagh, 2007) . bats are an ancient and heterogeneous group of ecologically important mammals, representing nearly a quarter of all mammalian diversity on earth. they belong to the order chiroptera and further classified in two suborders yinpterochiroptera and yangochiroptera. the first includes the non-echolocating pteropodidae family (megabats) and five echolocating rhinolophoidea microbat superfamilies. yangochiroptera contain thirteen echolocating microbat families (tsagkogeorga et al., 2013) . bats are thought to host a large plethora of viruses. these include, amongst the others, lyssaviruses, filoviruses, henipaviruses, and reoviruses (calisher et al., 2006) . before sars-cov epidemic, bats were not known to host covs. indeed, the first evidence of a bat cov was published in 2005 . after the sars epidemic, there was a boost in interest regarding searching for novel covs in various animals, including bats. to date, over 200 novel covs have been identified in bats and approximately 35 % of the bat virome sequenced to date is composed of covs (chen et al., 2014) . this data has been made available following the massive surveillance, coupled with the advent of next-generation sequencing (ngs) technology, which has been performed in wild animals banerjee et al., 2019) . just a small portion of these covs have been officially recognised by the ictv; many others are still pending for official designation. cov species detected in bats and officially recognised by the ictv are listed in table 3 and the following chapter reasonably discusses only officially recognized bat cov species. bats can carry and transmit covs into local bat populations via migration even though little is known about the migratory patterns of these animals. closely related covs can be detected in the same bat species living at locations separated by thousands of miles (drexler et al., 2010) and different cov species or genera can be found in different bat species living at the same roosting sites. however, some covs have been shown to be species-specific. accordingly, regional patterns of bat cov outbreaks at species level can be deduced from the population distribution of their respective bat hosts. although bats seem to develop clinical diseases induced by several viruses and bacteria (mühldorfer et al., 2011) , generally covs do not cause apparently overt disease in these mammals, also experimentally. this phenomenon seems to be related with peculiar characteristics of their immune system (ahn et al., 2019; brook et al., 2020) . based upon genomic data available so far, it is widely accepted that while birds represent the reservoir for covs belonging to genera gammacoronavirus and deltacoronavirus, bats are the natural reservoir for alpha-and betacoronaviruses. however, only betacoronaviruses of subgenera sarbecovirus, merbecovirus, nobecovirus and hibecovirus have been detected in bats so far. given that several betacoronaviruses from the subgenus embecovirus have been discovered in rodents, it was speculated that rodent covs may be the ancestors of currently circulating viruses belonging to this subgenus . covs have been detected at high frequency in bats in all continents, with alphacoronaviruses being more widespread than betacoronaviruses . subgenus colacovirus (genus alphacoronavirus) officially comprises the viral species bat coronavirus cdphe15, so far composed by two bat covs strains named cdphe15/usa/2006 and myotis lucifugus cov (myl-cov), which share a 98.2 % nucleotide identity across the whole genome. both strains have been detected in myotis lucifugus bats (vespertilionidae) also known as the northern american little brown bats. the former was detected in 2006 in colorado (genbank acc. no. kf430219), while the latter was reported in 2010 in canada. this virus was identified in the intestines and lungs and associated with minimal pathology or inflammation (subudhi et al., 2017) . subgenus decacovirus (genus alphacoronavirus) comprises the species rhinolophus ferrumequinum alphacoronavirus hub-2013 composed so far by btms-al-phacov/gs2013 and btrf-alphacov/hub2013 strains discovered in china in myotis spp. and rhinolophus ferrumequinum bats, respectively. these two viruses share very high sequence identities (higher than 98 %), which dramatically decrease in the s genes (only 85% nucleotide identity) (wu et al., 2016) . woo et al., 2006) shares an 86 % sequence identity with severe acute diarrhoea syndrome-coronavirus (sads-cov) of pigs . these two viruses are now included in the same viral species rhinolophus bat coronavirus hku2 (subgenus rhinacovirus, genus alphacoronavirus). viral strains btkynl63-9a, btkynl63-9b, btkynl63-15 and btkynl63-9a, identified in 2010 in triaenops afer bats from kenya, form the viral species nl63-related bat coronavirus strain btkynl63-9b that is part of the subgenus setracovirus (genus alphacoronavirus) along with human coronavirus nl63 (tao et al., 2017) . in this regard, a bat origin has been strongly suggested for two of the less-pathogenic hcovs causing mild respiratory symptoms in immunocompetent people, namely hcov-229e and hcov-nl63, both belonging to the alphacoronavirus genus. whereas hcov-229e (subgenus duvinacovirus) recognises as direct ancestor an alphacoronavirus from alpacas, which in turn derives from 229e-related covs identified in hipposiderid bats (corman et al., 2015) , hcov-nl63 is likely a recombinant virus originating from the distantly related 229e-related covs associated with hipposiderid bats and covs associated with triaenops afer bats (tao et al., 2017) (table 1 ). the s protein of hcov-nl63 is more closely related to that of 229e-related covs, whereas the rest of the genome with covs included in the nl63-related bat coronavirus strain btkynl63-9b species (tao et al., 2017) . different from the bovine coronavirus (bcov)-like viruses that cause enteric disease, in 2007 a novel alpaca cov was associated to respiratory disease in california, usa. full-length genome analysis showed that this respiratory alpaca cov was closely related to the alphacoronavirus hcov-229e (subgenus duvinacovirus) (crossley et al., 2012) . more recently, close relatives of hcov-229e were detected in african hipposiderid bats. interestingly, both bat and alpaca viruses displayed an intact accessory gene orf8 located at the genomic 3' end, while hcov-229e retained only a conserved trs preceding remnants of this orf, suggesting its loss after acquisition of a 229e-related cov by humans. therefore, hcov-229 is likely a descendant of the alpaca alphacoronavirus (corman et al., 2015) . strains forming the viral species bat hp-betacoronavirus zhejiang2013 (subgenus hibecovirus, genus betacoronavirus) were discovered in hipposideros pratti bats from china in 2013 (wu et al., 2016) . strain ro-batcov gccdc1 356 was identified from stools of rousettus leschenaultii, a species of fruit bats (pteropodidae) of southern asia, which were collected in yunnan province, china, in 2014 (huang et al., 2016) . ro-batcov gccdc1 356 shows a small intact orf of 276 nucleotides embedded between the n and ns7a genes. this orf has no homology to any known coronavirus, and the encoded protein exhibited 54.9 % amino acid identity with the p10 protein encoded by the first orf of segment s1 of bat fusogenic orthoreoviruses (genus orthoreovirus, species nelson bay orthoreovirus, also known as pteropine orthoreovirus). these viruses are double-stranded segmented rna viruses, belonging to the family reoviridae, and are able to cause severe pneumonia in humans (chua et al., 2007; lorusso et al., 2015) . ro-batcov gccdc1 356 is included in the viral species rousettus bat coronavirus gccdc1 within the subgenus nobecovirus, genus betacoronavirus. rousettus bat coronavirus hku9, belonging to subgenus nobecovirus, was also identified in rousettus leschenaultii and in other bat species (mendenhall et al., 2017) . this virus was first detected in 2007 in guangdong province in china (woo et al., 2007) . subsequent studies suggested that the virus was widely distributed and is circulating in different bat species (ge et al., 2012) . covs from the bthku9-like cluster were also detected in hipposidereos commersoni and rousettus aegyptiacus bats in kenya (tong et al., 2009) . being a fruit bat, rousettus leschenaultii has a wider flying range than most of the insectivorous bats in china, thus it may carry viruses over long distances. a comparison of the reported hku9-cov sequences showed a high genetic diversity within this viral species (luo et al., 2018a, b; lau et al., 2010; ge et al., 2012) . when mers-cov was first isolated in the middle east in 2012 and its genome sequenced, it was found that it was most closely related to ty-batcov hku4 discovered in tylonycteris pachypus and pi-batcov hku5 discovered in pipistrellus abramus, which were the only known members of subgenus merbecovirus at that time. these two viruses are now the prototype strains of tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 viral species, respectively, within subgenus merbecovirus, genus betacoronavirus. although mers related covs (mers-rcovs) were lately discovered, mers-cov was much closer in the s1 region to hku4-cov than to mers-rcov or hku5-cov. indeed, dipeptidyl peptidase 4 (dpp4), the receptor for mers-cov, is also the receptor for hku4, but neither for hku5 nor for early discovered mers-rcovs. however, hku4 prefers bat dpp4 over human dpp4, whereas mers-cov shows the opposite trend . so far, hku4-covs are only carried by tylonycteris spp. bats (t. pachypus and t. robustula) and are relatively conserved; hku5-covs are found in different pipistrellus spp. bats, including p. abramus, p. pipistrellus and p. minus . due to the current sars-cov-2 pandemic, attention should be given to the viral species severe acute respiratory syndrome-related coronavirus (sars-rcov,subgenus sarbecovirus, genus betacoronavirus) and middle east respiratory syndrome-related coronavirus (mers-rcov, subgenus merbecovirus, genus betacoronavirus), which enclose sars-cov and mers-cov, the first two highly pathogenic covs that were discovered in humans. in 2002, at the beginning of the sars epidemic, almost all early human index patients had animal exposure in a market place, in guangdong province, before developing disease. after sars-cov was identified, its rna and/or specific antibodies were found in masked palm civets (paguma larvata) and animal handlers in a market place. however, later investigations of farmed and wild-caught civets revealed that sars-cov strains found in market civets were transmitted to them by other wild animals (tu et al., 2004; kan et al., 2005) . subsequently, novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong lau et al., 2005) . these sars-rcovs showed genome sequence identity of 88-90 % among themselves and 87-92 % identity to human or civet sars-cov isolates. sars-rcovs were detected in rhinolophus spp. bats of other regions of china (tang et al., 2006; woo et al., 2006; yuan et al., 2010; ge et al., 2013) . sars-rcovs with higher genetic diversity with respect to chinese strains were also detected in rhinolophid bats from slovenia, bulgaria and italy in europe (drexler et al., 2010; rihtaric et al., 2010; balboni et al., 2011) . covs related to sars-rcov were also detected in hipposideros spp. and chaerophon spp. bats from ghana, kenya and nigeria (hu et al., 2015) . these evidences suggested that bats may be the natural hosts for sars-cov and that wild carnivores were only intermediate hosts. although these sars-rcovs showed high sequence identity to sars-cov, they were demonstrated to be unable to bind to the human cell angiotensin converting enzyme ii (ace2) receptor, the receptor of sars-cov, as a consequence of deletions in their s protein (ren et al., 2008) . besides, the theory of bat origin of sars-cov lacked a powerful support due to the failure of direct isolation of this virus from bats. thus, considering that no direct progenitor of sars-cov was found in bats and that rna recombination is the fuel for cov evolution, it has been proposed that sars-cov emerged through recombination of bat sars-rcovs. this hypothesis was made after the evidence of a single bat cave in yunnan, china, with very high covs diversity and considering that, within the identified covs, all genetic elements needed to form sars-cov have been identified in that single cave (ge et al., 2013) . recombination analysis also strongly supported the hypothesis that the civet sars-cov strain sz3 originated following a recombination event of two existing bat strains, wiv16 and rf4092 (hu et al., 2017) . moreover, wiv1, the closest relative to sars-cov that has been found in bats so far (more than 95 % nucleotide identity, higher than that of any other bat sars-rcovs (76-92 %)), likely arose through recombination of two other prevalent bat sars-rcov strains. the most frequent recombination breakpoints were within the s gene and upstream of orf8, which encodes an accessory protein. these genes were also involved in the crucial adaptation pathways of sars-cov from bats to wild carnivores, from wild carnivores to humans, and from human to human (cui et al., 2019) . wiv1 has been shown to have the capacity to bind to the human, civet and bat cell ace2 receptor (ge et al., 2013) . the isolation in cellculture of a highly related sars-cov strain, coupled with the evidence of a functional s protein capable of using the same ace2 receptor, provided robust and conclusive evidence for the bat origin of sars-cov. an additional sars-rcov strain has been shown, by reverse genetics studies, to have the capacity to bind to the human ace2 receptor (menachery et al., 2015) . quite the opposite, a direct bat cov highly related to mers-cov of humans was never detected. indeed, the genome sequences of mers-cov in human and dromedaries possess only around 65-80 % nucleotide identities to those of the other members of subgenus merbecovirus from different bats. human mers-covs were instead almost identical to mers-covs identified in dromedary camels (camelus dromedaries). lately, genomic sequence analyses indicated that covs now belonging to the mers-rcov species were found in several bat species from two bat families, vespertilionidae and nycteridae (lelli et al., 2013; de benedictis et al., 2014; corman et al., 2014a, b; anthony et al., 2017; moreno et al., 2017; wong et al., 2019) . however, none of these mers-rcovs is a direct progenitor of mers-cov, as their s proteins differ substantially from that of the human virus. the closest relative to mers-cov of humans and dromedary camels is mers-rcov strain neoromicia/5038 isolated from neoromicia capensis bats in south africa (geldenhuys et al., 2018, table 1 ). a short sequence (around 200 nucleotides) of viral rna identical to that of mers-cov was also detected in a taphozous perforates bat in saudi arabia (memish et al., 2013) . overall, although it is widely accepted that mers-cov ancestor is in bats, further studies are warranted in order to discover the precise mechanisms of its emergence in dromedary camels and humans. it was suggested that mers-cov ancestors had been circulating in bats for very long time. mers-cov has evolved to adapt to use human receptor and the dpp4-recognising bat coronaviruses like hku4 may follow up, thereby posing a serious risk to human health. recent mers-rcovs were shown to have the capacity to bind to the dpp4 as entry cell receptor as they acquired the s1 through recombination with hku4-like viruses (luo et al., 2018a, b) . as for the recent and threatening covid-19 outbreak in humans, we certainly know that sars-cov-2 belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from wild carnivores and horseshoe bats (genus rhinolophus) (gorbalenya et al., 2020; zhou et al., 2020; wu et al., 2020) . epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china (lorusso et al., 2020) . sars-cov-2 has been assigned to an existing species of hundreds of known viruses largely isolated from bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the 2002-2003 outbreak have been confirmed to cause sars in humans (gorbalenya et al., 2020) . importantly, it has also been confirmed that sars-cov-2 uses the ace2 receptor through the receptor binding domain (rbd) of the s protein (hoffmann et al., 2020; zhou et al., 2020) . likely, also sars-cov-2 has a bat origin. according to genome sequences available so far, the most closely related virus (96.2 % of nucleotide sequence identity) to sars-cov-2 is strain batcovratg13 identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al., 2020) . the receptor-binding spike protein of sars-cov-2 is highly divergent from other covs with less than 75 % nucleotide sequence identity to all previously described sars-rcovs, except for a 93.1 % nucleotide identity to batcovratg13 (zhou et al., 2020) . although sars-cov-2 uses the ace2 receptor, five out six critical amino acid residues in rbd were different between sars-cov-2 and sars-cov; the same residues were instead identical to those of pangolin sars-rcovs and, in turn, only one of these residues was identical to those of batcovratg13 (tang et al., 2020) , although this latter shows the highest nucleotide sequence identity with sars-cov-2 along the whole genome. thus, it was tempting to speculate that sars-cov-2 rbd region might have originated from recent recombination event in pangolins or that sars-cov-2 and sars-rcovs of pangolins represent the result of coincidental evolution (lam et al., 2020; tang et al., 2020) . overall, it remains to be solved whether also sars-cov-2 needed an intermediate (and amplification) host before being able to infect humans as it was the case for sars-cov and other hcovs. since a mammal reservoir has not yet been identified, a prudent use of specific antigens is strongly recommended for serological diagnosis of sars-cov-2 in animals as cross-reactions with viruses of the alphacoronavirus genus, widespread in animals, might occur (sun and meng, 2004) . analogously to bats, but with a lesser extent, also rodents have been recently demonstrated to play a significant role in the evolution of cov, in particular of those belonging to subgenus embecovirus of genus betacoronavirus. rodentia (rodents) is the largest order of mammals with more than 2000 species worldwide, representing a major source of zoonotic infectious diseases (han et al., 2015) . for decades, only one species of coronavirus, murine coronavirus (subgenus embecovirus, genus betacoronavirus), has been associated with rodents. the prototype virus, which was named mouse hepatitis virus (mhv), was first isolated in mice in 1949 (cheever et al., 1949) . a mhv variant was lately identified in rats in 1970 (parker et al., 1970) . rat coronavirus (rcov) causes epidemics of respiratory disease in laboratory rat colonies. the two prototype strains of rcov are sialodacryoadenitis virus (sdav) and parker's rcov (rcov-p) (bhatt et al., 1972; parker et al., 1970) . both strains infect the respiratory tract, and sdav can also infect the eye, salivary and lacrimal glands. young rats are especially susceptible to rcov with the infection occurring in the lower respiratory tract and developing into interstitial pneumonia (parker et al., 1970) . together with feline infectious peritonitis virus (fipv) and ibv, mhv has been one of the most strictly animal cov studied ever. mhv is a natural pathogen of mice, normally infecting the liver, gastrointestinal tract, and central nervous system, causing a wide range of disease, including hepatitis, gastroenteritis, and acute and chronic encephalomyelitis. importantly, it served as model for cov replication and pathogenesis, with emphasis for neuro-invasion and neurovirulence (weiss and navas-martin, 2005) . as for the additional structural protein he, some strains (such as jhm) of mhv contain the he protein, while others (such as a59) do not yokomori et al., 1989) . the role of rodents in the evolution of covs belonging to embecoviruses has been recently highlighted by means of the discovery of a novel betacoronavirus in norway rats (rattus norvegicus) in china. this virus forms a separate species named china rattus coronavirus hku24 (chrcov hku24) within the embecovirus subgenus. although designated as a novel species, this virus possessed genome characteristics that resemble to those of both betacoronavirus-1 and murine coronavirus, suggesting that chrcov hku24 represents the murine origin of betacoronavirus-1, with interspecies transmission from rodents to other mammals having occurred centuries ago (lau et al., 2015) . genus betacoronavirus consists of five subgenera, with bat covs being including in all but one of subgenus embecovirus, where rodent, human and bovine covs are included (https://talk.ictvonline.org/ taxonomy/). this supports the hypothesis that rodent covs were the ancestors of embecoviruses of other animals, while bats are the natural reservoirs for all other betacoronaviruses. importantly, rodent covs are not restricted to genus betacoronavirus. a deep virological screening was performed in 1465 rodents sampled in zhejiang province, china, during 2011-2013, with nearly 2% of rodents testing positive for cov . in particular, covs were detected in 10 striped field mice (apodemus agrarius), 4 norway rats, 14 lesser ricefield rats (rattus losea), 1 asian house rat (rattus tanezumi) and 1 chinese white-bellied rat (niviventer confucianus). amplicons of the replicase gene sequences were recovered from 21 (70 %) of the cov rna positive rodent samples described above and whole genome or nearly whole genome sequences (> 98 %) were recovered from 1 and 4 cov positive samples, respectively. by means of whole genome sequence analysis, authors were able to identify a divergent alphacoronavirus, which was lately officially designated as species lucheng rn rat coronavirus (lrnv) within the subgenus luchacovirus, and two novel betacoronaviruses termed longquan aa mouse coronavirus (lamv) and longquan rl rat coronavirus (lrlv) and assigned to the two established species betacoronavirus-1 and murine coronavirus, respectively . moreover, lrnv seems to be a recombinant virus as its n protein gene is more closely related to those of the genus betacoronavirus. overall, the discovery of rodent-associated covs belonging to subgenera that are distinct from those including bat covs warrants further investigations upon the role played by rodents in the evolution and emergence of these viruses. sars-cov replication has been studied in mice, syrian golden and chinese hamsters. the most severe symptoms of sars were observed in aged animals. indeed, aged mouse model of sars-cov has been generated (gretebeck and subbarao, 2015) . transgenic mice expressing human ace2 were also developed to closely mimic sars-cov infection in humans. some animal models have been tested and analysed on the genomic and proteomic level to study the pathogenesis of sars-cov. therefore, we have reason to believe that such models would work also for sars-cov-2. quite the opposite, studies have demonstrated that mice, guinea pigs and hamsters are not susceptible to experimental mers-cov infection, mainly because their homologous dpp4 molecules table 4 coronaviruses in domestic swine and associated diseases. do not function as receptors for mers-cov entry (cockrell et al., 2014) . the first mouse model of mers infection reported in 2014 involved transducing animals with recombinant adenovirus 5 encoding human dpp4 (hdpp4) molecules intranasally, and this resulted in replication of mers-cov in the lungs. this mouse model also showed clinical symptoms of interstitial pneumonia, including inflammatory cell infiltration, and thickened alveolar and mild oedema (song et al., 2019) . currently, six covs are circulating in swine (table 4 ). these include four alphacoronaviruses, transmissible gastroenteritis virus of swine (tgev) and its derivative porcine respiratory coronavirus (prcov) (subgenus tegacovirus), porcine epidemic diarrhoea virus (pedv) (subgenus pedacovirus) and sads-cov (subgenus rhinacovirus), one betacoronavirus, porcine haemagglutinating encephalomyelitis virus (phev) (subgenus embecovirus), and one deltacoronavirus, porcine deltacoronavirus (pdcov) (subgenus buldecovirus). tgev, pedv, sads-cov and pdcov are responsible for acute gastroenteritis in swine, with fatal infections in piglets born to seronegative sows, prcov causes a mild respiratory disease and phev is the causative agent of neurological and/or digestive disease in pigs (mora-díaz et al., 2019; wang et al., 2019). tgev was first described in uk in 1950s, representing the oldest known swine cov. tgev and prcov are closely related to canine coronavirus (ccov) and feline coronavirus (fcov) forming with these carnivore covs a unique species, referred to as alphacoronavirus-1. based on the analysis of the accessory protein gene orf3, it has been postulated that tgev has originated from ccov type ii (ccov-ii), since while ccov type i (ccov-i) exhibits an intact gene, both ccov-ii and tgev, which are strictly related in the s gene, have only remnants of orf3 (lorusso et al., 2008) . prcov, in turn, has derived from tgev through the deletion of ≈600 nucleotides at the 5' end of the s gene (corresponding to ≈200 amino acids at the n-terminus of the spike protein) and consequent change of the major tissue tropism from the enteric to the respiratory epithelium. this large deletion caused the loss of sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins, so that tgev but not prcov is able overcome the intestinal mucus barrier, having access to the gut mucosa . prcov shares some epitopes for neutralising antibodies with tgev, so that its extensive circulation in swine herds has resulted in a drastic reduction of tge outbreaks worldwide. pedv was introduced in the pig population in the 1970s, likely as a consequence of a spillover event from bats. the virus was first described in europe and had been primarily maintained as an endemic pathogen in european and asian swine populations until its introduction into north america in 2013. pedv is more strictly related to a scotophilus bat coronavirus 512 than to other known alphacoronaviruses, including tgev and human alphacoronaviruses hcov-229e and hcov-nl63. therefore, pedv and btcov/512/2005 likely have a common evolutionary precursor and a cov cross-species transmission may have occurred between bats and pigs (banerjee et al., 2019) . accordingly, pedv contains signature motifs at the 5′-untranslated region that are shared by bat covs, thus providing further support of the evolutionary origin of pedv from bats and potential cross-species transmission (huang et al., 2013) . currently, different pedv genotypes are described based on the s gene: i) g1a pedv, including classical european and asian strains with moderate virulence; ii) g2 pedv, also called "original us pedv", comprising highly virulent strains that originated in asia and are now widespread in the usa; iii) g1b pedv, which is represented by the so-called s-indel strains, i.e., strains presenting insertions and deletions in the s gene that are associated with mild clinical outbreaks. these strains are natural recombinant pedvs with a g2-like genomic backbone carrying an s1 region of g1a strains; iv) s1 n-terminal domain-deletion (ntd-del) strains that are g2-like strains containing a 194 to 216-aa deletion within the n-terminal domain of the s1 subunit, also associated to mild clinical forms (hou and wang, 2019) . recombinant strains between pedv and tgev have been also reported in europe (akimkin et al., 2016; belsham et al., 2016; boniotti et al., 2016) . sadv-cov, now referred to as swine enteric alphacoronavirus (seacov), is another virulent swine enteric alphacoronavirus that originated from bats, sharing an 86 % sequence identity with a bat alphacoronavirus hku2-cov. since viruses displaying a 96-98 % sequence identity to sads-cov were detected in rhinolophus spp. bats, sads-cov and hku2-cov likely descend from a common ancestor . accordingly, both viruses now belong to the unique species rhinolophus bat coronavirus hku2. in contrast, phev, which was first described in 1957 in nursery pigs with encephalomyelitis in ontario, canada, has not derived from bat covs, but its evolutionary history is tightly intermingled with other two closely related betacoronavirus, hcov-oc43 and the oldest known bcov, with which phev may have common ancestors (vijgen et al., 2006) and is included in the same viral species, betacoronavirus-1 (corman et al., 2018) . most probably, hcov-oc43 and phev descend from a rodent betacoronavirus through preliminary adaptation to bcov, from which they may have emerged in the context of a pandemic recorded historically at the end of the 19th century (corman et al., 2018) . pdcov was recently detected in 2012 in hong kong during cov molecular surveillance in avian and mammalian species. this swine deltacoronavirus seems to recognise another different ancestor, likely emerging from a host-switching event between avian and mammal covs. the most closely related pdcov relative has been identified in quail deltacoronavrus uae-hku30 and the virus has been proposed to be a recombinant between other two avian deltacoronaviruses, sparrow cov hku15 and bulbul cov hku11. all these deltacoronavirus are now members of the same species coronavirus hku15 (lau et al., 2018) . pigs were found to be susceptible to experimental infection with the betacoronavirus mers-cov (vergara-alert et al., 2017), while sars-cov rna was detected in pigs and wild boars wang et al., 2005) . in contrast, a recent experimental infection demonstrated that pigs are not susceptible to sars-cov-2 . few studies have been carried out to assess the circulation of covs in farmed or free-ranging wild boars (sus scrofa). antibodies against tgev/prcov were detected in some animals in slovenia (vengust et al., 2006) and croatia (roic et al., 2012) and pedv rna was demonstrated in south korea (lee et al., 2016) . a wild boar sold at a live animal market of guangzhou, china, was positive for sars-cov rna . the main covs infecting ruminants are reported in table 5 . the oldest known ruminant cov is bcov, which is also the prototype of the species betacoronavirus-1 (subgenus embecovirus, genus betacoronavirus). this virus is able to cause a variety of clinical forms, including enteric disease with high mortality rates in neonate calves, winter disease (a severe enteric form) in lactating cows (decaro et al., 2008b) , and a respiratory disease, also known as shipping fever, in cattle of all ages, with a higher prevalence in 2-3 month-old calves (decaro et al., 2008a) . it was postulated that the presence of genetic signatures differentiates enteric and respiratory bcovs (hasoksuz et al., 1999) , but it was ultimately evident that the same virus strain could be responsible for simultaneous appearance of enteric and respiratory disease in the same animals (chouljenko et al., 2001) . it has been postulated that bcov originated from a rodent cov (corman et al., 2018) . very recently, a novel cov, representing a new viral species, referred to as china rattus coronavirus hku24 (chrcov-hku24), was detected in norway rats in china. this virus was phylogenetically distinct from mhv and hcov-hku1 and displayed genome features that were intermediate between bcov and mhv. therefore, chrcov hku24 may represent the murine origin of bcov and rodents are likely an important reservoir for ancestors of subgenus embecovirus (lau et al., 2015) . bcov is paradigmatic of how covs are able to cross the interspecies barriers, establishing its derivatives as separate viral lineages affecting the respiratory and/or enteric tract of humans (hcov-oc43), swine (phev), horses (equine coronavirus, ecov), and dogs (canine respiratory coronavirus, crcov). a number of bcov-related viruses, all currently included in the unique species betacoronavirus-1, have been detected in the enteric and/or respiratory tract of domestic and wild ruminants. these bcov-like covs include viruses of domestic and domesticated ruminants that were reported in sheep and goats (reinhardt et al., 1995; yang et al., 2008) , water buffalo (bubalus bubalis) (decaro et al., 2008c) , llamas (lama lama) and alpacas (vicugna pacos) (cebra et al., 2003; jin et al., 2007) . in the wild, bcov-like covs were demonstrated in six species of the cervidae family, which are caribou/ reindeer (rangifer tarandus caribou), elk/wapiti (cervus elephus), samber deer (cervus unicolor), white-tailed deer (odocoileus virginianus), sika deer (cervus nippon yesoensis) and water deer (hydropotes inermis) (amer, 2018) . similar viruses were also found to circulate in the giraffe (giraffa camelopardalis) (hasoksuz et al., 2007) , several species of antelopes (alekseev et al., 2008; chung et al., 2011) , wisent (bison bonasus), himalayan tahr (hemitragus jemlahicus) (chung et al., 2011) , and dromedary camels (camelus dromedarius) (woo et al., 2014) . the last strain, detected in the united arab emirates and consequently named dromedary camel coronavirus uae-hku-23 (dccov uae-hku23), was slightly divergent from other bcov-like viruses (woo et al., 2014) . dromedary camels are susceptible to mers-cov infection, developing asymptomatic infections or mild upper respiratory disease, so that they are considered the natural host of mers-cov, with adult animals in many countries in the middle east as well as in north and east africa showing > 90 % seroprevalence to the virus (hemida et al., 2017b) . although human-to-human transmission has occurred outside middle east due to travel-associated patients with mers and has caused large clusters of human cases within healthcare facilities in saudi arabia, jordan and united arab emirates, it remains inefficient and sustained community transmission has not being documented so far, thus suggesting multiple virus introduction into the human population by infected dromedaries (hemida et al., 2017b) . more recently, a phylogenetic study of 173 mers-cov full-genome sequences revealed recombination signatures that defined five major phylogenetically stable lineages, all of which contained human and camel mers-cov sequences (sabir et al., 2016) . in the same study, an alphacoronavirus strictly related to hcov-229e was found in the respiratory tract of dromedary camels of saudi arabia (sabir et al., 2016) . although some studies ruled out the susceptibility of other domestic ruminants to mers-cov (reusken et al., 2013; adney et al., 2016) , a recent study detected specific antibodies and rna in sera and nasal secretions, respectively, of domestic ruminants raised in africa, including sheep, goats and cattle (kandeil et al., 2019) . llamas were found to be susceptible to experimental infections with mers-cov (vergara-alert et al., 2017). the only cov that has been so far known in horses is ecov, which is a bcov-descendant betacoronavirus (subgenus embecovirus). ecov was first isolated from the faeces of a diarrhoeic foal in 1999 (ecov-nc99) in north carolina, usa (guy et al., 2000) , and was initially believed to only affect foals. since 2010, the virus has been recognised in japan, europe and the usa as a new, clinically important, enteric virus of adult horses (pusterla et al., 2018) . despite mers-cov was successfully adapted to the in-vitro growth in equine cell lines (meyer et al., 2015) , serological and molecular table 5 coronaviruses in domestic and domesticated ruminants and associated diseases. sabir et al. (2016) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 investigations have demonstrated that horses are not naturally infected by mers-cov (meyer et al., 2015; hemida et al., 2017a) , nor they are susceptible to experimental infection (adney et al., 2016; vergara-alert et al., 2017) . however, surprisingly, mers-cov rna was detected in respiratory specimens of three donkeys of 42 from egypt (kandeil et al., 2019) , a finding that requires further confirmation. a molecular survey aimed to assess cov circulation in horses in saudi arabia and oman has detected two dccov uae-hku23 strains in enteric samples of horses (hemida et al., 2017a) . scarce data are available about cov circulation in donkeys. these equids are susceptible to ecov infection since positive rt-pcr results were obtained from a donkey in ireland (nemoto et al., 2019) . in addition, three donkeys (7.1 %) of 42 from egypt tested positive for mers-cov rna in their nasal secretions (kandeil et al., 2019) . covs of carnivores are listed in table 6 . three covs are known in dogs, i.e., two alphacoronaviruses of the subgenus tegacovirus, namely ccov-i and ccov-ii, and one betacoronavirus of the subgenus embecovirus, namely crcov. ccovs (species alphacoronav-irus-1) are commonly responsible for mild, self-limiting enteritis in pups . although they are neglected viruses and vaccination is not recommended due to the absence of an effective challenge model, two independent studies have demonstrated their significant involvement in the onset of acute canine enteritis (duijvestijn et al., 2016; dowgier et al., 2017) . the evolutionary history of ccovs is tightly intermingled with that of tgev and fcovs. ccov-i possesses a divergent spike protein and the intact form of an additional gene, orf3, whose remnants are present in ccov-ii and, at a lesser extent, in tgev. therefore, ccov-ii has likely emerged as a consequence of recombination between the original ccov-i and an unknown cov in the s gene and of progressive loss of orf3 (lorusso et al., 2008) . a further recombination occurred in the very 5' end of the s gene between ccov-ii and tgev, giving rise to back recombinant ccov-ii strains, also known as tgevlike ccovs, having a spike protein n-terminus of tgev in a ccov-ii backbone (decaro et al., , 2010 . consequently, the ccov taxonomy was revised, with classical and tgev-like strains being referred to as ccov-iia and ccov-iib, respectively. while ccovs are usually involved in mild forms of diarrhoea, there are some hypervirulent strains that are associated to severe, haemorrhagic, sometimes fatal gastroenteritis. in addition, ccov-iia strains, designated pantropic ccov, that are able to spread systemically and cause severe disease and the death of infected dogs have been reported in italy (buonavoglia et al., 2006; alfano et al., 2020) , other european countries (decaro et al., 2013) and south america (pinto et al., 2014) . genomic sequences from pantropic ccovs were analysed, but no obvious genetic signatures that may have caused the switch in pathogenicity were found (decaro and buonavoglia, 2011; decaro et al., 2013) . different from ccov-i and ccov-ii, the betacoronavirus crcov is associated with mild respiratory signs and has been proposed as an etiological agent of canine infectious respiratory disease (cird) together with other viral and bacterial agents . the virus was first detected firstly in uk in 2003 (erles et al., 2003) and subsequently in other european and extra-european countries (decaro et al., 2007 (decaro et al., , 2016 mitchell et al., 2017; maboni et al., 2019; piewbang et al., 2019; more et al., 2020) . being a bcov derivative, crcov possesses the same genomic organisation, with some differences in accessory orfs located between the s and e protein genes. in particular, while some crcovs possess a unique 8.8 kda protein gene directly downstream of the s protein gene, other canine bcov-like covs display the canonical set of bcov accessory genes but with truncated forms of the 4.8 kda protein gene . in cats, two alphacornavirus-1 genotypes are known, namely fcov type i (fcov-i) and fcov type ii (fcov-ii), the latter being generated as table 6 coronaviruses in domestic and domesticated carnivores and associated diseases. jakob, 1914jacob (1914 , pedersen et al. (1984) cat ( erles et al. (2003) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 a consequence of recombination events between ccov-ii and fcov-i that generated viruses with a ccov-ii genomic region, encompassing orf1b, orf2 (s gene), orf3abc, orf4 (e gene), and partial orf5 (m gene), in the context of an fcov-i backbone (pedersen, 2014) . both genotypes are involved in the development of feline infectious peritonitis (fip), a perivascular pyogranulomatosis of cats that may occur in two clinical forms, effusive and non-effusive fip, which are characterised by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip occurs as a consequence of a change in tissue tropism of an enteric fcov strain (feline enteric coronavirus, fecv), infecting enterocytes of the intestinal villi, that acquires the ability to infect monocytes/macrophages switching to the more virulent fipv, which is responsible for systemic infections and dysregulation of the proinflammatory cytokines (addie et al., 2009) . the changes responsible for the pathogenetic shift have been investigated for many decades, being suggested to be variably represented by point mutations located in the s gene (rottier et al., 2005) , deletion/insertion in the group-specific genes 3c (vennema et al., 1998; chang et al., 2010) , 7b (vennema et al., 1998) or 7a (kennedy et al., 2001a) . however, none of these differences appeared to consistently correlate with disease phenotype. more recent studies have identified specific genetic signatures in the s gene of fcov-i that are implicated in monocyte/macrophage tropism. two amino acid substitutions, m1058 l and/or s1060a, corresponding to nucleotide mutations a23531 t/c and t23537 g, respectively, in the viral genome, together distinguished fcovs found in the tissues of fip cats from those found in the faeces of healthy cats without fip in > 95 % of cases (chang et al., 2012) . however, subsequent studies concluded that these mutations are likely to be markers of systemic fcov infection rather than fip per se (porter et al., 2014; barker et al., 2017) . two alphacoronaviruses, both belonging to subgenus minacovirus, are currently known in mustelids, namely mink coronavirus 1 (mcov-1) and ferret coronavirus (frcov). mcov-1 has been recently identified as the etiological agent of mink epizootic catarrhal gastroenteritis (ecg), an infectious disease of farmed american (neovison vison) and european (mustela lutreola) mink first described in 1975 (larsen and gorham, 1975) and later affecting several million mink in different countries (vlasova et al., 2011) . the disease is observed at greater frequency in mink of ≥4 months and is characterised by seasonality, high morbidity (approaching 100 %) and low mortality (< 5 %). recent full-genome analysis demonstrated that mcov-1 is phylogenetically distant from ccovs and fcovs, being closely related to frcov (vlasova et al., 2011) . presently, the two viruses are considered separate species within subgenus minacovirus (https://talk.ictvonline.org/taxonomy/). frcov has been recognised as the causative agent of epizootic catarrhal enteritis (ece), first described in 1993 in domestic ferrets (mustela putorius furo) in the eastern part of the usa (williams et al., 2000) and subsequently reported in domestic and laboratory ferrets throughout the world (murray et al., 2010) . analogous to fcov, frcov exists in two different pathotypes: i) ferret enteric coronavirus (frecv) is associated to ece, a highly contagious diarrhoeal disease also known as green slime disease, which affects mainly young ferrets with morbidity and mortality rates similar to those of ecg; ii) ferret systemic coronavirus (frscv) is responsible for a systemic diseases of ferrets, which is characterised by pyogranulomatous perivasculitis and peritonitis resembling to those of fip (murray et al., 2010) . similar to fip, wise et al. (2010) have shown that frecv and frscv differ significantly in spike protein and that deletions in frcov 3c may also correlate with the severe pathotype of frscv. recombination in the s, 3c and e genes between different frcov has been also reported (lamers et al., 2016) . different covs were found to circulate in wild carnivores. ccovs were detected in wolves (canis lupus), red foxes (vulpes vulpes), eurasian otters (lutra lutra), common genets (genetta genetta) (alfano et al., 2019; rosa et al., 2020) . ccov-like viruses were also found in african wild carnivores, including spotted hyenas (crocuta crocuta) and silver-backed jackals (canis mesomelas) (goller et al., 2013) . fcovs have a wide circulation in non-domestic felids (kennedy et al., 2002 (kennedy et al., , 2003 , with fip cases being reported in servals (felis serval) (juan-salles et al., 1997) , cheetah (acinonyx jubatus) (kennedy et al., 2001b) , mountain lion (puma concolor) (stephenson et al., 2013) , and european wildcat (felis silvestris) (watt et al., 1993) . divergent alphacoronavirus-1 viruses were detected in chinese ferret badger (nyctereutes procyonoides) and raccoon dog (melogale moschata) (dong et al., 2007) . the same study reported the identification in asian leopard cat (prionailurus bengalensis) and chinese ferret badger of an unclassified cov, which was closely related to gammacoronaviruses in most parts of the genome, whereas the s gene displayed the highest sequence identity to alphacoronaviruses (dong et al., 2007) . with the discovery of deltacoronaviruses, these viruses were later included in this novel genus along with avian and porcine strains (woo et al., 2009; wang et al., 2014) . some domestic and wild carnivores are also susceptible to sars-cov infection. while the potential natural reservoirs are horseshoe bats, sars-like cov strains were found to be widespread in masked palm civets (paguma larvata) and raccoon dogs, which were suspected to be intermediate hosts (guan et al., 2003) . full-genomic comparative analysis has shown that sars-like covs isolated from palm civets are under strong selective pressure and are genetically most closely related to sars-cov strains infecting humans early in the outbreaks (song et al., 2005) . sequence analysis of the sars-cov-like virus in masked palm civets indicated that they were highly homologous to human sars-cov with nucleotide identity over 99.6 %, indicating the virus has not been circulating in the population of masked palm civets for a very long time (shi and hu, 2008) . a chinese ferret-badger (melogale moschata) was found to have neutralising antibodies against sars-cov (guan et al., 2003) , whereas sars-cov rna was detected in naturally infected cats and red foxes (vulpes vulpes), but not in domestic dogs . there was, however, a single dog testing positive for sars-cov (https://apps.who.int/iris/bitstream/handle/10665/ 70863/who_cds_csr_gar_2003.11_eng.pdf). among carnivores, sars-cov-2 is able to infect cats, ferrets and, at a lesser extent, dogs . in 2008, a highly divergent cov, tentatively named sw1, was discovered a deceased beluga wale (delphinapterus leucas) with pneumonia and hepatic necrosis (mihindukulasuriya et al., 2008) . the virus was only distantly related to ibv, so that it now represents the prototype of the single mammalian cov species belonging to the genus gammacoronavirus, namely beluga wale coronavirus sw1 (bwcov-sw1) (subgenus cegacovirus). few years later, related gammacoronaviruses were retrieved from faecal samples of three indo-pacific bottlenose dolphins (tursiops aduncus), which were named bottlenose dolphin cov (bdcov) hku22. comparative genome analysis showed that bdcov-hku22 and bwcov-sw1 have similar genome characteristics and structures, displaying a 98 % nucleotide sequence identity each to other (woo et al., 2014) . a novel betacoronavirus distantly related to mers-cov was detected in the faeces of european hedgehogs (erinaceus europaeus), an insectivorous mammal belonging to a related order of chiroptera, from germany. the virus was tentatively referred to as erinaceus cov (ericov) (corman et al., 2014b) and covs found in hedgehogs in france, england and italy had an identity from 92% to 98 % with the ericov (monchatreleroy et al., 2017; saldanha et al., 2019; delogu et al., 2020) . these hedgehog covs are are now included in a unique species, hedgehog coronavirus 1 (subgenus merbecovirus). the virus was not associated to any form of disease, so that western european hedgehog is a reservoir host of ericov in the absence of apparent disease, suggesting that hedgehogs in addition to bats may contribute to the evolution of merbecovirus (saldanha et al., 2019) . a slightly divergent merbecovirus was later found in amur hedgehogs (erinaceus amurensis) in china and was poposed as a prototype of a separate species, namely erinaceus amurensis hedgehog coronavirus hku31 (ea-hedcov hku31) . a novel coronavirus, named wénchéng shrew coronavirus (wesv) was detected in shrews (suncus murinus) in china . wesv is highly divergent from other alphacoronaviruses, exhibiting less than 71.1 % amino acid similarity to any known members of the genus alphacoronavirus in the coronavirus-wide conserved domains of the replicase polyprotein pp1ab and less than 61.3 % amino acid similarity to the other three coronavirus genera. however, taking into account the current ictv criteria, wesv is sufficiently divergent to be considered a distinct member of the genus alphacoronavirus, but not a new genus of the subfamily orthocornavirinae . covs have been known in veterinary medicine since many decades; some of these viruses, such as ibv, swine enteric covs, bcov and mustelid covs, can cause diseases that have a great impact on the farm industry. other covs, namely fipv, frscv and mhv, cause severe disease in companion (cats, ferrets) or laboratory (mice) animals. animal covs are paradigmatic on how covs evolve through accumulation of point mutations and homologous (and heterologous) recombination, generating different genotypes and pathotypes. these virus variants may have different antigenic properties, escaping the host immunity induced by vaccines, as is the case of ibv. alternatively, they may have a different tissue tropism in the same host that can increase or decrease the virus pathogenicity, as observed for the virus pairs fecv/fipv or frecv/frscv and tgev/prcov, respectively. in other circumstances, the cov evolution may result in the switch of the host range from one animal species to another one or from animals to humans. the former event is well documented in veterinary medicine, with a plethora of viruses being originated from ibv and bcov that adapted to different animal species. however, the most interesting scenario is the jumping and further adaptation of an animal cov to humans. there is increasing evidence that all hcovs currently known recognise an animal origin, with bat or rodent covs being the most probable ancestors. in most instances, it was suggested that other mammals served as intermediate hosts prior to final adaptation to humans, i.e., alpacas and cattle for the low-pathogenic hcov-229e and hcov-oc43, respectively, and wild carnivores and dromedary camels for the high-pathogenic sars-cov and mers-cov, respectively. other two hcovs, namely hcov-nl63 and hcov-hku1, were likely derived from bats and rodents, respectively, but whether this transmission required an intermediate mammalian host is presently unknown. the origin of sars-cov-2 should be zoonotic, since highly related sequences were detected in bats, but a definitive intermediate host has been not identified so far. what should we expect from the current pandemic? when hcov-oc43 crossed the species barrier to infect humans from domestic livestock around 1890, an epidemic of respiratory infection was recorded. even though, several years later, influenza was suspected to be the cause of it, in that pandemic involvement of central nervous system was more pronounced than in other influenza outbreaks. this evidence is further supported by molecular studies claiming that the most recent common ancestor of bcov and hcov-oc43 emerged around 1890 (vijgen et al., 2005) and by the fact that hcov-oc43 can be neuroinvasive (arbour et al., 2000) . likely, hcov-oc43 crossed species to infect dogs becoming established in this species as crcov . a similar scenario could be observed with sars-cov-2 with dogs and, at a greater extent, cats. apparently, cats represent, within the domestic animals which have been experimentally infected, the host, together with ferrets, which is able to sustain more efficiently sars-cov-2 replication . furthermore, based on structural studies and biochemical experiments, sars-cov-2 seems to have an rbd that binds with high affinity to ace2 also from ferrets and cats (andersen et al., 2020) . reasonably, a full comprehension of the animal cov molecular evolution, host range and pathobiology is beneficial to better understand the mechanism driving the emergence and adaptation to humans of zoonotic covs. the present review has highlighted that in the last 18 years, also thanks to the availability of novel sequencing technologies, we have witnessed a large number of novel covs being discovered in a large number of animals. truth to be told, it was difficult for us to summarise, in this single review, all covs detected in animals and the tight interaction existing between them and human covs. among animals, it is evident that bats are the group of mammals that harbor the largest number of covs and that many other animal covs recognise their ancestors in bat covs. in an excellent review (cui et al., 2019) written by the group coordinated by dr. zheng-li shi of the wuhan institute of virology, hubei, (china), city infamously known for being the epicenter and origin of the covid-19 outbreak, authors stated that "...given the prevalence and great genetic diversity of bat sars-rcovs, their close coexistence and the frequent recombination of covs, it is expected that novel variants will emerge in the future". this forecasting statement was not surprising to coronavirologists and it was not, importantly, surprising to those scientists that daily deal with the plethora of viruses existing at the human/animal health interface. although scientists were well aware of this hazard, no substantial actions were taken forward the limitations of strict and repeated contacts between humans and wildlife. indeed, whereas biological mechanisms underlying viral evolution are not under human control, social and cultural habits can be modified accordingly through a deep and pounding informative campaigns. if to the human habits we sum the impact of modern agricultural practices and urbanization and the decrease of vital space for wildlife, it is quite easy to understand that, if countermeasures are not taken, we will face novel serious health emergencies of animal origin in the following years with tremendous social and economic impact on our lives. as clearly demonstrated by the sars-cov-2 emergence, covs are the main characters of this intricate puzzle characterised by the interactions of viral biological mechanisms and human habits. our review was reasonably prepared also to highlight (once more!) how covs originate, evolve, jump, mutate and infect their host. could have the current covid-19 outbreak been avoided? answering this question is not relevant now, but actions to avoid the next viral spillover from animals to humans is certainly a priority. this task needs to be coupled with massive genomic surveillance in wild animals not limited to covs. massive sequencing of sars-cov-2 strains detected in humans and covs of wildlife will help further assess the origin of this novel human pandemic and plan future measures able to reduce the risk of emergence of new cov spillover events. however, additional tasks should be provisionally addressed in order to reduce the risk of future cov pandemic like the current one. these include: i) prevention of animal-to-human infections through a ban of the wet markets and a more friendly management of the environment; ii) studies on cov-host interactions to be performed both in vitro (cell cultures, ex-vivo explants of the respiratory tract) and in vivo (animals susceptible to sars-cov-2 infection); iii) development of new anticoronaviral drugs and evaluation of their efficacy in cell cultures and animal models. feline infectious peritonitis. abcd guidelines on prevention and management inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host new chimeric porcine coronavirus in swine feces bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences 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supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2020.108693. key: cord-356094-sbtigcfr authors: chen, huijie; muhammad, ishfaq; zhang, yue; ren, yudong; zhang, ruili; huang, xiaodan; diao, lei; liu, haixin; li, xunliang; sun, xiaoqi; abbas, ghulam; li, guangxing title: antiviral activity against infectious bronchitis virus and bioactive components of hypericum perforatum l. date: 2019-10-29 journal: front pharmacol doi: 10.3389/fphar.2019.01272 sha: doc_id: 356094 cord_uid: sbtigcfr hypericum perforatum l., also known as saint john’s wort, has been well studied for its chemical composition and pharmacological activity. in this study, the antiviral activities of h. perforatum on infectious bronchitis virus (ibv) were evaluated in vitro and in vivo for the first time. the results of in vitro experiments confirmed that the antiviral component of h. perforatum was ethyl acetate extraction section (hpe), and results showed that treatment with hpe significantly reduced the relative messenger ribonucleic acid (mrna) expression and virus titer of ibv, and reduced positive green immunofluorescence signal of ibv in chicken embryo kidney (cek) cells. hpe treatment at doses of 480–120 mg/kg for 5 days, reduced ibv induced injury in the trachea and kidney, moreover, reduced the mrna expression level of ibv in the trachea and kidney in vivo. the mrna expression levels of il-6, tumor necrosis factor alpha (tnf-α), and nuclear factor kappa beta (nf-κb) significantly decreased, but melanoma differentiation-associated protein 5 (mda5), mitochondrial antiviral signaling gene, interferon alpha (ifn-α), and interferon beta (ifn-β) mrna levels significantly increased in vitro and in vivo. our findings demonstrated that hpe had significant anti-ibv effects in vitro and in vivo, respectively. in addition, it is possible owing to up-regulate mrna expression of type i interferon through the mda5 signaling pathway and down-regulate mrna expression of il-6 and tnf-α via the nf-κb signaling pathway. moreover, the mainly active compositions of hpe analyzed by high-performance liquid chromatography/electrospray ionization–mass spectroscopy (esi-ms) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. this might accelerate our understanding of the antiviral effect of h. perforatum and provide new insights into the development of effective therapeutic strategies. the infectious bronchitis virus (ibv) is a prototype coronavirus containing a single-stranded positive-sense rna genome (cook et al., 2012) . ibv is the etiologic agent of infectious bronchitis (ib), which is a highly contagious, acute viral respiratory disease of chickens. ibv has been reported by many researchers all over the world (jungherr and terrells, 1948; jungherr et al., 1956; fabricant, 1998; yu et al., 2001; benyeda et al., 2009; sjaak de wit et al., 2011; westerbeck and machamer, 2019; wu et al., 2019) . ibv has led to severe losses in the poultry industry (jordan, 2017) , the direct losses are due to highly mortality, poor egg quality, and meat production, and the indirect losses result in increased costs and challenges in ibv prevention (liang et al., 2019) . at present, live attenuated vaccines are widely used for the prevention and control of ib. however, due to extensive genetic diversity of ibv strains, the vaccines are becoming increasingly inefficient, with poor cross-protection effects among different serotypes of vaccines (mo et al., 2013; chen et al., 2015; lin and chen, 2017; yan et al., 2018) . meanwhile, due to the lack of coordinated effort to prevent the ibv, and the lack of proper surveillance plus the introduction of foreign strains to combat the ibv in certain regions, the prevention and control of ibv has become very difficult. therefore, it is imperative to find an effective antiviral drug or agent for the prevention of ibv. in order to control drug residues, the chinese government has banned the use of antiviral drugs in food animals in china. therefore, the use of traditional antiviral herbs with no obvious side effects on the human body is still a major focus. some reports have confirmed that traditional chinese herbs could effectively inhibit the infection and replication of various viruses (li et al., 2009; wang et al., 2015; choi et al., 2016; sun et al., 2016; choi et al., 2017; yin et al., 2017; yi et al., 2018; luo et al., 2019) . hypericum perforatum l. belongs to the genus guttiferae, which contains approximately 400 species all over the world. the extract of h. perforatum contains several active compounds, including flavonoids, naphthodianthrones, and phloroglucinol derivatives (napoli et al., 2018; barnes et al., 2019) . several reports have shown that h. perforatum extract had antiviral effects, such as influenza a virus, porcine respiratory and reproductive syndrome virus (prrsv), and hiv (barnes et al., 2001; birt et al., 2009; pu et al., 2009a; pu et al., 2009b; pu et al., 2012) . like influenza a virus and prrsv, ibv also belongs to rna virus, but these prrsv and ibv belong to different viral families. since h. perforatum could resist influenza a virus and prrsv, could it resist ibv? in this study, we investigated the antiviral effects of h. perforatum extract against ibv utilizing several approaches in vitro and in vivo for the first time. moreover, the purpose of this work was to point out the antiviral active ingredients of h. perforatum and its anti-ibv mechanisms. for this purpose, the relative messenger ribonucleic acid (mrna) expression levels of ibv in cek cells, tracheas, and kidneys were measured. the positive green immunofluorescence signal of ibv in ceks was observed. in addition, hematoxylin-eosin (he) staining of tracheas and kidneys was performed, and the relative mrna expression of il-6, tumor necrosis factor alpha (tnf-α), ifn-α, ifn-β, mda5, mavs, and nuclear factor kappa beta (nf-κb) in vitro and in vivo were analyzed. finally, the antiviral principal chemical composition of h. perforatum extract was analyzed by high-performance liquid chromatography (hplc)/esi-ms. in summary, our findings for the first time showed that h. perforatum extract had significant antiviral effect on ibv, and it might up-regulate mrna expression levels of type i interferon via mda5 pathway and down-regulate mrna expression levels of il-6 and tnf-α through the nf-κb pathway. the hpe was found to be composed mainly of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. the ibv m41 strain (genbank: fj904723.1) was provided by key laboratory for laboratory animals and comparative medicine, northeast agricultural university and propagated in 10-dayold specific pathogen-free (spf) chicken embryos (harbin veterinary research institute, caas). 100 µl ibv m41 strain was inoculated into spf chicken embryos under aseptic operation. the survival status of chicken embryos was observed every 12 h, and the chicken embryos that died within 24 h were abandoned. then allantoic fluid from infected embryos was collected at 72 h post-inoculation and stored at −80°c. the crude extract of h. perforatum was prepared in the laboratory. in brief, the drug was pretreated with carbon dioxide supercritical extraction method, and the supercritical extraction extract (see) was obtained with a yield of 8.1% (wt/wt). then, 15 g of the pretreated medicinal material was weighed, 1.0 l of 85% methanol was added (raclariu et al., 2017) , ultrasonic extraction was performed twice for 30 min, and ultrasonic power was 400 w. the two extract solutions were combined and filtered, and concentrated under reduced pressure to obtain the crude extract with a yield of 22.4% (wt/wt). approximately 6 g of the crude extract of h. perforatum was dissolved in 200 ml of distilled water. an equal volume of ethyl acetate was added before shaking vigorously and separating in a separating funnel. the ethyl acetate layer (upper layer) and the water layer (lower layer) were obtained. it was then extracted with ethyl acetate until the upper layer had no color. all ethyl acetate extractions and water extractions were combined and evaporated to obtain extract of h. perforatum ethyl acetate (hpe) and extract of h. perforatum water (hpw). a stock solution of hpe (200 mg/ml), hpw (200 mg/ml) and see (200 mg/ml) was prepared with dimethylsulfoxide (dmso, sigma), respectively. immediately before each experiment in vitro, the working solution of h. perforatum extract was diluted in m199 medium without serum to obtain a final concentration. the hpe was dissolved in 5% dmso, while ribavirin (rt) was dissolved in physiological saline solution (pss, 0.86% nacl) in vivo experiments. cek cells were primary cultured from 18-day-old spf chicken embryo (harbin veterinary research institute, caas) and prepared according to the standard technique (schat and purchase, 1998; ghetas et al., 2015) . the cells were cultured in m199 medium (thermo fisher scientific, usa) supplemented with penicillin and streptomycin, and 10% serum at 37°c with 5% co 2 . then, ibv m41 strain was passaged in cek cells for adaption. 500 µl ibv m41 allantoic fluid was inoculated into cek cells in 5 ml cell culture flask, and the cytopathic effect (cpe) was observed. when the cells showed obvious typical cpe, the time and degree of the lesions were recorded and the first generation of ibv was collected by freezing and thawing cells repeatedly. then, the collected first generation ibv was inoculated into cek cells according to the above method, and the time and lesion degree of cpe were also recorded. the second generation ibv was collected according to the above method. after 10 times of passages of ibv in cek cells, there appeared typical cpe of cell exfoliation and fusion at same time post inoculation regularly. ibv was collected and stored at −80°c until use. the results of adaptation and replication in cek cells, such as cpe, reverse transcription polymerase chain reaction (rt-pcr), and ibv growth curve determined by tissue culture infective dose (tcid 50 ) at different time were tested. the cytotoxicity test was carried out according to the published method of thiazolyl blue tetrazolium bromide (mtt), and minor modifications were made (sui et al., 2010) . the monolayer of cek cells plated on 96-well culture plate were washed with d-hanks solution three times, and then hpe, hpw, and see with concentrations of 39. 06, 78.13, 156.25, 312.50, and 625 .00 µg/ml were added to the hole (three repeated holes per concentration). the control cells were incubated in the absence of experimental compounds, but incubated with the same concentration of dmso. the cek cells were cultured at 37°c for 48 h, then 20 μl mtt with concentrations of 5 mg/ml in m199 medium were added, and then the cells were incubated at 37°c for 4 h. after washing the cek cells with d-hanks, 200 μl dmso was added to each well, then the cek cells were incubated at 37°c for 10 min, and shaken gently at room temperature for 10 min to dissolved formazan precipitates, and od 570 was determined. using the mean values, the cell survival rate is calculated according to the following formula: in order to analyze the effects of hpe, hpw, and see on cells, cek cells cultured on 96 well plates were incubated with hpe, hpw, and see solutions at concentrations of 78.13, 39.06, 19.53 μg/ml at 37°c for 2 h, respectively, and then washed with d-hanks for three times. the cek cells were then infected with 100 tcid 50 ibv and cultured at 37°c for 30 h. the cell samples were frozen and thawed repeatedly for three times, and the total rna was extracted and reverse transcribed into complementary deoxyribonucleic acid (cdna). the relative mrna expression of ibv n gene was detected by real-time quantitative rt-pcr (qrt-pcr). in addition, according to the conventional method, the virus titer of cell samples was determined by tcid 50 . the specific operations were as follows: cek cells were cultured in 96-well tissue culture plates, and when the cells were full of monolayer, ibv virus was diluted to 10 −1 , 10 −2 , 10 −3 …, and 10 −12 times with serum-free m199, respectively. then the monolayer cek cells were washed with d-hanks solution three times, and the d-hanks solution was discarded. then the 100 µl ibv virus diluent as mentioned above was added to each hole, and each virus diluent repeated eight holes. the cells were treated by the same method with m199 culture as normal control. the culture plate was cultured in 5% co 2 and 37°c for 48 h. the cytopathic effect (cpe) was observed every 24 h, and the number of cpe pores and no cpe pores were recorded. according to the reed-muench method as previously described (guo et al., 2016; yang et al., 2018b) , the tcid 50 was calculated according to the number of cpe holes recorded. at the same time, the infected cek cells treated with 10 μg/ml rt and cek cells infected with ibv as control. in order to determine the impact of hpe, hpw, and see on ibv-infected cells, the cek cells cultured on 96 well plates were infected with 100 tcid 50 ibv at 37°c for 2 h, and then treated with hpe, hpw, and see solution at concentrations of 78.13, 39.06, 19.53 μg/ml at 37°c for 30 h. according to the above description, the relative mrna expression level and virus titer of ibv were detected. in order to study the direct effects of hpe, hpw, and see on the virus, 100 tcid 50 ibv was incubated with 78.13, 39.06, 19.53 μg/ ml hpe, hpw, and see solution at 37°c for 2 h, respectively. then cek cells were infected with the drug-treated ibv at 37°c for 30 h. as described above, the relative mrna expression levels and the virus titer of ibv were detected. in the above three experimental designs, the best antiviral way was selected for indirect immunofluorescence assay. after washing cek cells with pbs, fixed with 4% paraformaldehyde, then incubated with 0.1% glycine and quenched with 1% triton-x 100 for 10 min. after washing cek cells with pbs for 3 times, the cek cells were incubated with rabbit anti-ibv antibody (1:200) (prepared and stored in laboratory of pathology and anatomy, northeast agricultural university) for 1 h, and then incubated with fluorescent labeled goat antirabbit igg (1:500) (zhongshan, china) for 30 min in the dark. the fluorescent images were examined with a fluorescence microscope (ti-s, nikon, japan). at the same time, the infected cells treated with 10 μg/ml rt, and cek cells infected with ibv, and the mock cek cells were set as controls. one hundred and forty four 2-week-old spf chicks were randomly divided into hpe treated high dose group (hpe-th), hpe treated middle dose group (hpe-tm), hpe treated low dose group (hpe-tl), the ibv infected group (ibv), the ribavirin treated (rt) group, and normal control (nc) group with 24 chicks in each group. each group was completely isolated from the other groups, and housed in negative pressure-filtered air isolators under pathogen-free conditions. chicks from in three hpe-treated groups, the ibv-infected group, and the rt group were inoculated by nasal drops with 0.1 ml of 100 eid 50 of ibv m41 allantoic fluid per chick. chicks in the control group were inoculated with 0.1 ml sterilized negative allantoic fluid in the same manner. the chicks of the three hpe groups and the rt group were orally administered varying doses of three hpe (480 mg/kg, 240 mg/kg, 120 mg/kg) and ribavirin (60 mg/kg), respectively. both agents were administered twice daily for 5 days, beginning at 24 h after exposure to the virus. at the same time, the chicks of the ibv-infected group and the normal group were orally administered with pss. at 2, 4, 6, and 8 days postinfection, six chicks in each group were bled before euthanasia and necropsy at each designed day. the trachea and kidney tissues were collected and immediately extracted to obtain the total rnas. at the same time, tracheal and kidney in each group of chickens at 6 days postinfection were fixed in 10% buffer formaldehyde for histopathological assay. after washing in phosphate-buffered saline (pbs), the trachea and kidney tissues were fixed in 10% formaldehyde solution for 7 days, embedded in paraffin wax, cut into 3-μm-thick sections (leica rm4450, germany), and stained with he using standard histological staining procedure. the slides were examined by light microscopy (nikon, japan). the tissue slides were examined and evaluated according to the previous histopathologic scoring method for trachea (levisohn et al., 1986; todd et al., 1991) and kidney (reinhard et al., 1991; laudert et al., 1993; canales et al., 2012) with minor modification. for the trachea, the following morphological changes were included: cilia loss of mucous epithelium, extrusion of balloon-like cell and necrotic exfoliation of mucous layer, lymphocyte and heterophil infiltration in submucosal layer, hyperemia, and/or hemorrhage. for the kidney as followed: glomerular atrophy and fragmentation, tubular degeneration (granular and/or vacuolar degeneration) and necrosis (detached and broken epithelium), interstitial infiltration of lymphocytes and heterophils, hyperemia, and/or hemorrhage. the distribution and extent of the aforementioned lesions in the trachea and kidney of each chicken were scored as followed: 0, absence of lesion; 1, lesion represented in fewer than 10% of the involvement of tissue; 2, lesion represented in 10-50% involvement; 3, lesion represented in 50-90% involvement; and 4, lesion represented in more than 90% involvement. the pathological lesions of trachea and kidney were scored by 3 experienced veterinary pathologists (dr. guangxing li, ruili zhang, and xiaodan huang, northeast agricultural university, harbin, pr china), which were blinded to the identities of the experimental groups. the pathological injuries of trachea and kidney were quantitatively analyzed, and the statistical results were given in tables 2 and 3. treated cell samples were repeatedly frozen and thawed three times, and the equiponderant trachea and kidney tissues from each chick at various time points were prepared. then, the total rna was extracted using a universal rna extraction kit (thermo fisher scientific, usa) according to the manufacturer's instructions. all the concentration of rna and the a260/a280 ratio were determined with nanodrop 2000 spectrophotometer (thermo fisher scientific, usa), and the integrity of the extracted rna was detected by agarose gel electrophoresis. the total rnas were reverse transcribed into cdnas by pcr instrument (takara, japan) for qrt-pcr, and the cdnas were stored at −20°c. the relative quantification analyses was used to determine the levels of mrna expression of target genes, including n gene of ibv, il-6, tnf-α, il-1β, ifn-α, ifn-β, mda5, mavs, and nf-κb by an applied lightcycler 96 real-time pcr system (roche, switzerland). all primer pairs for real-time pcr detection are listed in table 1 . these primers were synthesized by genewiz biological technology co., ltd. (suzhou, china). the relative mrna expression levels of target genes were calculated by using the 2 −δδct method (yu et al., 2017 ). the amplification system was 2 μl cdna, 0.6 μl forward and reverse primers, 10 μl universal sybr green (rox) and 6.8 μl nuclease-free water, and the final volume of each system was 20 μl. the amplification reaction of qrt-pcr assays was conducted according to the following thermal profile: pre-incubation 1 cycles at 95°c for 600 s, followed by 2 step amplification: 42 cycles at 95°c for 15 s and 60°c for 30 s, followed by melting: 1 cycles at 95°c for 10 s, 65°c for 60 s, and 97°c for 1 s, followed by cooling 1 cycles at 37°c for 30 s. chromatography/diode array detector/ electrospray ionization-mass spectroscopy analysis of the hypericum perforatum ethyl acetate hplc/diode array detector/esi-ms analysis was performed on a waters 2695 hplc equipped with diode array detector and waters micromass zq esi-electrospray (waters, usa) operating in positive and negative ion mode. according to relevant reports (seger et al., 2004; jesionek et al., 2015) , the hplc condition was determined with a minor modification. hpe analysis was performed using an eclipse xdb-c18 column (100 mm x 4.6 mm, 5 μm, agilent technologies inc. usa). the mobile phase consisted of water (a) and acetonitrile (b) at a constant flow rate (1.0 ml/min). the solvent gradient elution method was as follows: 0-5 min, 5-95% b and 5-8 min, 95% b. the detection wavelength was 0-700 nm, the column temperature was room temperature, and the injection volume was 10 μl. the operating conditions of the mass spectrometer were dry gas temperature, 350°c, flow rate, 50 l/min; nebulizer pressure, 30 psi; sheath gas temperature, 250°c, flow rate, 10 l/min; fragmenter voltage, 100 v; capillary voltage, 3,500 v; mass range, 50 -1,100 d. determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the sample solution was prepared by dissolving 100.0 mg dried hpe in 25 ml mobile phase and filtered by 0.22 μm filter before hplc analysis. the content of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hpe was determined by hplc instrument and the chromatographic conditions described above. in the 25 ml analytical solvent, the hyperoside standard of 2.5 mg, or the quercitrin standard of 5.0 mg, or the quercetin standard of 2.0 mg, or the pseudohypericin standard of 1.0 mg, or the hypericin standard of 1.0 mg are dissolved to prepared stock solution, respectively. according to the guide of international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (singh, 2015; armutcu et al., 2018; hsi et al., 2019), the signal-to-noise ratio (s/n) of 3 and 10 were defined as the detection limit (lod), and the quantitative limit (loq), respectively. in order to detect the s/n, the stock solution of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were diluted to different concentrations, respectively. in order to obtain the standard curve, the stock solution of the above standard compounds was diluted into six appropriate dilution concentrations. quantification was conducted by using a six-point standard curve and an external standard method. intra-day and inter-day precision for hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were used to evaluate the repeatability and reproducibility of the established method. statistical analysis the number of the experiment repetition in all the experiment was three times. the experimental data was analyzed with spss 17.0 software (spss inc., chicago, il, usa). the results are expressed as the means ± standard deviation (sd). differences between groups were evaluated using the one-way analysis of variance (anova) of two tailed test. p < 0.05 were considered as statistically significant, and p < 0.01 were considered as highly significant. staining showed cell was survival (figure 1a) . at the same time, the cell survival rate measured by mtt method was close to 100%, which was further explained that 78 μg/ml of the drug had no significant effect on the cells (figure 1b ). when ibv was propagated in cek cells to the tenth generation, stable typical cytopathic effect (cpe) appeared at 36 h after ibv infection (figure 2a) . at the beginning, cells infected with ibv became round and refractive, then some cell exfoliated from the flask and left empty hole, some cells fused together and became multinuclear giant cells, indicating that ibv adapted to cek cells. the results of virus growth curve showed that ibv could infect and replicate in cek cells and reached the highest titer of 10 −5.8 tcid 50 per 100 μl at 36 h post-inoculation (figure 2b ). at the same time, the existence of ibv m41 was identified by rt-pcr ( figure 2c ). antiviral effect of supercritical extraction extract, hypericum perforatum ethyl acetate, and hypericum perforatum water in vitro the relative mrna expression level of ibv-n gene was detected by qrt-pcr and the virus titer of ibv was determined by tcid 50 to analyze the antiviral effect of hpe, hpw and see (figure 3) . it could be seen from figure 3 that under the maximum non-toxic concentration of the drug, the inhibition of hpe on ibv was significantly greater than that of hpw and see. hpw had a very weak effect on ibv and see had no effect on ibv. therefore, it was determined that the antiviral part of h. perforatum extract was ethyl acetate layer. as can be seen from figure 3a , in the three experimental designs, with the increase of hpe concentration, the mrna level of ibv decreased significantly in a dose-dependent manner. in addition, at the same drug concentration, the relative expression level of virus mrna was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and then hpe pre-treated virus before infection. it can be known from the figure 3b , with the increase of hpe concentration, the titer of ibv decreased gradually in a dosedependent manner. while, at the same drug concentration, the virus titer was the lowest when hpe directly treated the infected virus cells, followed by hpe pre-treated cells before infection, and followed by hpe pre-treated virus before infection. at the same time, the antiviral impact of hpe directly treated the ibv-infected cells at the concentration of 78.13 µg/ml, was similar to rt at the concentration of 10 µg/ml. therefore, hpe was selected for subsequent experiments in vivo and in vitro, furthermore the hpe directly treated the ibv-infected cells was selected in vitro experiments. the results of reverse transcription polymerase chain reaction identification of ibv m41 in cek cells. in which, m stands for deoxyribonucleic acid marker, n stands for polymerase chain reaction product for ibv n gene, w stands for negative water control. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 in order to further confirm the inhibitory effect of hpe on ibv-infected cells, the fluorescent signal of virus was detected by ifa (figure 4) . from the figure 4 , it can be seen that the cek cells infected with ibv produced a strong fluorescence signal at 30 h after infection. on the contrary, the fluorescence signal of cek cells infected with ibv and treated with hpe was weakened, and the fluorescence signal decreased in a dosedependent manner with the increase of hpe concentration. this further confirmed that hpe has a very good inhibitory effect on ibv infected cells. in order to study the effect of hpe on gene mrna expression induced by ibv infection in cek cells, the mrna expression level of related genes were determined, including mda5, mavs, ifn-α, ifn-β, nf-κb, il-6, and tnf-α. as can be seen from figure 5 , hpe treatment significantly affected the mrna levels of mda5, mavs, ifn-α, and ifn-β after ibv infection at 30 and 36 h, and the mrna expression levels of these genes changed similarly. these data suggested that hpe could increase mrna expression level of type i interferon in the late stages of ibv infection, which is possibly related to mda5 signaling pathway. in addition, hpe treatment significantly reduced the mrna expression levels of nf-κb, il-6, and tnf-α, which were up-regulated after ibv infection at 30 and 36 h. these data demonstrated that hpe could decrease the mrna expression of pro-inflammatory genes, which is possibly related to nf-κb signaling pathway. at 6 days after infection, the symptoms that appeared in the ibv infected group were sneezing, tracheal wet rales, mouth breathing, coughing, ruffled feathers, and frequently shaking. but the symptoms that appeared in the hpe and rt treatment group such as sneezing, tracheal wet rales, mouth breathing, coughing, feather wrinkling, and frequent trembling were mild. and the trachea and kidneys of different groups of spf chickens were conducted for histopathology assay. as can be seen from figure 6 , most of the tracheal cilia in the ibv infected group fell off, the mucosal epithelial cells exfoliated and disappeared, the submucosal structure was loosely arranged, and there was a certain amount of serous exudation, inflammatory cells, and red blood cells. in the hpe treatment group, with the gradual increase of the dosage, the shedding of tracheal cilia and epithelial cells was gradually decreased, there were a small number of inflammatory cells and erythrocytes, and the exudation of inflammatory serous fluid decreased. as shown in figure 7 , in the ibv infected group, a large number of renal tubular epithelial cells were degenerated and necrotic, the nucleus were concentrated, fragmented and dissolved, the renal tubular epithelial cells fell off into the lumen, dissolved or disappeared, the structure of renal tubules was figure 5 | the effects of hypericum perforatum ethyl acetate on the messenger ribonucleic acid (mrna) expression level of related genes in vitro. chicken embryo kidney (cek) cells were infected with 100 tcid 50 infectious bronchitis virus (ibv) at 37°c for 2 h, and then incubated with 78.13 μg/ml hpe. the cek cells were treated with m199, 100 tcid 50 ibv, and 78.13 µg/ml hpe as the control, respectively. subsequently, total rna was extracted from cek cell samples at 24, 30, and 36 h after treatment. the relative mrna expression of melanoma differentiation-associated protein 5 (a), mitochondrial antiviral signaling gene (b), interferon alpha (c), interferon beta (d), nuclear factor kappa beta (e), il-6 (f), and tumor necrosis factor alpha (g) were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < 0.05 and highly significant at **p < 0.01 when compared with the ibv-infected cell. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 incomplete, and there were a certain number of inflammatory cells and red blood cells in the interstitium. the wall of renal capsule was ruptured and a certain amount of inflammatory cells and serous exudation could be seen in the lumen of renal capsule. in the hpe treatment group, with the increase of the dosage, the exfoliation and dissolution of renal tubular epithelial cells decreased gradually, the structure of renal tubule was gradually complete, the number of red blood cells and inflammatory cells in the stroma was less, and the structure of renal capsule wall was more complete. there was little infiltration of inflammatory cells in the renal vesicle cavity. as can be seen from tables 2 and 3, the pathological scores of trachea and kidney in ibv group were highly significantly higher than those in nc group (p < 0.01). compared with the ibv group, the pathological scores of trachea and kidney in the hpe-th group and the hpe-tm group decreased highly 1.9 ± 0.2 0.8 ± 0.2 1.6 ± 0.3 1.7 ± 0.3 6.0 ± 0.4** hpe-tl 6 3.2 ± 0.3 3.1 ± 0.2 2.7 ± 0.3 0.8 ± 0.3 9.8 ± 0.5 hpe-tm 6 1.7 ± 0.2 1.8 ± 0.3 1.5 ± 0.3 0.8 ± 0.2 5.8 ± 0.4** hpe-th 6 1.6 ± 0.3 0.7 ± 0.3 0.9 ± 0.2 0.7 ± 0.3 3.9 ± 0.4** the difference was considered highly significant at **p < 0.01 vs. ibv infection group. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 significantly (p < 0.01), and the score was similar to that of the rt group. at the same time, with the increase of hpe dose, the score decreased in a dose-dependent manner. this indicated that hpe had better anti-ibv effect. to confirm the inhibitory effect of hpe, ibv mrna levels in trachea and kidney of different groups were measured by realtime qrt-pcr. as shown in figure 8 , compared with nc group, the expression level of mrna in trachea and kidney of spf chicks infected with ibv increased significantly. compared with ibv group, the expression level of mrna in trachea and kidney of spf chicks treated with hpe decreased significantly. with the decrease of hpe concentration, the expression level of mrna increased in a dose-dependent manner. with the increase of the days of ibv infection, the effect of hpe-th and hpe-tm group on the level of mrna expression was similar to that of rt group. these data indicated that infected chickens treated with hpe exhibited an overall reduction in viral mrna levels. it is well known that type i interferon, including ifn-α and ifn-β, plays a very important role in antiviral activity. as can be seen from figures 9 and 10 , the relative mrna expression of il-6, tnf-α, ifn-α, ifn-β, mda5, mavs, and nf-κb in the trachea and kidney were up-regulated after ibv infection, which was consistent with previous studies (he et al., 2016; chhabra et al., 2018) . in hpe treatment group, the mrna expression levels of ifn-α and ifn-β in the trachea were up-regulated in the early stage of ibv infection, and the changes of ifn-α and ifn-β were consistent with the mrna expression of mda5 and mavs in the trachea. meanwhile, in the hpe-treated group, the mrna expression levels of ifn-α and ifn-β in the kidney were up-regulated in the late stage of ibv infection. in addition, the changes of ifn-α and ifn-β were similar to the mrna expression levels of mda5 and mavs in the kidney. these data suggested that hpe may up-regulate mrna expression levels of ifn-α and ifn-β in trachea and kidney via the mda5 signaling pathway. 3.7 ± 0.2 3.9 ± 0.1 2.5 ± 0.4 1.7 ± 0.2 11.8 ± 0.5 nc 6 0.6 ± 0.1 0.6 ± 0.1 0.9 ± 0.2 0.1 ± 0.1 2.2 ± 0.2** rt 6 0.9 ± 0.1 1.4 ± 0.2 1.4 ± 0.3 0.6 ± 0.2 4.3 ± 0.3** hpe-tl 6 3.4 ± 0.3 3.8 ± 0.3 2.3 ± 0.4 1.6 ± 0.3 11.1 ± 0.6 hpe-tm 6 1.2 ± 0.2 2.0 ± 0.4 1.5 ± 0.4 0.8 ± 0.2 5.5 ± 0.5** hpe-th 6 0.8 ± 0.2 1.2 ± 0.4 1.0 ± 0.1 0.7 ± 0.2 3.7 ± 0.4** the difference was considered highly significant at **p < 0.01 vs. ibv infection group. figure 8 | the effects of hypericum perforatum ethyl acetate (hpe) on infectious bronchitis virus (ibv) messenger ribonucleic acid (mrna) expression levels of trachea (a) and kidney (b). spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea and kidney at 2, 4, 6, and 8 day post-infection. the relative mrna expression of ibv was determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < 0.05 and highly significant at **p < 0.01. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 at the same time, it is well known that inflammatory factors play an important role in inflammation. it also plays a very important role in ibv infection. as can be seen from figures 8 and 9 , in the hpe-treated group, the mrna expression levels of il-6 and tnf-α in the trachea and kidney were down-regulated, and the changes were consistent to nf-κb mrna expression levels, indicating that hpe may be down-regulated the mrna expression levels of il-6 and tnf-α by nf-κb signaling pathway. therefore, hpe may exert an anti-ibv effect by increasing the mrna expression levels of type i interferon and reducing mrna expression levels of proinflammatory factors il-6 and tnf-α, and this may be related to the mda5 signal pathway and nf-κb signal pathway. (figure 11) . the mass spectra of these compounds were carefully examined (figure 12 ) and compared with the standard and reference data (seger et al., 2004; cao et al., 2011) and found to have five peaks in the hpe (table 4) . these peaks corresponded to hyperoside (peak 1), quercitrin (peak 2), quercetin (peak 3), pseudohypericin (peak 4), and hypericin (peak 5). determination of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin in the hypericum perforatum ethyl acetate the calibration curves of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin were as followed: y = 20, 579x + 25,971, y = 18,877x + 41,654, y = 250,933x−272,745, y = 76,343x + 16,472, and y = 73,815x + 3,490 .7, where y is peak area, x is sample concentration, and the concentration ranges were 10-60, 40-140, 10-60, 1-6, and 6-36 μg/ml, respectively. and the calibration curve was in accordance with a linear regression (r 2 = 0.9993-0.9997). the lod (s/n = 3) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of 0.64 to 0.78 μg/ml. the loq (s/n = 10) of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin was in the range of 0.16 to 0.22 μg/ml. by measuring the peak area and retention time of standard compounds to evaluate the intra-day and inter-day precision, their relative sd was less than 1%. the results showed that the hpe contained 0.92% hyperoside, 1.37% quercitrin, 10.16% quercetin, 2.55% pseudohypericin, and 3.46% hypericin. ibv has a variety of known strains, these strains continue to mutate, and more mutants continue to recombine, resulting in more diverse and complex genotypes and serotypes of ibv (bande et al., 2015; feng et al., 2015; laconi et al., 2019) . due to the poor cross-protection of vaccines between different serotypes, it is difficult to prevent and control ibv infection. therefore, development of an effective antiviral therapy is a crucial strategy for treating ibv infection. in this study, the anti-ibv effect of the drug was studied by three kinds of methods, qrt-pcr, determination of tcid 50 , and indirect immunofluorescence (ifa). qrt-pcr is a method of measuring the total amount of products after each reaction cycle with fluorescent chemicals in dna amplification. ifa is a method to detect the presence of specific antigens in cells after the cells were treated with fluorescent-antibodies. tcid 50 is the amount of infection in which 50% of the cells are infected by the virus. the anti-virus effect of drugs is often detected by qrt-pcr (schnepf et al., 2009; li et al., 2011; zhang et al., 2018) , ifa (xu et al., 2008; zhang et al., 2017a) and tcid 50 (xu et al., 2008; zhang et al., 2013) in antiviral research. and these antiviral drug reports are similar to our research, so we also used these methods to detect the anti-ibv effect of h. perforatum. although qrt-pcr and ifa could not detect live viral particles, they were used as a supplement in this study, and in this study we used the method of tcid 50 , which is a general method for the detection of live virus (leclercq et al., 2014; petiot et al., 2018) . therefore, the results of antiviral studies in vitro are also credible in this study. in addition, although the best way to culture ibv is to use chicken embryos or chickens, the ibv replication in cek cells was tested in this study. because if the virus doesn't replicate well, viral particles can also be reduced or even die, which in turn affects the antiviral effect of the drug. in this study, we showed that ibv can replicate well in cek cells (figure 2) , which is consistent with the research report (zhang et al., 2018) , and hpe had potential utility as antiviral agents against ibv. our results showed that the hpe can reduce virus mrna expression level (figures 3a and 8) and virus titer ( figure 3a) . additionally, the see had no inhibitory effects and the hpw had poor inhibitory effects (figure 3) . the results suggest that the hpe, which contains an enrichment of the main effective substances, was the bioactive fraction of h. perforatum extracts. moreover, the hpe had anti-ibv activity, such as reducing the fluorescence signal produced by ibv (figure 4) , and alleviating the pathological injury of trachea and kidney caused by ibv in chickens (figures 6 and 7) . when came to evaluation of antiviral effects, ribavirin was used as control drugs. the inhibitory effect of 78.13 µg/ml hpe was comparable to that of 10 µg/ml ribavirin in vitro, and 480 mg/kg hpe was comparable to that of 60 mg/ kg ribavirin in vivo. in the efficacy of antiviral effects, as in the reporting of others herbs (choi et al., 2016; sun et al., 2016; choi et al., 2017; yi et al., 2018; luo et al., 2019) , the effects of the herbal extract were lower than ribavirin even under several times higher dosage against ibv mrna level, virus titer, and histopathological injury caused by ibv. however, it is logical since ribavirin is pure compared to the extracts containing many inactive compounds. some studies have shown that h. perforatum extract have an antiviral effect on influenza a virus and hiv (barnes et al., 2001; birt et al., 2009; pu et al., 2012) , suggesting that hpe have the potential to be developed and used as antiviral drugs. in this study, we found that h. perforatum extract had significantly antiviral effect on ibv in vitro and in vivo, respectively. in addition, anti-ibv effect of hpe might correlate figure 9 | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of genes in trachea. spf chickens were infected with ibv, followed by oral administration of hpe and ribavirin, respectively. total rna were subsequently extracted from trachea at 2, 4, 6, and 8 day postinfection. the relative mrna expression of (a) melanoma differentiation-associated protein 5, (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il-6, and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < 0.05 and highly significant at **p < 0.01. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 figure 10 | effect of hypericum perforatum ethyl acetate (hpe) on the messenger ribonucleic acid (mrna) expression of gene in kidney. specific pathogen-free (spf) chickens were infected with infectious bronchitis virus (ibv), followed by oral administration of hpe and ribavirin (rt), respectively. total rna were subsequently extracted from kidney at 2, 4, 6, and 8 day post-infection. the relative mrna expression of (a) melanoma differentiation-associated protein 5, (b) mitochondrial antiviral signaling gene, (c) interferon alpha, (d) interferon beta, (e) nuclear factor kappa beta, (f) il-6, and (g) tumor necrosis factor alpha were determined by quantitative reverse transcription polymerase chain reaction. the differences between means were considered significant at *p < 0.05 and highly significant at **p < 0.01. frontiers in pharmacology | www.frontiersin.org october 2019 | volume 10 | article 1272 with mda5 and nf-κb signaling pathway. belonging to acid inducible gene i (rig-i)-like receptors (rlrs) in pattern recognition receptors (prrs), it is well known that mda5 plays a crucial role in avian respiratory disease progression (yu et al., 2017) , whose function is to identify various pathogenassociated molecular patterns (pamps), and activates mitochondrial antiviral signaling gene (mavs, also called ips-1/visa/cardif) (lin et al., 2006) , activated mavs can recruit downstream interferon regulatory factor-3/7 (irf3/ irf7) (shi et al., 2015) , leading to the rapid production of type i ifns (xu et al., 2005; soulat et al., 2008) . nf-κb system is a master transcription factor in the recognition signaling and host responses to immune attacks (deng et al., 2018) . it is well known that nf-κb mediates inflammation, and several cytokine are also linked to nf-κb signaling to enhance the inflammatory responses, such as tnf-α, il-6, and il-1β (salminen et al., 2008) . several reports have pointed out the mda5 signaling pathways and innate immune cytokines were activated after infection with ibv m41 strain (he et al., 2016) . mda5 signaling pathway was disrupted by cleavage of the adaptor protein mavs in the js/2010/12 strain of ibv infection (yu et al., 2017) . the type i ifn response plays a critical role in resisting saibk2 strain of ibv (yang et al., 2018a) . mda5 signaling pathways and innate immune cytokine (nf-κb and irf3) were induced after ibv-m41 strain infection (zhang et al., 2017b) . on the other hand, the report had shown that h. perforatum extract significantly downregulated the concentration of il-6 and tnf-α in lung tissue for mice infected with an influenza a virus (pu et al., 2012) . in this study, we found that mrna expression levels of mda5, mavs, ifn-α, ifn-β, nf-κb, tnf-α, and il-6 were significantly up-regulated after ibv infection in vitro and in vivo (figures 5, 9, and 10) . meanwhile, we found that hpe may be up-regulate mrna levels of ifn-α and ifn-β by mda5 signaling pathway, and down-regulate mrna expression levels of il-6 and tnf-α through the nf-κb signaling pathway (figures 5, 9, and 10) , suggesting that hpe may be inhibit ibv by affecting innate immune cytokines. because of remarkable antiviral effect on ibv, we analyzed the active components of hpe. the mass spectrum of hyperoside contained a negative quasi-molecular ion at 463.74 m/z [m-h] − (figure 12b ), in accordance with the previous literature (cao et al., 2011) . the mass spectrum of figure 12b figure 12c) . in addition to a positive quasi-molecular ion at 543.29 m/z [m+na] + ( figure 12d ) and a negative quasi-molecular ion [m-h] − at 519.08 m/z (figure 12e) , confirming the presence of the pseudohypericin. finally, the mass spectrum of hypericin shows a positive quasi-molecular ion [m+h] − at 505.44 m/z ( figure 12f ) and a negative quasi-molecular ion [m-h] − at 503.33 m/z ( figure 12g) . according to the results in figure 3 , the hpe is the main active fraction, containing abundant levels of hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin (table 4 and figure 11 ). our study provides, for the first time, clear evidence that the extract of h. perforatum, containing hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, possess anti-ibv activities. furthermore, its anti-ibv effect may be associated with reduced mrna expression levels of the proinflammatory cytokines il-6, tnf-α by nf-κb signaling pathway, and related to up-regulate mrna expression levels of type i interferon through the mda5 signaling pathway, and could be useful for the development of new antiviral agents. however, further studies are required to elucidate its detail mechanism of action. all 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of licorice, a widely-used chinese herb the infectious bronchitis coronavirus envelope protein alters golgi ph to protect spike protein and promote release of infectious virus construction and immunogenicity of novel chimeric virus-like particles bearing antigens of infectious bronchitis virus and newcastle disease virus visa is an adapter protein required for virus-triggered ifn-beta signaling in vitro inhibition of classical swine fever virus replication by sirnas targeting npro and ns5b genes attenuation, safety, and efficacy of a qx-like infectious bronchitis virus serotype vaccine induction of innate immune response following introduction of infectious bronchitis virus (ibv) in the trachea and renal tissues of chickens porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type i interferon andrographolide inhibits mechanical and thermal hyperalgesia in a rat model of hiv-induced neuropathic pain frontiers in pharmacology | www.frontiersin.org october chinese herbal medicine compound yi-zhi-hao pellet inhibits replication of influenza virus infection through activation of heme oxygenase-1 characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens avian infectious bronchitis virus disrupts the melanoma differentiation associated gene 5 (mda5) signaling pathway by cleavage of the adaptor protein mavs in vitro inhibition of vesicular stomatitis virus replication by purified porcine mx1 protein fused to hiv-1 tat protein transduction domain (ptd) chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus cellular immune response in chickens infected with avian infectious bronchitis virus (ibv) astragalus polysaccharides inhibit avian infectious bronchitis virus infection by regulating viral replication the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 chen, muhammad, zhang, ren, zhang, huang, diao, liu, li, sun, abbas and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-256444-grw5s2pf authors: lai, michael m.c.; cavanagh, david title: the molecular biology of coronaviruses date: 1997-12-31 journal: advances in virus research doi: 10.1016/s0065-3527(08)60286-9 sha: doc_id: 256444 cord_uid: grw5s2pf publisher summary this chapter discusses the manipulation of clones of coronavirus and of complementary dnas (cdnas) of defective-interfering (di) rnas to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. the nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense rna. its size ranges from 27 to 32 kb, which is significantly larger when compared with other rna viruses. the gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. this soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. viruses. the coronavirus polymerase gene alone (20-22 kb) is about the same size as the whole of the picornavirus (-8 kb) and vesicular stomatitis virus (-11 kb) genomes added together. the gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. this soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet (latin corona, hence the name of the genus). coronaviruses are responsible for a number of economically important diseases. avian infectious bronchitis virus (ibv) was the first coronavirus to be isolated, from the domestic fowl, and propagated in the 1930s. in addition to respiratory disease, which can predispose chickens to possibly lethal secondary bacterial infections, some strains also cause nephritis (king and cavanagh, 1991; cook and mockett, 1995) . porcine transmissible gastroenteritis virus (tgev) causes devastating disease in newborn pigs, with mortality often approaching 100% (enjuanes and van der zeijst, 1995) . intriguingly, there are also naturally occurring mutants [i.e., porcine respiratory coronavirus (prcv)] of tgev which cause only mild respiratory disease and no enteritis. several other coronaviruses also cause enteritis: bovine coronavirus (bcv), turkey coronavirus (tcv; bluecomb virus), feline coronavirus (fcv), canine coronavirus (ccv) and porcine epidemic diarrhea virus (pedv). fcv may also cause feline infectious peritonitis. an fcv has been isolated from a cheetah and bcvs from wild sambar deer and waterbuck (tsunemitsu et al., 1995) . these bcvs caused enteritis when inoculated into domestic calves. humans are known to suffer from two very different coronaviruses, human coronavirus (hcv) oc43 and hcv 2293, both of which are a cause of the common cold. there is evidence for the presence of coronaviruses in tissues taken from multiple sclerosis (ms) patients (reviewed by cavanagh and macnaughton, 1995) . this inflammatory, demyelinating neurological disease is associated with autoreactive t lymphocytes sensitized to myelin components of the central nervous system. recently, talbot and colleagues (1996) have demonstrated that many cd4' t-cell lines derived from ms patients showed a human leukocyte antigen-(hla)-dr-restricted, crossreactive pattern of antigen activation after in vitro selection of either myelin basic protein or hcv-229e proteins, suggesting that molecular mimicry between hcv and myelin may be a n immunopathological mechanism in ms. other coronaviruses [some strains of murine hepatitis virus (mhv) and porcine hemagglutinating encephalomyelitis virus (hev)] are well-known causes of neurological diseases, and mhv has been studied for many years in this context (dales and anderson, 19951 , although many mhv strains cause primarily hepatitis. the 1970s and early 1980s was the period in which coronavirus virion proteins and nested-set arrangements of mrnas were identified and the discontinuous nature of coronavirus transcription was initially demonstrated. the first published sequence of a coronavirus gene appeared in 1983, starting an era in which the whole of the genomes of four coronaviruses were clonedin piecesand sequenced. this decade has seen the manipulation of these clones, and of complementary dnas (cdnas) of defective-interfering (di) rnas, to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. this review is largely concerned with these areas. some topics are notable by their absence, space not permitting their inclusion. for example, the elucidation of the molecular basis of the antigenic properties of the large surface (spike) glycoprotein and its role in tissue tropism has been omitted. for these topics and all others both within and without the compass of our review for which a concurrently comprehensive and in-depth treatise is desired, the reader is referred to the book edited by siddell (1995a) . individual chapters in that book will be referenced a t the appropriate places in this review. all coronaviruses belong to one genus, coronavirus, within the family coronaviridae . initially, serological analysis was used to differentiate coronavirus species and showed that they could be divided into four antigenic groups (holmes, 1990) . the species and group divisions were subsequently refined by monoclonal antibody analysis and nucleotide sequencing, which revealed the close relatedness between tcv and bcv, resulting in the current classification of three antigenic groups (table i) . the same groupings emerge regardless of which structural protein sequences are compared (siddell, 1995b) . within group 1, tgev, fcv, and ccv are particularly closely related, all the members of group 2 being tightly clustered. the sole member of group 3, ibv, not only differs extensively from all other coronaviruses but also exhibits extensive variation within the species. the coronaviridae had remained a monogeneric family for a quarter of a century, until an accumulation of observations which showed that many of the features thought to be characteristic of the coronaviridae applied equally well to the genus torovirus, which had not been officially assigned to a family (figs. 1 and 2, table 11 ). therefore, in 1993, the international committee for the taxonomy of viruses (ictv) formally expanded the coronaviridae to include torovirus . the bringing together of coronavirus and torovirus was not the end of the taxonomic story; another family, arteriviridae, shared important characteristics in relation to the genome, structure, and strategies of transcription and translation (table i1 ) (plagemann and moennig, 1992; snijder and spaan, 1995) . however, the distinct morphology of the arteriviruses (fig. 11 , and their underlying differences from the coronaviruses in the size of the genome (fig. 2) and structural proteins (table ii) , precluded their inclusion in the coronaviridae. the common features uniting the two families (table 11 ) are at the heart of a proposal that an order be created to contain coronaviridae and arteriviridae to reflect their common features and, probably, their evolutionary relationships. the name nidovirales, from the latin nidus, meaning nest, has been designated for the order, as all members produce mrnas in an extensive nested-set arrangement. the remainder of this review is restricted largely to the coronaviruses. coronaviruses are enveloped, more or less spherical, approximately 120 nm in diameter, with a prominent fringe of 20-nm-long1 petalshaped surface projections (spikes) composed of a heavily glycosylated type i glycoprotein, spike protein ( s ) (fig. 1) . a subset of the coronaviruses (table i ) has an additional layer of short spikes (caul and egglestone, 1977; dea and tijssen, 1988) , which consist of hemagglutininesterase (he) protein, also a type i glycoprotein. these small spikes are not essential for viral infectivity. both the large and small spikes are anchored in the envelope, which is a lipid bilayer formed by virus budding from intracellular membranes. the envelope is associated with, in addition to the s and he proteins, a smaller type i11 integral membrane protein (m), which spans the envelope three times. an even smaller protein [envelope (e) or small membrane (sm) protein] has recently been shown to be an integral membrane protein of the viral envelope. inside the envelope is a ribonucleoprotein (rnp) core, which comprises the rna genome and a single species of nucleocapsid protein n. electron microscopic observation of viral rnp showed a long helix of 14 to 16 nm (macnaughton et al., 1978; sturman and holmes, 1983) . a very recent study of intact and detergent-treated tgev virions (risco et al., 1996) by negative-staining, ultrathin sectioning, freezefracture, immunogold mapping and cryoelectron microscopy showed a surprising new feature of coronavirus particles, namely, a spherical, probably icosahedral, core inside the virion (fig. 3) . these internal cores comprise not only the n protein and rna but also the m protein, m being the major core shell component. disruption of the cores released helical nucleocapsids. the presence of an icosahedral core in the coronavirus virion had heretofore been unsuspected. this core structure was also detected with mhv virion (risco et al., 1996) . this surprising new finding gives us cause to reconsider our view of coronavirus architecture. thus, the precise structure of the core and rnp inside the virion is not certain. toroviruses and coronaviruses have a similar morphology and virion composition ( fig. 1, table 11 ) but are distinguishable in a number of ways (table 11 ) (weiss and horzinek, 1987; horzinek, 1993, 1995; koopmans and horzinek, 1994) , necessitating their inclusion in separate genera. the morphology of the arteriviruses is substantially different from that of coronaviruses and toroviruses, particularly in having an icosahedral rnp core ( fig. 1 ) ; hence, a separate family is maintained for arteriviruses. however, the recent discovery of the icosahedral core for coronavirus (risco et al., 1996) may have blurred this distinction. the s glycoprotein is the outermost component of the virion, and is responsible for the attachment of the virus to cells (collins et al., 1982; godet et al., 1994; kubo et al., 1994) and for instigating the fusion of the virus envelope with cell membranes. it is the primary target for the host's immune responses; neutralizing antibodies are induced mainly by s (collins et al., 19821 , and immunization in animals with s alone can induce protection from some coronaviruses (ignjatovic and galli, 1994; torres et al., 1995) . within a coronavirus species, sequence variation is usually exhibited more by s than by any other structural proteins; the variation of the s protein sequence probably confers a selective advantage in immune animals. these and other aspects have recently been reviewed in detail . the s protein is large, ranging from some 1160 (ibv) to 1452 amino acids (fcv). there are many potential n-linked glycosylation sites (21 to 351, most of which have glycans attached. the s preproprotein has a n-terminal signal sequence and a membrane-anchoring sequence near the c terminus (fig. 4) . the s protein may be cleaved into s1 and s2 subunits; the extent of its cleavage varies greatly among the species (cavanagh, 1995) . a high proportion, up to loo%, of the s protein is cleaved in some coronaviruses (ibv, mhv, bcv, tcv, pedv) (cavanagh, 1983a) ; none is cleaved in others (tgev, fcv, ccv) (garwes and pocock, 1975) ; and very little of the s protein of hcv-2293 and hcv-oc43 is cleaved, although the s of oc43 is completely fig 1. models of the virions of a coronavirus, a torovirus, and an arterivirus. the he protein is present only in antigenic group 2 coronaviruses (see table i ). reproduced with permission from cavanagh et al. (1994) . present in only a subset of coronaviruses (table i) . may have an isometric core in addition (risco et al., 1996) . no such protein described. table i ) and with respect to the number and position of nonstructural protein genes. the polymerase genes encode two orfs, l a and lb, which overlap. l, leader sequence; he, hemagglutinin-esterase; s, spike; e, small membrane protein; m, integral membrane protein; n, nucleocapsid protein; an, poly(a) tail; g, and gl, small and large glycoproteins, respectively. risco et al. (1996) for tgev. this model illustrates the observation that internal cores (ic), possibly icosahedral, were observed inside virions of tgev. the cores comprise the helical ribonucleoprotein (nc) (genome rna + n protein) and the m protein. reproduced with permission from risco et al. (1996). cleaved if trypsin is present (hogue and brian, 1986) . the extent of s cleavage depends on the cell type (frana et al., 1985) . cleavage generates two glycopolypeptides, n-terminal s1 and c-terminal s2, the latter being acylated . s1 is probably linked to the s2 subunits by noncovalent linkage: trypsin treatment of mhv virions caused cleavage of all s proteins without disrupting the spikes ; however, s1 can be released from virion by either urea or mild alkali treatment (cavanagh and davis, 1986; weismiller et al., 1990) . among the coronavirus genus as a whole, the s2 polypeptide is much more conserved than s1. regions of up to 30% amino acid identity (particularly in the transmembrane domain) exist between the s2 polypeptides of coronaviruses in the different antigenic groups, whereas and (b) (schmidtet al., 1987) . the amino acid numbering has been normalized with respect to that of the longest known mhv s protein, that of mhv4 (jhm) ( s . e. . (a) the protein has an amino-terminal signal peptide (sp) and a transmembrane (tm) sequence near the c terminus. the glycosylated propolypeptide is cleaved a t a basic connecting peptide (cp) to yield glycopolypeptides s1 and s2. the locations shown are those of three mutations present in mutants of mhv4 recovered from a persistently infected neural cell line, the mutants requiring a ph of 5.5-6.0 for membrane fusion (gallagher et al., 1991) . (b) s of another mhv-jhm (schmidt et al., 1987) , which has a 141-amino acid deletion with respect to (a). bacterial expression products containing residues 33-40 and 1264-1276 bound mab 11f and 10g, respectively, both of which neutralize virus infectivity and inhibit membrane fusion. the arrow indicates the positions of amino acid substitutions in jhm mab 11f-resistant mutants (grosse and siddell, 1994) . a peptide comprising residues 900-908 bound another mab that neutralized virus and inhibited fusion (luytjes et al., 1989). there is almost no conservation of the s1 sequence. furthermore, comparison of s1 sequences among strains of a given species, or between species of a given group, reveals hypervariable regions, which include frequent deletions, mutations, or recombination (cavanagh et al., 1988; s. e. parker et al., 1989; banner et al., 1990; gallagher et al., 19901, suggesting that this region is externally exposed and not essential for the structure. the s2 polypeptide has two regions with a seven-residue periodicity, forming heptad repeats (fig. 4) indicative of a coiled-coil structure (de groot et al., 1987) . indeed, current evidence suggests that the mature s protein forms an oligomer; for tgev, it is probably a trimer (delmas and laude, 1990 ). however, a dimer structure has been proposed for ibv s protein (cavanagh, 1983~) . therefore, the oligomeric s protein is envisaged as being anchored in the membrane by a n a-helical region near to the c terminus of s2. just beyond the outer membrane surface is the shorter (minor) repeat structure predicted to be an a! helix of 5-7 nm. the major repeat indicates a helix of 10-13 nm, which may form the narrow stalk of the spikes (de groot et al., 1987) . all coronavirus s2 proteins have a highly conserved eight-residue sequence kwpww/yvwl, the last five residues of which probably form the beginning of the membrane-spanning domain. terminating 10 residues upstream of kwp is a leucine-zipper motif, the length varying from three to five heptad repeats (britton, 1991) . the highly conserved sequences of s2 may play a role in forming the stalk, which has a more rigid structure. in contrast, the s1 domain is predicted to form the globular portion of the spikes, consistent with its highly variable nature. the s protein has two important biological activities for the virus: a. induction of membrane fusion. this activity may be required for viral entry into cells or for cytopathic effects. expression of the recombinant s gene has provided unequivocal evidence that the s protein alone is sufficient to cause membrane fusion, as shown by syncytium formation (de groot et al., 1989; pfleiderer et al., 1990; yo0 et al., 1991; taguchi, 1993) . several regions of the s protein, widely separated in a linear sense, have been implicated in the membrane fusion process by the following observations: (1) s2 of bcv expressed in insect cells caused fusion (yo0 et al., 1991) . (2) a monoclonal antibody that inhibited cell fusion was shown to bind to the s2 domain of mhv ( fig. 4) (luytjes et al., 1989) . (3) changes at three s2 residues (1067, 1094, and 1114 in the mhv4 s protein; fig. 4 ) were associated with a change from a requirement for a neutral ph to an acidic ph for fusion (gallagher et al., 1991) . (4) two bacterial expression products containing residues 33-4061) and 1264-1276 6 2 ) ofthe jhm strain ofmhvinducedmonoclonal antibodies 11f and 10g, respectively, both of which inhibited fusion ( fig. 4) . ( 5 ) chemical modifications of the cysteine residues, specifically residue 1163 in the ectodomain of s2, reduced the fusion activity of the jhm strain of mhv (gallagher, 1996) . this result also suggests strain-specific differences in the conformation of the s protein, since the fusion activity of the a59 strain of mhv was not affected by this modification. (6) some mutations to cysteine residues within the transmembrane domain of s adversely affected fusion, suggesting that the transmembrane domain is involved in conformational changes that are associated with fusion activity (bos et al., 1995) . these results combined suggest that the s2 ectodomain contains the major determinants for membrane fusion; however, s2 does not contain hydrophobic domains typical of fusion proteins. thus, several disparate regions, including some in the s1, may contribute to the fusion activity, probably because some of these regions are juxtaposed in the threedimensional structure or can affect the overall conformation of the spikes. interestingly, monoclonal antibody-resistant mutants of the jhm strain ofmhvselected with antibody 11f had mutations not a t the antibody-binding site (residues 33-40 of s l ) , but at a distant site, i.e., residues 1109-1116 in the s2 domain (grosse and siddell, 1994) (fig. 4) , suggesting that s is folded such that regions which are widely separated in the linear sense are juxtaposed to form functional domains. early studies of coronvirus-induced cell-cell fusion suggested that only cleaved s was able to promote cell fusion . more recent studies in which mhv s proteins with mutated s 1 3 2 connecting peptides were expressed have shown that cleavage is not essential for fusogenic activity, although cell-cell fusion is more efficient when the s protein is cleaved (stauber et al., 1993; taguchi, 1993; bos et al., 1995) . furthermore, naturally occurring mutants of mhv, derived from persistently infected mouse cells, which are defective in s cleavage, have delayed fusion activity (gombold et al., 1993) . expression of the feline infectious peritonitis virus (fipv) s protein, which is not cleaved a t all, also resulted in syncytia formation (de groot et al., 1989) . these results indicate that s protein cleavage is not required for but can enhance membrane fusion. whether membrane fusion activity, as manifested by syncytia formation, is required for viral infectivity has not been established. there are mhv strains (e.g., mhv-2) that do not cause syncytia formation in cultured cells; however, these viruses may be able to cause virus-cell membrane fusion within the infected cells. b. receptor binding. monoclonal antibodies (mab) against the s protein of most coronaviruses can neutralize viral infectivity; thus, it is assumed that the s protein mediates virus binding to the receptors on target cells. indeed, the s protein or a portion of it can bind to the viral receptor molecules in uitro. this has been demonstrated for mhv and tgev s proteins (godet et al., 1994; kubo et al., 1994) . the binding domain has been mapped to the n-terminal 330 amino acids of mhv s1 protein. site-directed mutagenesis of this region showed that mutations of the residues at position 62 and positions 212, 214, and 216 abolished the binding of the protein to the receptor (suzuki and taguchi, 19961 , suggesting that the receptor-binding site might comprise discontiguous regions in the linear sense. the s2 subunit is not involved in receptor binding (taguchi, 1995) . the receptor-binding sites of tgev s protein have been mapped to a 223-residue region (aa 506-729) of the s1 (godet et al., 19941 , which overlaps with a n epitope for a neutralizing mab. this neutralizing mab was able to block the binding of the 223-residue polypeptide to the receptor; conversely, the receptor did not block the binding of the mab to this polypeptide, suggesting that the receptor-binding determinants and the neutralizing epitopes are distinct and are part of a domain of s whose configuration is independent of the remainder of the s protein. s proteins of bcv and hcv-oc43 bind to 9-o-acetylneuraminic acid (schultze et al., 1991a) ; this binding is required for viral infection. the significance of this binding will be discussed in section v,b on virus attachment. intriguingly, several coronavirus s proteins share some sequence identity with the receptor for the fc fragment of mammalian immunoglobulins (fcy receptor). thus, mab to the fcy receptor could immunoprecipitate s protein from the mhv-infected cells, and s could bind to the fc fragment of immunoglobulin. this molecular mimicry was first demonstrated for mhv and, more recently, for bcv and tgev as well (oleszak and leibowitz, 1960; oleszaket al., 1992 oleszaket al., ,1995 . it may play a role in modulating viral pathogenicity. this potential function is significant because expression of the s protein in the infected cells induces not only humoral antibodies but cellular immunity as well (welsh et al., 1986) ; the potential binding of s to the fc fragment of immunoglobulin may modulate these immune responses. the m protein is one of only two of the structural proteins [the other being the e protein (see below)] that are essential for the production of coronavirus-like particles. the sequence of the m protein reveals that the m polypeptides comprise 225-230 amino acids, except for some members of the tgev group, which have an additional 30 or so residues a t the amino terminus, forming a cleavable membrane insertion signal. the amino-terminal 20 or so residues of the mature m protein of all the coronaviruses are hydrophilic, exposed at the virion surface, and have a small number of glycosylation sites. glycans are of the n-linked type for ibv and the tgev group and o-linked for the mhv group (rottier, 1995) . the remainder of the n-terminal half of the molecule forms three helical membrane-spanning domains, although a mutant m protein which lacked all three of the membrane-spanning domains did associate with membranes in uitro (mayer et al., 1988) . the structure of the c-terminal half is uncertain, but it is believed to be largely situated on the inside of the viral envelope, based on protease susceptibility cavanagh et al., 1986b) and sequence-based predictions (armstronget al., 1984; rottier et al., 1986) . however, some m molecules of tgev virions have the c terminus exposed a t the virion surface (laviada et al., 1990; risco et al., 1995) . moreover, mab specific for the c-terminal46 amino acids of m neutralized tgev virions in the presence of complement and caused antibody-mediated, complementdependent cytolysis of tgev-infected cells (risco et al., 1995) . studies with mutant mhv m proteins expressed from vaccinia virus recombinants had shown that some had the n terminus and others the c terminus a t the luminal side of the endoplasmic reticulum, equivalent to the outer surface of virions (locker et al., 199213) . some molecules of one mutant m protein had both termini at the luminal surface, and other molecules had both termini at the cytoplasmic surface (locker et al., 199213, 1994) . thus, the precise topology and the structural role of the m protein are still not certain. recent studies have shown that some m proteins are also associated with the rnp core of tgev and constitute the outer shell of the internal core (risco et al., 1996) . this core-associated m can be clearly separated from the viral envelope. therefore, m may play a dual structural role in forming both the envelope and the internal core of the virion. several properties of the m protein suggest that it is involved in virus particle assembly: (1) the m protein binds to the purified nucleocapsid in uitro (sturman et al., 1980) . (2) when the m protein was expressed alone, it was localized in the golgi complex, near the location where virus particles bud (tooze et al., 1984; tooze and tooze, 1985) . however, recent studies showed that the site of m protein retention in the golgi was slightly different from that for viral particle budding (klumperman et al., 19941, suggesting that additional factors are involved in virus particle assembly. this will be discussed in section v,h on virus assembly. the m protein of tgev has a n additional biological activity: induction of a-interferon (charley and laude, 1988; laude et al., 1992) . thus, it may play a role in viral pathogenesis. monoclonal antibodies against the m protein do not neutralize viral infectivity, suggesting that m is not involved in receptor binding. however, some of these antibodies can neutralize viral infectivity in the presence of complement (collins et al., 1982; laviada et al., 1990) , indicating that part of the m protein is exposed on the virion surface. the he glycoprotein-or perhaps one should say the he gene-of coronaviruses is something of a n enigma. only coronaviruses belonging to the mhv group possess the he gene (table i) . even there, not all virus strains within a species express the he protein (luytjes et al., 1988; . as with many of the so-called nonstructural protein genes of coronaviruses, the product of the he gene is not essential for viral replication, certainly not in the cell types used in the laboratory. the he protein was first detected in bcv (king et al., 1985) and some mhv strains; however, acceptance of it as a legitimate virus-encoded protein was delayed because in one of the mhv strains studied most thoroughly, a59, virions lacked he. the he gene of a59 was later shown to lack the initiation codon of the he open reading frame (orf); thus, the he gene is a pseudogene in this (luytjes et az., 1988) and several other mhv strains . a complete, functional he gene was subsequently identified in the jhm strain and several others . the he glycoprotein, of approximately 65 kda (424 amino acids in bcv), has been detected in virions of hev, mhv, hcv-oc43, bcv, and tcv. when analyzed under nonreducing conditions, the he protein migrates as a dimer of approximately 140 kda (king et al., 1985) . the mature protein is believed to exist in the virion as a dimer, anchored by the c terminus, forming a fringe of short spikes visualized by electron microscopy (caul and egglestone, 1977; dea and tijssen, 1988) . it is not known whether each spike consists of more than one he dimer. those coronaviruses which contain he in their virions cause hemagglutination much more efficiently than those that do not. similar to the s protein, he alone can mediate hemagglutination and hemadsorption (king et al., 1985; hogue and brian, 1986; vlasak et al., 1988b; deregt et al., 1989; pfleiderer et al., 1991; schultze et al., 1991a) ; however, he seems to have weaker activity than s (schultze et al., 1991a) . he binds to 9-o-acetylated neuraminic acid (vlasak et al., 198813; schultze et al., 1991a) , which is also a target for s binding. some he-specific mab can neutralize bcv infectivity (deregt and babiuk, 1987; deregt et al., 1989) . thus, he protein of bcv may participate in virus binding to the receptor. the relative importance of he and s in hemagglutination and tissue tropism of bcv is not known. as its name implies, the he protein also has esterase activity; specifically, it is a neuraminate-o-acetylesterase. it hydrolyzes the 9-0acetylated sialic acid on erythrocytes, thereby reversing hemagglutination induced by the he or s protein; thus, he is considered a receptordestroying enzyme (vlasaket az., 1988a,b; yokomori et az., 1989; . the putative esterase active site is fgds, encoded by amino acids 19-22 of the mature he polypeptide of bcv (m. d. parker et al., 1989; kienzle et al., 1990) . in these respects, it resembles the hemagglutinin-esterase-fusion (hef) glycoprotein of influenza c viruses, which also has hemagglutinating and 9-o-acetylated sialic acidhydrolyzing esterase activities. moreover, the he protein of coronavi-ruses shares some 29% amino acid identity with the hef of influenza c virus, including conservation of the position of the putative esteraseactive site fgds and many cysteine residues (luytjes et al., 1988; s. e. parkeret al., 1989; kienzle et al., 1990; zhanget al., 1991) . unlike the hef protein of influenza c virus, which is cleaved into two subunits (nakada et al., 19841 , the coronavirus he protein is not cleaved and lacks most of the c-terminal subunit of the hef of influenza c virus. because of the close relatedness between the coronavirus he protein and the influenza c virus hef protein, and because the he gene is present in only one coronavirus group, it was proposed that the he gene was acquired by a coronavirus as a result of recombination between an ancestral coronavirus and influenza c virus (luytjes et al., 1988) . interestingly, the torovirus berne virus also has an he pseudogene (gene 4; fig. 2 ) (snijder and horzinek, 1995) , the amino acid sequence of which has approximately 30% identity with the c-terminal part of the coronavirus he. the functional significance of he for coronaviruses is not known. among coronaviruses, only bcv requires he for infectivity; however, the presence of he may affect the pathogenicity of some coronaviruses, as evidenced by the findings that passive administration of he-specific mab in mice altered mhv pathogenicity and that mhvs with an he have different neuropathogenicity from those without he . conceivably, the presence of he in an mhv may allow the virus to utilize an alternative receptor independently of the s protein. however, this is not the case, as evidenced by the finding that an mab specific for the murine biliary glycoprotein molecule, which is the major mhv receptor recognized by s, inhibited the infectivity of an he-containing mhv (gagneten et al., 1995) . thus, the he protein does not enable a virus to bypass the primary mhv receptor and may provide only an auxiliary function for virus binding to target cells. until recently it was thought that coronaviruses possessed three (s, n, m) or four (including he) structural proteins. it is now clear that coronaviruses, but not toroviruses, possess an additional virion protein, the e protein. it plays an essential role in virion assembly. it has been shown that the e and m proteins are the only two viral proteins absolutely required for virion assembly (bos et al., 1996; vennema et al., 1996) . this protein has been demonstrated for ibv (smith et al., 1990; liu et al., 19911, tgev (godet et al., 1992) and mhv (yu et al., 1994) . when the deduced e proteins of the other coronaviruses are taken into account, it transpires that the e proteins vary from 84 t o 109 amino acids, corresponding to molecular weights of 9100 to 12,400 (siddell, 1995~) . siddell has highlighted a number of features common to all the e proteins, namely, a hydrophobic region of some two dozen residues, starting near the n terminus; a cysteine-rich region immediately downstream from this; a conserved proline residue in the middle of the molecule, and otherwise very low amino acid identity in the genus as a whole; and an abundance of charged residues in the cterminal half of the protein (siddell, 1995~) . it is now well established that this protein is associated with highly purified virion preparations (liu et al., 1991; godet et al., 1992; yu et al., 1994) . liu and inglis calculated the ratio of s:n:m:e proteins in virions of ibv-beaudette strain to be 1:11:10:2, indicating an amount of e protein similar to that of s protein (liu et al., 1991) . in contrast, godet et al. estimated that the s:m:e protein ratio in virions of tgev was 20:300:1 (godet et al., 19921, and vennema et al. (1996) have suggested an m:e ratio of approximately 1oo:l for virions of mhv. it is not clear why there is such a wide range of variations. the e protein in the cells is localized in the perinuclear region, with some migrating to the cell surface (godet et al., 1992; yu et az., 1994) . experimental evidence suggests that the e protein is anchored in the membrane by sequence in the n-terminal half of the molecule. thus antibodies specific for epitopes in the c-terminal half of the tgev e protein produced cell-surface fluorescence in paraformaldehyde-fixed, tgev-infected cells (godet et al., 1992) , but the precise topology of the protein has not been elucidated. the role of the e protein in virion assembly will be discussed in section v,h on virus assembly and release. the e proteins of ibv and mhv are translated from the third and second orfs, respectively, of mrnas 3 and 5 of the respective viruses. both of these are polycistronic mrnas (see figs. 5 and 7 and section v,g,2). in contrast, in all other viruses, the e protein is derived from a monocistronic mrna. the mechanism of translation of the ibv and mhv e proteins is discussed in section v,g. the n protein is a 50-to 60-kda phosphoprotein which, together with the genomic rna, forms a helical nucleocapsid (rnp). the rnp of coronaviruses have been reported variously as being from 9-11 to 14-16 nm in diameter (see laude and masters, 1995, for references) . the n protein in rnp provides only limited protection to the rna genome against ribonucleases. the n proteins vary from 377 to 455 amino acids in length, are highly basic, and have a high (7-11%) serine content, potential targets for phosphorylation. sequence conservation within the genus is low. thus, the n proteins of ibv and tgev have only 29% identity with that of bcv. even within the mhv group, the n proteins of mhv and bcv share only 70% identity, whereas the m proteins of these two viruses have 86% identity (lapps et al., 1987) . based on sequence comparison, three structural domains in the n protein have been identified (parker and masters, 1990) . the middle domain is an rna-binding domain (masters, 1992; nelson and stohlman, 1993 ) which binds to both coronaviral and nonviral rna sequences in uitro (robbins et al., 1986; stohlman et al., 1988; masters, 1992) ; however, it does not contain any motifs characteristic of other rna-binding proteins. under specific binding conditions, the mhv n protein binds to the leader rna sequence, particularly nucleotides 56-67 . furthermore, an anti-n mab immunoprecipitated all of the mhv rna molecules which had the leader sequence . the n protein of ibv also bound to the 3' untranslated region of the ibv rna in uitro (zhou et al., 1996) . these rna-binding properties are consistent with the fact that the n protein interacts with the viral genomic rna to form nucleocapsid. this interaction is necessary for the formation of virus particles, as n alone cannot be incorporated into virus particles, whereas the n-rna complex can (bos et al., 1996; vennema et az., 1996) . however, the specificity of the rna-n protein interaction required for nucleocapsid formation has not been elucidated. the n protein also binds to membranes and phospholipid (anderson and wong, 1993) . this may be another property which facilitates the formation of virus particles. the finding that the n protein binds to the 5' and 3' ends of viral rna suggests that the n protein may also modulate viral rna synthesis because the ends of the rna are likely involved in the regulation of rna synthesis. in an in uitro rna replication system, the addition of mhv n-specific antibodies inhibited viral rna synthesis (compton et al., 1987) , suggesting that the n protein is a component of the rnasynthesizing machinery. the ability of n to bind to the membrane (anderson and wong, 1993) may enable the formation of the rna replication or transcription complex, in view of the fact that viral rna synthesis occurs in the membrane fraction of infected cells dennis and brian, 1982) . the three structural domains of the n protein are separated by spacer regions, which are not conserved (masters, 1992) . the functions of the n-and c-terminal conserved domains are not yet clear. using a targeted recombination approach (koetzner et al., 1992; masters et al., 1994) to generate recombinant viruses that have a chimeric n gene containing parts of bcv and mhv sequences, peng et al. (1995a) have shown that there is strict sequence specificity within the conserved structural domains for viable recombinants. since the n protein constitutes the nucleocapsid, mutations within the n protein will likely affect the stability or viability of the virus. indeed, several temperaturesensitive and thermolabile mutants of mhy have deletions or mutations within the n protein (koetzner et al., 1992; peng et al., 1995b) . viruses with site-specific mutations of the n gene have been generated by targeted recombination techniques; interestingly, revertants of these mutants often have second-site mutations located a t different domains, suggesting that there are interactions between different domains of the n protein (peng et al., 199513) . the role of phosphorylation in the n protein has not been elucidated. the coronavirus contains a positive-sense, single-stranded rna genome, which is the largest viral rna genome known, ranging from 27.6 to 31 kb. the large size of the viral rna requires the virus to develop special mechanisms of rna synthesis to counter the deleterious effects of the possible errors during rna synthesis. the virion rna functions as an mrna and is infectious. it contains approximately 7-10 functional genes, 4 or 5 of which encode structural proteins. the genes are arranged in the order 5'-polymerase-(he)-s-e-m-n-3', with a variable number of other, mostly nonstructural and largely nonessential, genes interspersed among them (fig. 5) . this gene arrangement also applies to toroviruses and arteriviruses (fig. 2) . the 5' terminus of the coronavirus genome is capped, and the rna starts with a leader sequence of 65-98 nucleotides, which is also present a t the 5' end of mrnas, followed by a 200-to 400-nucleotide untranslated region (utr). at the other end of the genome is a 3' utr of 200-500 nucleotides followed by a poly(a) tail. almost two-thirds of the entire rna is occupied by the polymerase gene, which comprises two overlapping orfs, la and lb. at the overlap region is a specific seven-nucleotide "slippery" sequence and a pseudoknot structure, characteristic of the ribosomal frameshifting signal (brierley et al., 1987 (brierley et al., , 1989 lee et al., 1991; , which is required for the translation of orf lb. the architecture of the nonstructural protein genes interspersed between the known structural protein genes varies significantly among different coronavirus species (fig. 5 ) . for example, in hcv-229e, gene 3 contains two orfs, whereas in the related virus pedv these two orfs are fused (duarte et al., 1994) . in hcv-oc43, gene 4 is missing altogether (mounir and talbot, 1993) . finally, in ibv, two orfs are inserted between m and n genes. the variability of gene structure indicates the plasticity of coronavirus rna and the frequent occurrence of recombination and also suggests that there is no strong conservation pressure on these nonstructural proteins. there is a stretch of consensus sequence, ucuaaac (for mhv), or a related bcv (hcv-oc43) fig 5. comparative genome structure of the different coronaviruses. the complete sequences are available for mhv, ibv, tgev, and hcv-229e. the gene 1 sequences of the remaining viruses have not been completed. gene 1 sequences are interrupted and shortened to highlight the remaining genes. the vertical lines represent mrna start sites; thus, each region between two vertical lines represents a separate gene ("transcription unit"). the structural protein genes are marked by various symbols, and nonstructural protein genes are represented by unfilled boxes. the gene arrangements of ns protein genes and e protein gene are very heterogeneous in terms of transcription unit and the relative size and position among different strains of the same virus species; only the representative one is presented. the numbering system for the genes of hcv-229e deviates from the published one to be consistent with the other viruses. hcv-oc43 does not have a gene 4. sequence, a t sites immediately upstream of most of the genes. these sequences represent signals for transcription of subgenomic mrnas (see section v,e). finally, a pseudoknot structure has been shown to be present a t the 3' end of the coronaviral rna (williams et al., 1995) . a characteristic feature of the coronaviridae, and of the arteriviridae as well, is that all known member species generate a 3'-coterminal nested set of five or more mrnas (see fig. 7 ). each coronavirus and arterivirus subgenomic mrna has the leader sequence a t its 5' end. curiously, no leader rna sequence is present in the torovirus rnas (fig. 2) . in 1990, the coronavirus study group published its recommendations for the nomenclature of coronavirus genes, mrnas, and proteins (cavanagh et al., 1990) . at that time it was reluctant to apply the term "nonstructural" t o the potential products of genes which were suspected of not being structural proteins. this caution was a consequence of our lack of knowledge of those gene products, a situation which has improved greatly in the last 5 years or so. this has resulted in the term "nonstructural (nsl" being applied more widely to several gene products. every gene that encodes the ns proteins has been deleted in a t least some naturally occurring virus isolates; thus, most of the ns genes are not essential for viral replication. however, some of the ns proteins may play a role in viral tissue tropism or pathogenicity. the polymerase is encoded by gene 1, which accounts for approximately two-thirds of the genome (fig. 2) . the complete polymerase gene of four coronaviruses (ibv, mhv, hcv-229e, and tgev) covering each of the three coronavirus groups has been sequenced lee et al., 1991; bonilla et al., 1994; eleouet et al., 1995) . although the polymerase genes vary in size from approximately 18 to 22 kb, the encoded proteins have many structural features in common. the degree of amino acid identity for this gene product is greater than is observed for any other coronavirus gene product. the polymerase gene is predicted to encode a protein of approximately 740-800 kda. proteins of this size have not been detected in coronavirus-infected cells, in part because of co-translational polypro-tein processing. the pol gene encodes two orfs, l a and lb, which overlap by a few dozen nucleotides (figs. 2 and 6). the second, orf lb, is in the -1 reading frame with respect to the upstream orf l a and is translated following ribosomal frameshifting in the overlap region. this will be examined in more detail in section v,g. there is greater amino acid identity among the l b than the l a orfs. for example, l a and l b of ibv, the least typical coronavirus in terms of protein sequences, have amino acid identityhimilarity of approximately 30/50% and 55/79%, respectively, compared with those of mhv, hcv, and tgev. it is the l a orf which accounts for the mhv polymerase gene being approximately 1-2 kb longer than those of ibv, hcv, and tgev. a number of functional domains within pol have been predicted following computer-based motif analyses hodgman, 1988; gorbalenya et al., 1989a,b; lee et al., 1991) ; some of these functional domains have been confirmed by experimental analysis. the location of these motifs is illustrated in fig. 6 . three motifs have been identified in orf l a , indicating the presence of one or two papain-like cysteine proteases (plp): a chymotrypsinlpicornaviral3c-like protease (lee et al., 1991) . (a) the polymerase gene comprises two orfs, l a and lb, which overlap, the l b orf being translated after ribosomal frameshifting. (b) the positions of motifs: plp 1 and 2, papain-like protease; x domain, highly conserved between ibv and mhv; 3clp, picornavirus-3c-like protease; md, membrane-associated domain; gfl, growth factorlike; pol, rna-dependent rna polymerase; mb, metal-binding motif; hel, helicase. (c) genetic complementation groups fu and baric, 1994) . (d) processing scheme for part of the l a orf (denison et al., 1992 (3clp) and a cysteine-rich growth factor-related protein (gfl). mhv, hcv-229e, and tgev have two plp domains (1 and 21, with plpb corresponding to the single plp domain of ibv. sequence corresponding to a cysteine protease of streptococcus pneumoniae has been identified in l a of ibv. upsteam of plpb is a region termed the x domain, a region of particularly high conservation between ibv and mhv and similar to one near the thiol protease of alpha-and rubiviruses . there is no functional evidence so far to link the gfl with known growth factors, but the predictions of most of the protease domains have been confirmed by experimental analysis. the first plp domain of mhv is responsible for the cleavage of p28/p30 and p65 from the n terminus of the mhv orf l a polyprotein ( fig. 6 ) (baker et al., 1989 (baker et al., , 1993 bonilla et al., 1995 bonilla et al., , 1997 . this plp was inhibited by zinc chloride but not by leupeptin (baker et al., 1989; denison et al., 1992) . deletion analysis defined this proteinase domain to be within the sequence encoded by the 3.6-4.4-kb region from the 5' end of the genome. site-directed mutagenesis showed that residues cys-1137 and his-1288 were essential for protease activity (baker et al., 1993) . some amino acid sequences between the p28 cleavage site and the plp domain were also essential for the cis cleavage that generates p28 (baker et al., 1993; bonilla et al., 1995) . the function of plp2 has not been demonstrated. the 3clp domain extends for approximately 300 amino acids and is homologous to proteases encoded by picornaviruses and several other virus genera. the putative 3clp domain of hcv-229e has been expressed as a /?-galactosidase fusion protein in escherichia coli and shown to have autocatalytic proteolytic activity, releasing an active 3clp protein (ziebuhr et al., 1995) . an antiserum against this fusion protein immunoprecipitated a 34-kda protein from hcv-229e-infected cells. similar activity has been demonstrated for the 3clps of mhv and ibv (tibbles et al., 1996) . this protease cleaves not only its own boundaries but also several downstream sites within orf l a and orf lb, probably both in cis and in trans. computer analysis predicted that the catalytic center of the ibv 3clp would include cys-2922, his-2820, and glu-2843 (gorbalenya et al., 1989a,b) . site-directed mutagenesis confirmed the role of the cys and his residues but showed that the glu residue was not essential (liu and brown, 1995) . the same approach confirmed that the predicted qs(g) dipeptide bonds in the l b orf are the targets for the protease activity of the 3clp of ibv (discussed further in section v,g). similar conclusions were reached for 3clp activity of mhv and hcv-229e grotzinger et al., 1996) . the importance of cys-3495 in the 3clp of mhv has been demonstrated (seybert et al., 1997) . in uitro transcription and translation of a cdna containing the putative 3clp of mhv produced polypeptides of 38 and 33 kda, which were subsequently processed to products of 32 and 27 kda . the 27-kda protein possesses the 3c-like protease activity (lu et al., 1996) . the 3clp domain is flanked by predicted membrane-spanning domains, which may be important for the proteolytic activity (tibbles et al., 1996) (fig. 6) . poor expression of the ibv 3clp protein in vitro led to the discovery that this protease was ubiquinated and subsequently degraded by an adenosine triphosphate (atp)-dependent protease present in reticulocyte lysate (tibbles et al., 1995) . this is the third example of a viral protein subject to turnover in this manner and involves a different virus class from the previously reported examples, in a picornavirus (oberst et al., 1993) and an alphavirus . the ubiquitin-mediated, atp-dependent proteolytic pathway is a major cellular, nonlysosomal, protein degradation system, which may cause rapid turnover of the coronaviral polymerase. the functional domains associated with rna synthesis are located within the more conserved l b orf. these include domains for an rnadependent rna polymerase, a nucleoside triphosphate (ntp)-binding/ helicase domain, and a zinc-finger nucleic acid-binding domain (metal binding domain) (fig. 6) . computer analyses identified the polymerase domain h o d p a n , 1988; gorbalenya et al., 1989a,b) . unlike the gdd motif present in many viruses, the corresponding sequence in coronaviruses is sdd. whether the polymerase gene products contain activities other than proteases and polymerases is not known. the coronaviruses exhibit great heterogeneity with respect to the number and genome location of ns protein genes and in regard to the number of orfs within a gene (fig. 5) . the functions of these ns proteins are still unknown. there are two genes located between the polymerase and s genes of these viruses (fig. 5) . gene 2-1 encodes the he protein, while gene 2 encodes an ns protein of unknown function. the gene 2 protein com-prises approximately 260 amino acids (30 kda) (luytjes et al., 1988; shieh et al., 1989; labonte et al., 1995) . the bcv and mhv homologs share 45% amino acid identity, while the homolog of hcv-oc43 has 92% identity with that of bcv. this gene product has been detected in the cytoplasm of mhv-, bcv-, and hcv-oc43-infected cells and may be phosphorylated (bredenbeek et al., 1990; zoltick et al., 1990; coxet al., 1991; labonte et al., 1995) . computer analysis of its sequence suggested the presence of a nucleotide-binding site (luytjes et al., 1988) . however, no function has been assigned to this protein, and it is not required for virus replication in culture (schwarz et al., 1990) . interestingly, the c terminus of the torovirus orf l a product (polymerase) has 31-36% sequence identity with the gene 2 product of mhv (snijder et al., 1991) . this evolutionary relationship between coronavirus and torovirus suggests that the gene 2 product is probably involved in viral rna synthesis, since it is expressed as part of the torovirus polymerases. there are two to three orfs in this region, and their structure and the mechanism of expression of gene products vary markedly among different coronavirus species. they can be expressed as two different genes, i.e., expressed from two separate mrnas (e.g., mrnas 4 and 5 of mhv and bcv and mrnas 3 and 3-1 of tgev) or localized in one gene, thus requiring internal initiation of translation from a single polycistronic mrna (e.g., mrna 3 of the ibv and fcv groups). in ibv, it contains three orfs (3a, 3b, and 3c); orf 3c encodes the e protein, which is a viral structural protein, while 3a and 3b encode ns proteins. the gene products of both orfs 3a and 3b (approximately 7 kda) have been detected in small quantities in virus-infected cells (liu et al., 1991) . in tgev, this region contains two orfs, being separated from the e protein gene. these two orfs are encoded by mrnas 3 and 3-1, respectively, the predicted protein products being approximately 8 and 27 kda, respectively. in a related nonenterogenic strain, prcv, however, there are multiple deletions in this region, essentially inactivating one or both of the orfs (rasschaert et al., 1990; wesley et al., 1991) . it has been suggested that the absence of the 3a product, in addition to a shorter s protein, might be associated with their lack of enteropathogenicity. however, vaughn et al. (1995) have recently described two prcv strains which have an intact 3a gene (vaughn et al., 1995) . canine coronavirus has gene 3 orfs equivalent to those of tgev, exhibiting high amino acid identity (>80%), although the second orf is truncated by a stop codon (horsburgh et al., 1992) . two other members of the tgev group exhibit a variation on the same theme. pedv and hcv-229e lack a homolg of orf 3a of tgev and ccv. pedv has an orf corresponding to 3b of tgev, while hcv-229e has two orfs corresponding to the single orf of pedv (duarte et al., 1994) . members of the group i coronaviruses also exhibit great heterogeneity in this region. mhv-jhm produces mrna 4, which encodes a 15-kda protein. this protein has been detected in virus-infected cells (ebner et al., 1988) . in contrast, hcv-oc43 contains only 11 amino acids in this region (mounir and talbot, 1993) . gene 5 of mhv has two orfs, 5a and 5b. the latter encodes the structural e protein and is the predominant product made from mrna 5 . it is not clear whether orf 5a is translated at all. at least one strain of mhv lacks the 5a orf ; also, hcv-oc43 has the 5a orf but is unable to produce a corresponding mrna (mounir and talbot, 1993) . in summary, there is great heterogeneity with respect to the number, size, and mechanism of expression of orfs between the s and e genes. these ns proteins probably are not required for viral replication. the lack of necessary function may account for the heterogeneity which arose during evolution. ibv is unique in that it has two orfs (5a and 5b), which encode proteins of 7.4 and 9.5 kda, respectively. these proteins have been detected in very small amounts in virus-infected cells (liu and inglis, 1992a) . the function of these orfs is not clear. tgev has an additional gene 7, which encodes a 9.1-kda protein (garwes et al., 1989; tung et al., 1992) , in the region corresponding to the 3' end untranslated region of other viruses (fig. 5) . this protein is hydrophobic and is associated with the endoplasmic reticulum and cell surface membranes (tung et al., 1992) , but its nuclear localization has also been reported (garwes et al., 1989) . fcvs and ccv have two orfs in the same region, the first being analogous to the single orf of tgev. the second (7b) orf encodes a 14-kda soluble protein containing the sequence ktel (vennema et al., 19921 , which is similar to the endoplasmic reticulum retention signal, kdel. the protein is partially retained in the endoplasmic reticulum but is also slowly secreted out of the cells. the functions of these proteins are not known. coronaviruses have relatively restricted host ranges, infecting only their natural hosts and closely related animal species. occasionally, cross-species infection of coronaviruses occurs, such as the experimental infection of monkey by mhv, which causes central nervous system demyelination (murray et al., 1992; cabirac et al., 1994) , and the occasional infection of humans by bcv, which causes diarrhea. bcv also infects turkeys and tgev infects dogs, suggesting some flexibility in their host range. the expansion of viral host range can be achieved by passing the coronavirus in a heterologous cell line, as demonstrated by the emergence of an mhy variant with the ability to infect originally nonpermissive cell lines, such as human cells, after serial passages (baric et al., 1997) . in animals, coronaviruses have restricted tissue tropism; for example, most hcv strains cause only respiratory infections. different strains of a coronavirus may have distinct tissue specificity; for example, tgev infects both the gastrointestinal tract, causing fatal diarrhea, and respiratory tract tissues without causing primary respiratory symptoms, whereas prcv, which is closely related to tgev, infects the respiratory tract of pigs but replicates poorly in the intestinal tract (cox et al., 1990) . the species and tissue specificity of a coronavirus infection is a t least partially dictated by the nature and distribution of cellular receptors and other related molecules that regulate virus entry, as evidenced by the viral replication that results when viral rna is directly introduced into cell types of other animal species. thus, coronaviruses have the potential to replicate in many cell types. the complete coronavirus replication cycle takes place in the cytoplasm. it has been shown that mhv can replicate in enucleated cells and in the presence of actinomycin d and a-amanitin, suggesting that nuclear functions are not required for viral replication wilhelmsen et al., 1981) . there are, however, reports of the inhibition of replication by actinomycin d of some coronaviruses, including feline enteric coronavirus (lewis et al., 19931, ibv (evans and simpson, 19801, hcv-229e (kennedy and johnson-lussenberg, 19781 , and mhv in some cell lines (dupuy and lamontagne, 1987) . thus, nuclear functions may be required for viral replication under certain conditions. this issue has not been resolved. the first step in viral infection is the binding of the virus to target cells. hemagglutination and hemadsorption have been used as assays for studying virus-cell interaction, although the erythrocyte itself is not a target cell for coronavirus infection. several coronaviruses, including hev, ibv, bcv, and some strains of mhv and hcv, can cause hemagglutination (sugiyama and amano, 1980; schultze et al., 1990; zhang et al., 1994a) . the binding residue on the cell surface is a 9-o-acetylated neuraminic acid of glycoproteins or glycolipids (schultze et al., 19901 , although different coronaviruses may prefer different structural isoforms of 9-o-acetylated neuraminic acid. for bcv, the virus binding to erythrocytes is mediated through either the s or he protein, both of which have hemagglutinating activities, the s protein having the stronger activity (king et al., 1985; schultze et al., 1991a,b) . the he protein of bcv and hev also recognizes 9-o-acetylated neuraminic acid, and its esterase activity is also specific for this molecule; thus, he protein has both receptor-binding and receptor-destroying activities (vlasak et al., 1988a,b; schultze et al., 1991b) . expression of the he protein of mhv on the cell surface conferred a hemadsorption activity ; however, even viruses that lack he protein (e.g., ibv) can cause hemagglutination, suggesting the role of s protein in hemagglutination. thus, the he and s proteins of various coronaviruses may have comparable functions, enabling the virus to bind the sialic acid residues; however, only the he protein confers the receptordestroying activity. the residue necessary for hemagglutination by ibv is a2,3-linked n-acetylneuraminic acid . curiously, the hemagglutinating activity of ibv is not evident until the virus particle is treated with neuraminidase, suggesting that the s protein itself is covered by sialic acid. although virus binding to erythrocytes provides a good model system for studying virus-cell interactions, it may not necessarily reflect the actual mechanism of virus attachment to target cells. the classical study of virus attachment to target cells involved the in uitro binding of mhv to macrophages from genetically susceptible m i c w l m. c. lai and david cavanagh and resistant mouse strains (shif and bang, 1970) . this study showed that mhv bound equally well to cells from resistant and susceptible mice, even though macrophages from resistant mice were resistant to virus infection. similar observations have been made on splenic lymphocytes, thymocytes (krzystyniak and dupuy, 19811 , and glial cells (wilson and dales, 1988) ; thus, it appears that genetic resistance is not exerted at the level of virus binding in these cases. similarly, established tissue culture cell lines, including murine and primate cells, irrespective of their degree of susceptibility or resistance to mhv, bound mhv to the same extent (van dinter and flintoff, 1987; kooi et al., 1988 kooi et al., , 1991 . thus, virus may bind to a ubiquitous molecule on the cell surface, which, however, may not lead to virus infection. whether this ubiquitous molecule is a sialic acid-containing glycoprotein has not been established. the binding of bcv to its target cells, such as mdck cells, is mediated by 9-o-acetylneuraminic acid residues similar to those on erythrocytes. removal of the sialic acid by neuraminidase abolished virus attachment, while resialization restored it . hcv-oc43 binds to a similar sialic acid residue but prefers a form slightly different from that for bcv (kunkel and herrler, 1993) . the he protein of bcv can also mediate virus binding to target cells, and this binding may be required for viral infection, as suggested by the finding that mab against he inhibited bcv infectivity (deregt and babiuk, 1987; deregt et al., 1989) . one inhibitor of the esterase activity of he protein, diisopropylfluorophosphate, also inhibited bcv infection (vlasak et al., 1988a) . the s protein of bcv probably also participates in virus binding to target cells, as suggested by the finding that the mab against s protein can neutralize bcv infectivity . the relative importance of s and he proteins is not clear. in contrast, none of the mab against the he protein of mhv inhibited mhv infection . despite the finding that the binding of he and s proteins to target cells is necessary for bcv infection, the binding of bcv or hcv-oc43 to n-acetylneuraminic acid in itself is not likely the basis of viral cell tropism because sialic acid is a common cell surface carbohydrate residue; thus, an additional, more cell type-specific molecule is probably required for viral infection. the finding that mhv and other coronaviruses bound to resistant as well as susceptible cells indicates that this binding may represent an initial step in the virus attachment process, which is not sufficient for viral infection. it is likely that a more specific binding between virus and cells is required for the establishment of viral infection. this binding involves a specific virus receptor molecule on the cell surface. a. mhv receptor. the mhv receptor was the first coronavirus receptor to be identified. it is the murine homolog of a member of the carcinoembryonic antigen (cea) family (dveksler et al., 1991; williams et al., 1991) and belongs to the biliary glycoprotein (bgp) subfamily. the terminology of mhv receptors in the literature is somewhat controversial, the following terms being used interchangeably: mmcgm1, mhvr-1, and bgpa. it has an immunoglobulin-like structure, consisting of four immunoglubulin-like loops, the n-terminal loop being the virus-binding domain (dveksler et al., 1993b) . the sequence of the c terminus (cytoplasmic domain) of the receptor is not essential. glycosylation of the protein also is not necessary for its receptor function in uiuo (dveksler et al., 1995) . the functional significance of the receptor in viral infection in uiuo was demonstrated by the finding that an mab against the mhv receptor inhibited viral infection in mice (smith et al., 1991) . subsequently, several additional members of cea family were found to serve as mhv receptors, including an mmcgm2-like protein (also termed mhvr-2 and bgpb), which is the product of an alternatively spliced form of mmcgml rna and is expressed in both the liver and brain, in contrast to the liver-specific expression of mmcgml (yokomori and dveksler et al., 1993a) ; an allelic gene product of the bgp gene in sjl mice, a mouse strain resistant to mhv infection (yokomori and lai, 199213; dveksler et al., 1993a) ; bgp-2, which is the product of a new member of the murine bgp gene (nedellec et al., 1994) ; and a novel pregnancy-specific glycoprotein (psg)-like protein, which is expressed in the mouse brain, in contrast to placenta-specific expression of other psg molecules . all these molecules contain a consensus motif in the virus-binding domain (n-terminal loop). thus, several different cea family members, which are differentially expressed in different cells and tissues, can potentially serve as an mhv receptor. different strains of mhv may use different cearelated molecules as receptors at different efficiencies (compton, 1994; . the prototype mhv receptors (mhvr-1) are expressed in the liver, gastrointestinal tract, b cells, macrophages, and endothelial cells but not in t cells (coutelier et al., 1994; godfraind et al., 1995) , consistent with the target cell specificity of mhv. however, the mhv receptor is also expressed in other tissues, e.g., kidney, which are not targets for mhv infection. also, sjl mice express a functional mhv receptor (yokomori and lai, 199213; dveksler et al., 1993a) but are resistant to mhv infection (knobler et al., 1984) . thus, receptor expression is not sufficient for viral infection. it is not yet clear which molecules are used by mhv as receptors in cross-species infection (e.g., rats and monkeys) (murray et al., 1992; cabirac et al., 1994) . recently it was shown that bgp and cea molecules of human origin could serve as receptors for some mhv strains . the expression of the receptor molecules on the cell surface is necessary for virus infection, and the expression level of the receptor may determine the relative susceptibility or resistance to viral infection in some cells. during persistent viral infection of cultured murine cells, the expression level of the receptor is offen reduced, resulting in the relative resistance of the cells to viral superinfection, which could be overcome by the expression of an exogenous receptor (sawicki et al., 1995; chen and baric, 1996) . thus, there is a rough correlation between receptor expression and the susceptibility of a cell type to virus infection. under certain circumstances, virus may infect cells by a receptorindependent mechanism; for example, mhv-infected murine cells may fuse with human cells, which do not have mhv receptors, and cause the latter cells to become infected (gallagher et al., 1992) . it has been shown that mhv infects polarized epithelial cells through the apical, but not the basolateral, surface (rossen et al., 1995a (rossen et al., , 1997 . it is not clear whether the virus receptor is differentially expressed on the two different surfaces. b. receptors for tgevand hcv-229e. the receptors for tgev and hcv-229e have been identified as aminopeptidase n (apn) of the porcine and human species, respectively (delmas et al., 1992; yeager et al., 1992) . prcv also uses porcine apn as a receptor; thus, virus binding to the receptor is not sufficient to explain the differences in tissue tropism between tgev and prcv. apn is a member of the membrane-bound metallopeptidase family and is widely distributed on diverse cell types; it is highly expressed on the brush border membrane of enterocytes. some of the antibodies against human apn can block hcv-229e binding (yeager et al., 1992) ; however, the catalytic site of the protease activity of apn is not required for receptor function, and the inhibitors of apn do not block viral infection . similar to mhv, tgev infects polarized cells through the apical, but not the basolateral, surface (rossen et al., 1994) . again, it is not clear whether this is restricted by the differential expression of apn on the different sides of the cells. tgev has also been shown to bind to a 200-kda protein on the surface of the enterocytes on the villi of the small intestine (weingartl and derbyshire, 1994) . pcrv does not bind to this molecule. both the temporal expression (mainly in the newborn) and spatial distribution patterns (on the villi of the gastrointestinal tract) of the 200-kda protein correspond to the pattern of susceptibility of piglets to tgev infection. thus, the expression pattern of this molecule appears to have a better correlation than the porcine aâ�¬" with the tissue tropism of tgev. this 200-kda molecule may be an alternative receptor used by tgev. the relative functional significance of this molecule and aâ�¬" as tgev receptors is not yet clear. the fipv strains of fcv and canine coronavirus apparently utilize the aâ�¬" of feline and canine species, respectively, as receptors (benbacer et al., 1997) . cross-species utilization of feline aâ�¬" by coronaviruses of different species (canine, feline, and human) has also been reported (tresnan et al., 1996) . fipv, however, is unique among coronaviruses in that it causes an antibody-dependent enhancement (ade) phenomenon (weiss and scott, 19811 , which is the result of the binding of the virus-antibody complex to fc receptors on the surface of macrophages, leading to enhanced virus uptake and spread. this ade phenomenon has been attributed to the s protein-antibody complex (vennema et al., 1990b; corapi et al., 1992; olsen et al., 1992) . the fc receptor may be a co-factor or an alternative receptor for fipv entry into macrophages. in this regard, the s protein of mhv has been shown to have limited sequence homology with the murine fc receptor and to have the ability to bind to the fc fragment of immunoglobulin oleszak et al., 1992) . whether the fc receptor plays a role in mhv infection is not clear. however, mhv does not exhibit ade. c. receptors for other coronaviruses. sialic acid (n-acetyl-9-0acetylneuraminic acid)-containing glycoproteins are probably a component of the cell surface molecules required for bcv and hcv-oc43 infection because the removal of sialic acids inhibits bcv infection and resialylation restores virus infectivity ; however, it is unlikely that it is the primary receptor molecule used by these viruses since the distribution of these molecules is more widespread than the susceptible target cells. the identity of the specific receptor for these viruses has not been determined. for hcv-oc43, it has been shown that the virus binds to a major histocompatibility complex class i molecule (collins, 1994) . however, the receptor function of this molecule has not been established. the mechanism of coronavirus entry into target cells has been controversial. early electron microscopic studies visualized virus (mhv and ibv) particles inside lysosome-like vesicles near plasma membranes, suggesting that virus enters cells by endocytosis ("viropexis") (david-ferreira and manaker, 1965) ; however, other studies suggested that virus enters cells by direct fusion between virions and the plasma membrane (doughri et al., 1976) . lysosomotropic drugs, such as ammonium chloride and chloroquine, inhibited mhv-3 virus entry (krzystyniak and dupuy, 1984) . also, mhv-specific antibodies did not lyse virusinfected cells during the virus-entry process, as would be the case if the virus fused with the cell membrane (krzystyniak and dupuy, 1984) . these results suggested that mhv-3 enters cells by an endocytotic pathway. similar studies using the a59 strain of mhv, however, showed that ammonium chloride delayed, but did not inhibit, the viral infection of l-2 cells (mizzen et al., 1985) . the effects of ammonium chloride were interpreted to be inhibiting virus uncoating in this case. recent studies by the same group have further shown that only a small proportion of adsorbed virus enters cells by the endocytotic pathway since ammonium chloride, chloroquine, and dansylcadaverine, all of which inhibit receptor-mediated endocytosis, did not have significant effects on mhv entry (kooi et al., 1991) . the majority of mhv particles enter cells by virus-cell fusion at the plasma membrane. this interpretation is consistent with the finding that the optimum ph for mhvinduced cell fusion is 7.4 (weismiller et al., 1990; kooi et al., 19911 , rather than the acidic ph expected for a virus that enters cells by an endocytotic pathway (e.g., vesicular stomatitis virus). the optimum ph for bcv-and ibv-induced cell fusion is also neutral (payne and storz, 1988; li and cavanagh, 1992) . these findings suggest that coronavirus enters cells by virus-cell fusion at the plasma membrane. on the other hand, virus internalization by endocytosis may be a nonproductive mechanism which does not depend on virus-receptor interaction, since some mhv-resistant cell lines can internalize mhv particles as efficiently as susceptible cell lines (kooi et al., 1991) . most surprisingly, even vero cells, an african monkey kidney cell line which presumably does not have an mhv receptor, can internalize virus (kooi et al., 1991) . therefore, it is likely that mhv enters cells by both acidic-phdependent (endocytosis) and -nondependent pathways (kooi et al., 1991) . the exact mechanism of virus entry may depend on cell types and virus strains. interestingly, an mhv variant which has mutations in the s protein has an acidic optimum ph of 5.5-6.0, in contrast to the ph of 7.5 for the parental virus (gallagher et al., 1991) . this virus variant probably enters cells by an endocytic pathway, a fact supported by the finding that infection of this variant virus is inhibited by ammonium chloride or chloroquine. what triggers virus internalization after virus-receptor binding is not clear. it has been shown that a conformational change in the s protein could be induced at ph 8.0 and incubation at 37â°c . whether this represents the expected conformational change following virus-receptor binding is not clear. irrespective of the mechanism of virus internalization, fusion between the viral envelope and cell membrane must occur, either at the cell surface or in the endosome, for viral infection to take place. virus-induced cell-cell fusion has been used to investigate the ability of a virus to induce fusion. early studies with mhv indicated that virus-induced fusion from without (caused by virions at the cell surface) or fusion from within (caused by de ~o u o synthesized s protein on the cell surface) required cleavage of the s protein (frana et al., 1985; sturman et al., 1985) . work on bcv supported this view (payne and storz, 1988; storz et al., 1991) . however, more recent experiments involving the expression of s protein (de groot et al., 1989; stauber et al., 1993; taguchi, 1993) and studies of mhv fusion mutants (gombold et al., 1993) have indicated that uncleaved s can cause syncytium formation, though less efficiently than the cleaved s. of course, coronaviruses such as tgev, which have no cleaved s protein, are infectious, in fact, highly so. since fusion of the virion envelope with a cell membrane is an essential part of the infection process, these results suggest that tgev must be able to cause viruscell fusion. thus, virus-cell fusion and cell-cell fusion may have different requirements, and, for at least some coronaviruses, s cleavage is not required for the fusion of a virion with a cell membrane. nevertheless, cleaved s may be more efficient at inducing fusion for some coronaviruses. the concentration of s at the surface of a virion may be higher than at the cell surface, such that even the uncleaved s can induce virion-cell fusion, even though it cannot cause cell-cell fusion. virusreceptor interaction may also trigger a signal transduction pathway to facilitate the internalization of the virus-receptor complex. one study showed that tyrosine kinase is activated in macrophages immediately following mhv-3 infection (dackiw et al., 1995) . it is not yet known whether this is required for virus entry. the mechanism of virus uncoating, i.e., the release of virion rna from the nucleocapsid, after the virus has been internalized remains unclear. one study suggested that virus uncoating may involve an endosomal neutral phosphatase, which preferentially dephosphorylates the nucleocapsid protein (mohandas and dales, 1991) . furthermore, while immature oligodendrocytes were sensitive to jhm virus infection, differentiated oligodendrocytes were resistant, probably due to a block in virion uncoating (beushausen et al., 1987) . the factors responsible for the differences in these two types of cells may involve protein kinases (wilson et al., 1990) . additional cellular factors may be required for viral penetration and uncoating. various murine cell lines, all of which express virus receptors, show different degrees of susceptibility to infection by different mhv strains (kooi et al., 1988; asanaka and lai, 1993; yokomori et al., 1993) . cell-cell and virus-cell fusion studies indicated that virus infection is blocked at different stages of virus entry, including penetration and uncoating, in different cell lines (van dinter and flintoff, 1987; asanaka and lai, 1993) . these cell lines could be grouped into at least three complementation groups with respect to the virus entry process (flintoff, 1984; asanaka and lai, 1993) . thus, virus penetration and uncoating appear to require separate cellular factors. it has been suggested from the studies using recombinant viruses between the a69 and jhm strains of mhv that viral s protein may interact with these cellular factors . the nature of these factors is not yet clear. following virus uncoating, the first macromolecular synthetic event is predicted to be the synthesis of an rna-dependent rna polymerase(s) from the incoming viral genomic rna, as is the case for all positive-strand rna viruses. the polymerase is translated from gene 1 at the 5' end of the genomic rna, most likely directly from the incoming genomic rna. the process of primary translation has not been observed experimentally. however, inhibitors of protein synthesis applied early in the infection blocked rna transcription (mahy et al., 1983; perlman et al., 1986; sawicki and sawicki, 19861 , indicating that protein synthesis, most likely the translation of a viral polymerase, is necessary for viral rna synthesis. this virus-specific polymerase is responsible for the synthesis of negative-strand rna from the incoming genomic rna and subsequent transcription of mrnas from the negative-strand template. the nature of polymerase is discussed in section iv,a. since the genomic-sized rna is used for both packaging into virus particles to become virion rna and as an mrna for protein translation, the distinction between rna transcription and rna replication is often blurred. in this review, we will use the term "transcription" to describe the synthesis of subgenomic mrnas as well as genomic rna used for translation; the term "replication" will be used to describe the synthesis of the genomic rna destined to be packaged into virions. coronavirus rna synthesis occurs via an rna-dependent rna transcription process; thus, rna synthesis can occur in the presence of actinomycin d (with the exception of some coronaviruses, as discussed in section v,a). the majority of the virus-specific rnas in the cells are mrnas, which are transcribed from a negative-strand rna template. for clarity of discussion, the structure of the mrnas will be discussed first. coronavirus mrnas consist of six to eight species of different sizes, depending on the coronavirus species and strains (lai, 1990) . the largest mrna is equivalent to the genomic rna, and the remainder are subgenomic in size. these rnas are designated mrnas 1 through 7, in order of decreasing size, according to the recommendations of the coronavirus study group in 1989 (cavanagh et al., 1990) . some mrnas have been given a hyphenated name, e.g., mrna 2-1, because they were discovered after the original set of mrnas was named. they have a nested-set structure, and all of them contain sequences starting at the 3' terminus and extending to various distances toward the 5' end (stern and kennedy, 1980b; lai et al., 1981; leibowitz et al., 1981) . the smallest mrna contains only the 3' terminal orf, while each next larger mrna contains one additional orf. the structure of the mrnas in relation to the genome structure is shown in fig. 7 . thus, except for the smallest mrna, all of the mrnas are structurally polycistronic. in general, each orf in the genome is represented by an mrna, whose sequence starts from a consensus signal upstream of the orf, and only the 5' most orf of each mrna can be translated; thus, each mrna is functionally monocistronic. however, there are exceptions: some mrnas, e.g., mrna 5 of mhv and mrna 3 of ibv, are translated into two or three proteins by different mechanisms (see section v,g). several additional minor mrna species have been detected, some of which could only be detected by reverse transcription-polymerase chain reaction (la monica et al., 1992; schaad and baric, 1993) . these minor rnas probably represent rna transcripts from weak or atypical mrna start signals (see below). most do not contain a complete orf at the 5' end; thus, they are probably not functional. furthermore, in mhv, several mrnas, e.g., mrnas 2-1, 2-2, and 3-1, are transcribed only in some virus strains la monica et al., 1992) . the syntheses of these mrnas appear to be differentially regulated by the sequence at the 5' end of the viral genome la monica et al., 1992) . coronavirus mrnas have another unique structural feature: their 5' ends have a leader sequence of approximately 60-90 nucleotides, which is derived from the 5' end of the genomic rna (lai et al., , 1983 spaan et al., 1983) . the leader sequences of all the mrnas are identical for a given strain of virus, except for slight variations at some of the leader-mrna fusion sites, and are identical to the sequence present at the 5' end of the genomic rna. at the mrna start sites on the viral genomic rna, there is a short stretch of sequence that is nearly homologous to the 3' end of the leader rna (budzilowicz et az., 1985) . this sequence constitutes part of the signal for subgenomic mrna transcription (makino et al., 1991) . sequence comparison of viral genomic and mrnas suggests that subgenomic mrnas are derived by fusion of the 5' end genomic rna sequence (leader) to the mrna start sites on the viral genomic rna. the mrna start sites are usually located between the genes; hence, they are termed intergenic (ig) sequences. however, some of the igs may overlap the coding region of the upstream gene. the core sequence of the ig for mhv is ucuaaac or a slightly variant form of this sequence at various ig sites (joo and makino, 1992) . other virus species also have similar ig sequences. the leader sequence of mhv ranges in length from 72 to 82 nucleotides, the variation resulting from the heterogeneity of the 3' end sequence, which contains two to four copies of a pentanucleotide (ucuaa) repeat. the homologous nucleotides (ucuaa) at the 3' end of the leader and ig sites serve as fusion sites for the leader and mrnas. some of the mhv mrnas are heterogeneous, consisting of several subspecies, each containing different copy numbers of the ucuaa repeat . this fact suggests that the fusion between the leader rna and the mrnas is not very precise. the length and sequence of the leader rna in other coronaviruses vary. however, the 3' end of the leader sequence always contains a pentanucleotide ucuaa or a closely related sequence. mrnas of coronaviruses other than mhv are usually homogeneous in their structure, probably a reflection of the fact that leader rna at the 5' end of the genome and ig sites in these viruses contain only a single copy of the the ucuaa-like sequence (hofmann et al., 1993a) . the copy number of this pentanucleotide repeat apparently plays an important role in the regulation of mrna transcription. coronavirus rna synthesis is mediated by rna-dependent rna synthesis via a negative-strand rna intermediate (complementary to the genomic rna). coronavirus negative-strand rna represents no more than 1-2% of the total intracellular virus-specific rna sawicki and sawicki, 1986) . bothgenome-sized and subgenomic negative-strand rnas, which correspond in number of species and size to those of the virus-specific mrnas, have been detected (sethna et al., 1989; hofmann et al., 1990) . the relative molar ratios of the various subgenomic negative-strand rna species are comparable to those of the positive-strand subgenomic mrnas. the 5' end of the negativestrand rna contains poly(u) sequences, which are shorter than the poly(a) sequences present on the positive-strand rnas . at the 3' end of the negative-strand rna is the complementary sequence of the leader rna (anti-leader) (sethna et al., 1991) . structurally speaking, the subgenomic negative-strand rnas appear to be mirror images of the positive-strand subgenomic mrnas. all of the negative-strand rnas in the infected cells are present in the form of double-stranded rna, no free negative-strand rna is detected . in virus-infected cells, virus-specific mrna synthesis can usually be detected a few hours after infection and throughout most of the viral replication cycle (stern and kennedy, 1980a; leibowitz et al., 1981; keck et al., 1988a) . the molar amounts of the different mrna species vary; smaller mrnas are generally more abundant than larger ones, but this rule does not always hold true. nevertheless, the relative ratio of different subgenomic mrna species remains constant throughout, suggesting that the synthesis of the various subgenomic mrna species is regulated coordinately. some viruses may show slight variations in the amounts of individual mrna species present during infection (hiscox et al., 1995a) . later in infection, there appears to be an enhanced synthesis of the genomic-sized rna (keck et al., 1988a) . the kinetics of negative-strand rna synthesis follows a pattern similar to that of positive-strand mrna synthesis; however, the peak of negative-strand rna synthesis appears to occur earlier than for positive-strand rna sawicki and sawicki, 1986) . thereafter, negative-strand rna synthesis drops significantly, in contrast to that of positive-strand rna synthesis, and negative-strand rna appears to be stable sawicki and sawicki, 1986) . a similar pattern of kinetics of negative-strand rna synthesis is also seen in the accumulation of the negative-strand rna of a di rna, which very rapidly reaches a steady-state level after transfection . therefore, the negative-strand rna probably functions as a template for multiple rounds of positive-strand rna synthesis. this conclusion is supported by the study of a ts mutant defective in negative-strand rna synthesis (schaad and baric, 1994) . however, the ability to synthesize negative-strand rna seems to be maintained throughout the viral life cycle, as evidenced by the finding that a transfected di rna can replicate even when transfected late in the infection (jeong and makino, 1992) . since all subgenomic rnas consist of a leader rna derived from the 5' end of the genome and a body sequence derived from various downstream sequences, they must be synthesized by fusion of two discontiguous sequences either during or after transcription. an early study showed that the leader sequence of each mrna can be exchanged freely between two coinfecting viruses, suggesting that the leader rna and mrnas are transcribed independently and can conjoin in a random fashion (makino et al., 1986b) . more recent studies using di rna constructs that contain an inserted mrna start signal (see below) established that the leader rna and mrnas are usually derived from two separate rna molecules (jeong and makino, 1994; zhang et al., 1994b) . these studies unequivocally showed that coronaviral mrna synthesis is carried out by either a discontinuous transcription or transsplicing process, which fuses sequences from two different rna molecules. several transcription models have been proposed, each of which is consistent with some of the experimental data. these models are not mutually exclusive, as components of each model may operate at different stages of the viral replication cycle. before presenting these models, we will discuss several findings pertinent to coronaviral rna transcription. 1. coronavirus replication takes place entirely in the cytoplasm. nuclear functions are believed not to be required for rna synthesis wilhelmsen et al., 1981) ; thus, viral rna transcription does not involve the conventional rna splicing machinery present in the nucleus. 2. early ultraviolet (w) transcriptional mapping studies indicated that in the late stage of viral replication, the w target size of each subgenomic and genomic mrna is approximately equivalent to the physical size of the respective mrna (jacobs et al., 1981; stern and sefton, 1982a) ; thus, each mrna is transcribed independently rather than derived by the processing of a large precursor rna. however, early in infection, the w target sizes of the subgenomic mrnas were found to be equivalent to that of the genomic rna (yokomori et al., 199213) ; thus, at least early in infection, the synthesis of a genomic-length rna is required for subgenomic mrna synthesis, although it is not clear whether this requirement is for a positive-or a negative-stranded, full-length rna. a more recent analysis of the uv target sizes of subgenomic mrnas of mhv suggested that, even late in the infection, the w target sizes of some subgenomic mrnas are slightly larger than their physical lengths but smaller than genomic size (den boon et al., 1995) . similar observations were made for equine arteritis virus (an arterivirus). this recent result is consistent with either of two interpretations: (a) the subgenomic mrnas are derived from a slightly longer rna template or (b) they are derived from a mixture of templates of different sizes (genomic as well as subgenomic). the difference in w target size between the early and late stages of viral rna replication suggests that different mechanisms of rna synthesis may operate at the different stages of the viral replication cycle. 3. the molar ratios of different subgenomic mrna species and those of subgenomic negative-strand rnas are similar (sethna et al., 1989; hofmann et al., 1990) , suggesting that subgenomic mrnas and subgenomic negative-strand rnas are derived from each other or under the same transcriptional regulation. 4. the leader rna at the 5' end of each mrna is identical in each mrna and to the leader rna at the 5' end of genomic rna. furthermore, there is sequence homology between the 3' end of the leader rna and the mrna start sites on the genomic rna (budzilowicz et al., 1983 , where the leader sequence is fused to the mrnas. there is some sequence divergence between the leader rna and some of the mrna start sites; in these cases, the leader rna of the resulting mrnas usually mimics the sequence of the mrna start site rather than the leader at the 5' end of the genome. this finding was used to suggest the possible presence of rna proofreading activity during coronavirus transcription (lai, 1986 (lai, ,1990 ; van der most et al., 1994). the following transcriptional models (fig. 8) address the possible mechanism of fusion between the leader sequence and mrnas. most of the experimental evidence came from mhv studies. the exceptions will be noted. a. leader-primed transcription. this model proposes that the virion genomic rna is first transcribed into a genomic-length, negativestrand rna, which, in turn, becomes the template for subsequent subgenomic mrna synthesis. the leader rna is transcribed from the 3' end of the negative-strand rna and dissociated from the template. the free rna subsequently associates with the template rna at various mrna start sites and serves as a primer for transcription of mrnas. it is proposed that the discontinuous transcription step takes place during positive-strand rna synthesis. several pieces of evidence are compatible with this model: detected in the cytoplasm of mhv-infected cells (baric et al., 1985) . some of these are dissociated from the template rna and, thus, may serve as a potential source of primers in this transcription model. these rnas have distinct sizes which are reproducible from cell to cell (baric et al., 1987) ; however, they are not exactly the same size as the leader sequence present in the subgenomic mrnas. thus, these free leader rnas must be processed before they are incorporated into mrnas. a temperature-sensitive mutant of mhv, which synthesizes leader rna but not mrnas at the nonpermissive temperature, has been isolated . the isolation of this mutant suggests that mhv mrna synthesis is discontinuous, requiring different viral proteins for the synthesis of leader rna and mrnas. thus, a distinction can be made between leader rna synthesis and mrna synthesis. of the viruses is derived from the other coinfecting virus (makino et al., 1986b) . this result suggests that the leader sequence and body sequence of each mrna are derived from two separate pools. this phenomenon is reminiscent of the rna reassortment that occurs in rna viruses with segmented rna genomes. this result is best explained by the possibility that free leader rnas participate in viral rna synthesis. in an in vitro transcription system utilizing cytoplasmic extracts from mhv-infected cells, exogenous leader rnas can be utilized for mrna synthesis (baker and lai, 1990) . the exogenous leader rna was incorporated into the subgenomic mrnas at a site that matched precisely that of the endogenous leader rna present in the viral subgenomic mrnas, regardless of the length of the exogenous leader rna used, suggesting that the exogenous leader rna sequence was processed before being incorporated into mrnas. furthermore, the truncated leader rna which lacked the 3' end ucuaa sequence could not be incorporated into mrnas, suggesting the importance of this sequence in transcription (baker and lai, 1990) . 5. the leader rna sequence, specifically the copy number of the ucuaa repeats at the 3' end of the leader rna, can affect the transcription of some viral subgenomic mrnas. for example, whereas an mhv strain containing two ucuaa repeats transcribes mrna 2-1, a strain with three ucuaa repeats does not, despite identical sequences in the mrna start sites of these two viruses la monica et al., 1992) . this finding suggests that the leader rna plays an essential role in the regulation of mrna transcription. according to this model, the free leader rna binds to the mrna start site (ig) of the full-length negative-strand template via the complementary sequences between the 3' end of the leader (positive-strand) and the ig site of the template rna (negative-strand) and serves as the primer for rna transcription. the free leader rna (primer) may be longer than the leader sequence in the subgenomic mrnas. there are certain mismatched nucleotides between the leader and template at some mrna start sites; in the latter case, sequences in the mature mrnas usually match those of the template instead of the leader. therefore, the free leader rna probably undergoes 3' end cleavage before transcription starts to remove the leader nucleotides that are not complementary to the template rna (lai, 1986 (lai, ,1990 van der most et al., 1994) . transcription is then initiated from the 3' end of the processed leader rna. this model is consistent with most of the sequence data of mrnas. it also explains the curious finding that some mrnas of mhv are heterogeneous in the copy number (from two to four) of the pentanucleotide (ucuaa) repeats at the leader-mrna fusion site . this heterogeneity is best explained by the imprecise binding between the leader rna and template rna due to the presence of multiple copies of ucuaa (lai, 1990) . indeed, bcv, which contains only one copy of ucuaa in both the 5' leader and ig sites, does not show this type of heterogeneity in its mrnas (hofmann et al., 1993a) . some recent data, however, cannot be explained by this rna sequence-homology-driven transcription model. a particular mhv strain (mhv-bc), which has four copies of the ucuaa in the leader rna, synthesizes some subgenomic mrnas that are very heterogeneous in length and in leader-mrna fusion sites . the sequence data of its mrnas showed that the leader rna of this virus is randomly fused to sites where no sequence homology exists between the leader and fusion sites (zhang et al., 199413) . a similar though less conspicuous heterogeneity in the leader-mrna fusion sites has also been observed in another mhv strain in a di rna-based transcription system (see section v,e,5) (van der most et al., 1994) . these findings suggest that the sequence complementarity between the leader rna and ig sites may not be the driving force for mrna transcription. thus, a modified version of the leader-primed transcription model proposes that the ucuaaac sequence provides a recognition signal for viral polymerases and viral or cellular transcription factors. these proteins bind to the leader and ig sites of the template rna, and the subsequent rna-protein and protein-protein interactions result in the formation of a transcription complex to initiate mrna transcription and effect leader-mrna fusion zhang and lai, 1995) . the salient feature of this model is that the discontinuous transcription step occurs during positive-strand rna synthesis; thus, transcriptional regulation is exerted mainly during positive-strand rna synthesis. this is consistent with current knowledge of the regulation of mhv rna synthesis. it has been shown that mhv mrna transcription requires multiple cis-acting rna sequences (see section v,e,5). in contrast, the initiation of negative-strand rna synthesis requires only the 3' end 55-nt plus poly(a) . thus, most of the regulatory elements appear to regulate positive-strand rna synthesis. since the free leader rna is the centerpiece of this transcription model, it readily explains why the leader rna from a different virus can be utilized freely in trans during mixed infections (makino et al., 1986b) . however, this model does not explain the finding that subgenomic replicative-intermediates (ri) and replicative-form (rf) rnas were detected and were functional during viral rna synthesis (sawicki and sawicki, 1990; schaad and baric, 1994 ) (see section v,e,4,b). it is possible that the subgenomic mrnas synthesized can be transcribed into subgenomic negative-strand rnas, which, in turn, become the templates for mrna transcription at a later stage in the viral replication cycle. this would explain why the uv target sizes for mrnas are nearly equivalent to the physical sizes of mrnas late in the infection and yet are equivalent to the genomic-sized rna early in the infection (yokomori et al., 1992b) . in contrast to the leader-primed transcription model, this model proposes that the discontinuous transcription step occurs during negative-strand rna synthesis, generating subgenomic negativestrand rnas, which then serve as templates for subgenomic mrnas in uninterrupted transcription. this model was proposed to account for the detection of subgenomic negative-strand rnas (sethna et al., 1989; hofmann et al., 1990) and subgenomic ris (sawicki and sawicki, 1990) in virus-infected cells. in this model, ig (mrna start site) sequences on the genomic rna serve as termination or pausing signals for negative-strand synthesis (konings et al., 19881 , and the nascent subgenomic negative-strand rna then jumps to the leader rna sequence at the 5' end of the genomic rna by an unknown mechanism to continue rna synthesis. as a result, the nascent negative-strand subgenomic rna fuses with the negative-strand leader sequence, generating a subgenomic negative-strand rna that contains an anti-leader sequence at its 3' end and a poly(u) sequence at its 5' end sethna et al., 1991) . structurally, these negativestrand rnas are mirror images of the subgenomic mrnas and, thus, can potentially serve as a template for uninterrupted transcription of subgenomic mrnas. in this model, the regulation of subgenomic mrna transcription would be exerted on negative-strand instead of positive-strand rna synthesis. this model is consistent with the following observations: 1. subgenomic negative-strand rnas have been detected in virusinfected cells (sethna et al., 1989; hofmannet al., 1990) . these rnas have structures that are mirror images of those of the completed subgenomic mrnas. the relative molar ratios of the different subgenomic negative-strand rnas are similar to those of the corresponding viral mrnas (sethna et al., 1989; hofmann et al., 1990 ). in the infection (sawicki and sawicki, 1990) . the smaller ris were precursors of the smaller mrnas and the larger ris generated the larger mrnas, suggesting that each subgenomic mrna was transcribed from the corresponding subgenomic-sized negative-strand template (sawicki and sawicki, 1990) . another study, which analyzed the subgenomic rfs of a temperature-sensitive mutant of m w , also suggested that subgenomic negative-strand rnas are functional (schaad and baric, 1994) ; although, in this study, ris were not directly examined. 3. the uv targets for subgenomic mrna synthesis at the later stage of viral replication are subgenomic in length (jacobs et al., 1981; stern and sefton, 1982a; yokomori et al., 1992b) , roughly corresponding to the physical lengths of each subgenomic mrna, suggesting that the templates for these mrnas are subgenomic. 4. in di rna systems (see section v,e,5), when multiple ig sequences were present, the sequences in the 3' end often had a higher transcription efficiency than those at the 5' end, consistent with the proposal that igs serve as transcriptional termination sites, which impede the elongation of the negative-strand rnas (van marle et al., 1995; krishnan et al., 1996) . however, in some cases, the higher transcription efficiency of the 3' proximal ig was observed only when the neighboring igs were very close together, suggesting a spatial constraint rather than sequential interference (joo and makino, 1995) . this model, however, cannot explain why the w targets for subgenomic mrna synthesis early in infection are of genomic size (yokomori et al., 1992b) and why, later in the infection, the targets for these same mrnas are still larger than the respective subgenomic mrnas but not longer than genomic size (den boon et al., 1995) . it also cannot explain why the nature of the leader sequence can regulate differential transcription of various mrna species, such as mrna 2-1 of mhv, inasmuch as the leader sequence on the template rna is localized downstream of the transcription termination site for negative-strand rna synthesis. finally, it is difficult to explain why the leader rnas are derived in trans. c. trans-splicing of nascent rna transcripts. this model proposes that the fdl-length positive-or negative-strand rnas are spliced posttranscriptionally to generate subgenomic rnas. it was initially considered unlikely because of the findings that coronavirus replicates in the cytoplasm rather than in the nucleus wilhelmsen et al., 1981) , where the splicing machinery is present, and that uv target sizes of subgenomic mrnas are equivalent to the physical sizes of subgenomic mrnas (jacobs et al., 1981) . furthermore, there are no consensus splicing donor and acceptor sequences in the coronavirus genomic rnas. however, the trans-splicing model is compatible with recent findings that early in infection, the w targets for subgenomic mrna synthesis are of genomic length (yokomori et al., 1992b) , and that both the leader rna and ig sequence of mhv negative-strand rna bind to a cellular factor, heterogeneous nuclear rnp (hnrnp) al, which is involved in alternative rna splicing h.-p. li and m. m. c. lai, unpublished observation) . a modified splicing model thus can be proposed as follows: a full-length negative-strand rna is first synthesized. components of the splicing machinery derived from the nucleus or cytoplasm then bind to the leader sequence and ig sites on the negative-strand rna and form a splicing complex. the leader and ig can be derived from different rna molecules. splicing between the leader and ig generates a subgenomic negative-strand rna. once the spliced subgenomic negative-strand rnas are generated, they are used as templates for subsequent mrna synthesis. later in infection, even the subgenomic negative-strand rnas may be able to participate in rna splicing to generate smaller subgenomic negativestrand rnas because they themselves also contain the leader and ig sequences. this model may thus explain why the w target for mrna transcription is of genomic length early in infection (yokomori et al., 1992b) and may shed light on the recent puzzling finding that later in infection, the w target sizes are still larger than the actual sizes of the subgenomic mrnas (den boon et al., 1995) . it also explains the functional roles of subgenomic ris (sawicki and sawicki, 1990) . this potential splicing, however, must be different from conventional rna splicing because it occurs in the cytoplasm, and the splicing donor and acceptor sequences must also be different from the conventional ones. since some of the splicing factors are probably derived from the nucleus, this model predicts that nuclear functions are involved in mhv rna transcription. based on the findings that some coronaviruses, including bcv, tgev, and ibv (sethna et al., 1989; hofmann et al., 1990; zhao et al., 1993) , contain subgenomic mrnas in the virion, probably as a result of nonspecific rna packaging, it was proposed that these virion-associated subgenomic mrnas can be used directly as templates for the synthesis of subgenomic negative-strand rnas, which, in turn, serve as templates for the synthesis of additional subgenomic mrnas (sethna et al., 1989) . this model may explain the presence of subgenomic negative-strand rnas and ris in the infected cells, but it cannot explain the genomiclength nature of the w target sizes for mrna synthesis early in infection (yokomori et al., 1992b) , nor can it explain how leader rnas from different virus strains can be randomly incorporated into mrnas of a different virus. furthermore, the virion-associated subgenomic mrnas have not been detected in all coronavirus species. the available data cannot unequivocally rule out any of the proposed transcription models. the primary difficulty in experimental analysis is that once the subgenomic mrnas are synthesized, by whatever mechanism, they are transcribed into negative-strand rnas because the cis-acting signal for negative-strand rna synthesis in mhv resides in the 55 nucleotides at the 3' end plus poly(a) (lin et al., 1994) , which is present in every subgenomic rna. thus, it is difficult to separate the primary and secondary events of transcription. it is possible that these transcription models are not mutually exclusive. for example, early in infection, a leader-primed transcription or trans-splicing mechanism may operate, generating subgenomic mrnas, which are then amplified into subgenomic negative-strand rnas; the latter serve as templates for further amplification of subgenomic mrnas thereafter. the subgenomic negative-strand rna can be used for either uninterrupted transcription or leader-primed transcription to generate positive-strand subgenomic rnas. a combination of these models would be consistent with most of the experimental data. this twostep model of primary and secondary transcription (jeong and makino, 1992) may explain the apparent differences in the possible mechanism of transcription between early and late stages of viral infection. because of the large size of coronavirus rna, no infectious cdna or rna clones are now available for reverse genetics studies. this difficulty has hampered progress in the study of the molecular biology of coronaviruses. di rnas of several coronaviruses (see section vi,e) have been molecularly cloned and used as a substitute for the genomic rna to study the cis-and trans-acting signals involved in viral rna synthesis. although natural di rnas do not contain an mrna start signal and, consequently, cannot transcribe an mrna, the insertion of such a signal into the di rna allows an mrna to be transcribed from the transfected di rna in the virus-infected cells, thus enabling studies of the regulatory sequences for transcription. following is a summary of information that has been obtained using this approach. it should be cautioned, however, that regulation of rna transcription probably depends on overall rna conformation and that the cis-acting sequence required for rna synthesis very often varies with the di rna vector used; therefore, the results obtained from di rna studies may not be directly applicable to the viral genome. a full understanding of the regulation of viral rna synthesis still awaits the development of an infectious cdna clone. the following cis-acting signals for coronavirus rna transcription have been determined primarily from mhv di rna studies (with some from bcv di) (fig. 9) . a. ig sequence. the ig sequence can be considered to be the promoter element for transcription. it also serves as the mrna start site and the site of fusion between the leader rna and body sequence of mrnas. a seven-nucleotide core sequence, ucuamc, is sufficient to initiate mrna synthesis (makino et al., 1991) . extensive site-specific mutagenesis studies have shown that most of the single-nucleotide mutations within this core sequence could be tolerated, although the transcription efficiency of some of these mutants was lower (joo and makino, 1992; van der most et al., 1994) . these seven nucleotides represent the minimum promoter; deletion of a nucleotide results in complete ablation of mrna transcription. the effects of the sequences near the promoter on transcription are contradictory: in certain situations, the nature of the neighboring sequences did not affect transcription (makino and joo, 19931 , but under other circumstances, it did (jeong et al., 1996) . thus, the strength of the promoter appears to depend on the context of the overall rna sequence and structure. the relative flexibility of sequence requirement of the promoter sequence in the di rna system appears to differ significantly from that seen in the viral genomic rna. in the mhv genome, there are more than 20 stretches of sequence resembling the ucuaaac sequence, in addition to the six promoters for the known subgenomic mrnas (joo and makino, 1992 ). yet, most of these did not promote mrna synthesis from the viral rna genome to any appreciable extent, in contrast to their ability to promote transcription in the di rna vector system (joo and makino, 1992) . in the viral genome, the single-nucleotide substitution of a g residue in the core promoter sequence completely abolished mrna synthesis (shieh et al., 19891, whereas this is tolerated in the di rna (joo and makino, 1992) . thus, there appear to be significant differences between the sequence requirement for mrna synthesis in the di rna and in the natural viral genomic rna. when there are multiple ig sequences in the di rna, the order of the ig sequences may influence transcriptional efficiency. an ig located at the 3' end generally has an advantage in initiating mrna synthesis (van marle et al., 1995; krishnan et al., 1996) . the sequences near the igs may suppress transcription (jeong et al., 1996) . b. the leader sequence at the 5' end of the dz rna. the leader sequence at the 5' end of the viral genomic rna becomes the leader sequence of subgenomic mrnas; thus, it fills a structural role for mrna synthesis. however, the leader rna of the subgenomic mrnas is not derived exclusively from the leader rna of the same (di) rna; in fact, most are derived in trans from a separate rna molecule, such as helper virus rna (jeong and makino, 1994; liao and lai, 1994; zhang et al., 199413) . nevertheless, mrna transcription from an ig site in the di rna still requires the presence of a leader rna sequence at the 5' end of the di rna as a cis-acting sequence . deletion of this cis-acting leader abolished transcription. furthermore, the sequence of this leader rna, particularly its 3' end sequence, can affect the efficiency of transcription from certain ig sequences on the di rna (zhang et al., 199413) . for example, the leader rna containing two pentanucleotide (ucuaa) repeats transcribes an mrna from the ig 2-1 site more efficiently than the leader rna with three ucuaa repeats. thus, the cis-acting leader rna plays a role similar to that of an enhancer. these findings suggest that the leader rna serves two functions : (1) it supplies the leader rna to the subgenomic mrnas, and (2) it serves as an enhancer-like sequence to regulate transcription. this finding also suggests that there is either a direct or an indirect interaction between the leader and ig sequences. some additional sequences downstream of the leader may also enhance transcription from an ig site in the di rna ; however, the precise sequence requirement is not known. this sequence requirement shows some virus sequence specificity, since it cannot be replaced with other viral rna sequences . it may be needed to maintain overall rna conformation for the recognition of the ig sequence. c. the 3' utr. in an mhv di rna construct, partial deletion of the 3' utr completely abolished transcription from an upstream ig site in the di rna (lin et al., 1996) . this stretch of 3' utr is probably involved in positive-strand rna synthesis, since the length of this required sequence (305 nt) is significantly longer than that required for negative-strand rna synthesis (55 nt). the 3' utr requirement for mrna transcription is surprising, since positive-strand rna synthesis starts from the 5' end; thus, the 3' end sequence is the last to be transcribed. this 3' utr sequence requirement is similar to that for rna replication (kim et al., 1993b; lin and lai, 1993 ) (see section v,f). this finding suggests that the 3' end may interact with the 5' end and possibly with ig sequences during transcription. a nine-nucleotide sequence, uuuauaaac. this sequence, located immediately downstream from the ucuaa repeats at the 3' end of the leader rna in the viral genome, plays a significant role in rna transcription. it is deleted from the genome of one of the mhv strains and is often deleted in naturally occurring di rnas . in this particular mhv strain (mhv-20, the leader-mrna fusion sites are very heterogeneous and do not always occur at the usual ucuaaac sites (zhang et al., 199413) . this nine-nucleotide sequence can serve as an mrna start signal, allowing transcription of an almost genomic-length mrna . in the di rnas, the presence or absence of this nine-nucleotide sequence influences transcription efficiency from the downstream ig site and, most importantly, affects the source of the leader rna incorporated into subgenomic mrnas (zhang et al., 1994b) . when this nine-nucleotide sequence is present, the leader sequence in the subgenomic mrnas is contributed both from the di rna in cis and from helper virus rna in trans. when this sequence is missing, the leader rna is derived exclusively from the helper virus rna (zhang et al., 1994b) . thus, this nine-nucleotide sequence appears to regulate the mechanism by which the leader rna is fused t o the subgenomic mrnas. these results combined suggest that multiple rna regions are involved in the regulation of mrna transcription. however, a recent study appears to contradict the need of cis-acting sequences other than the igs for mrna transcription. when a negative-strand rna containing only an ig sequence of tgev and a reporter gene was transcribed in situ from a transfected cdna by using a recombinant vaccinia virus-t7 rna polymerase expression system, this rna was transcribed in the presence of tgev, generating an mrna with a correctly fused tgev leader sequence (hiscox et al., 1995b) . the leadercontaining mrna could have been generated by either of the transcription mechanisms described (section v,e,4,a or section v,e,4,b) above. this study suggests that this negative-strand ig site is sufficient for transcription. however, it is possible that this activity represents a basal level of transcription and that other cis-acting sequences may enhance the efficiency of transcription. the application of inhibitors of protein synthesis at any time during the viral life cycle inhibits viral rna synthesis, suggesting that continuous protein synthesis is required for rna synthesis sawicki and sawicki, 1986) . a similar observation has been made using an inhibitor of cysteine proteases, which inhibits a specific step of the processing of gene l a products of mhv (see section v,g,2), suggesting that continuous production of polymerase is required for viral rna synthesis. the precise nature of the viral proteins involved has yet to be determined. temperature-sensitive mutants of mhv that are defective in rna synthesis at the nonpermissive temperature have been divided into at least five complementation groups, indicating that at least five proteins are involved in viral rna synthesis (leibowitz et al., 1982a; baric et al., 1990 ) (see fig. 6 ). all of these complementation groups are mapped within the gene 1 region (including both l a and lb). sequence analysis showed that gene l b contains an rna polymerase motif (gorbalenya et al., 198913; lee et al., 1991) . polymerase activities have been demonstrated in membrane fractions of bcv-and mhv-infected cells dennis and brian, 1982) , and several in vitro rna synthesis systems have been reported (compton et al., 1987; baker and lai, 1990) ; however, the nature of polymerases in these systems has not been identified. in one study, it was demonstrated that the antibodies against the n protein could inhibit rna synthesis, suggesting that n protein may be involved in rna synthesis (compton et al., 1987) . in addition to viral proteins, cellular factors may also be involved in rna synthesis. several cellular proteins have been shown to bind to the regulatory elements of mhv rna, including the 5' and 3' ends of the genomic rna and the 3' end of the negative-strand rna and ig sites yu and leibowitz, 1995a,b; zhang and lai, 1995) . the binding sites for the cellular proteins at the 5' end of genomic rna and the 3' end of negative-strand rna are complementary . the protein p35, which binds to the negative-strand leader sequence and the ig site, is particularly interesting. site-specific mutations of the ig site affected the binding of this protein and the efficiency of rna transcription to the same extent, suggesting that the binding of this protein is required for rna transcription . this protein recently has been identified as hnrnp a1 (h.-p. li and m. m. c. lai, unpublished observation) . the mutations at the 3' end of the viral genomic rna that abolished the binding of cellular proteins also inhibited both negative-strand and positive-strand rna synthesis, although the correlation between protein binding and rna replication was not absolute (yu and leibowitz, 1995a) . thus, cellular proteins probably play a significant role in viral rna replication and transcription. curiously, viral proteins in the infected cell extract could not be cross-linked to the viral rna in vitro, suggesting that viral proteins may interact with viral rna only indirectly through cellular proteins. this is in contrast to the finding that the purified n protein can bind to the leader rna sequence in vitro stohlman et al., 1988) . the reason for this discrepancy is not clear. the genomic-sized rna in coronavirus-infected cells theoretically consists of two populations: the messenger rna (mrna l), which is translated to yield gene l a and l b products, and the genomic rna, which is destined to be packaged into virion. early studies demonstrated that, late in the infection, most (95%) of the genomic-sized rna in the cells was associated with the viral nucleocapsid, while the remainder (5%) was present in polysomes perlman et al., 1986) . presumably, early in infection, most of the genomic-sized rna would be associated with polysomes to serve as mrnas for the synthesis of polymerase; however, this has not been demonstrated. it is not clear whether there is any difference in structure and mechanism of synthesis between these two rna populations. since genomic rna requires uninterrupted synthesis from the fulllength template, whereas mrnas involve discontinuous transcription, the two types of genomic-sized rna (mrna 1 and virion genome rna) may be synthesized by two different mechanisms. a recent study suggests that at least some of the mhv genomic-sized rnas are indeed synthesized by a discontinuous transcription, using the u c u m repeat in the leader rna and the nine-nucleotide uuuauaaac immediately downstream of the leader rna as the transcription start site . this raised the possibility that mrna 1 and virion genomic rna are distinguishable. however, it cannot be inferred from this study that the fate of the genomic-sized rna products derived from discontinuous transcription is different from the fate of those derived from uninterrupted rna synthesis. the possible involvement of discontinuous transcription in generating genomic-sized rna may explain several interesting findings regarding mhv genomic rna 1. the copy number of the ucuaa repeat in the leader sequence of the genomic rna, which ranges from two to four copies in different mhv strains, rapidly evolves during virus passage (makino and lai, 1989a; la monica et al., 1992) . starting with a pure virus population, the copy number in the viral genomic rna rapidly becomes heterogeneous during serial passages in tissue culture, and a new virus population with a different copy number of ucuaa repeats emerges (makino and lai, 1989a) . since this sequence variation is seen in the leader region but not in the ig regions, where uninterrupted rna synthesis probably occurs, this finding is best explained by the discontinuous transcription mechanism involving the 5' leader region. the imprecise fusion of the leader rna to the mrna start sites would result in heterogeneity of the copy number of the ucuaa repeats lai, 1990) . such heterogeneity is not observed when the virus, e.g., bcv, contains only one ucuaa copy in the leader rna (hofmann et al., 1993a) . the ucuaa region at the 5' end of the genomic rna is a hot spot of rna recombination during mixed infection of mhvs, resulting in recombinant mhvs with a crossover site at the 3' end of the leader rna sequence . this result is best explained by the discontinuous rna synthesis at the 5' end of the genomic rna. 3. if the generation of di rnas is viewed as an anomaly of rna replication, the structure of naturally occurring di rnas reveals an insight into the mechanism of rna replication. most of the naturally occurring mhv di rnas have a copy number of the ucuaa repeat different from that of the parental virus, and most lack a ninenucleotide sequence downstream of the ucuaa repeats . as discussed above, this is a reflection of discontinuous transcription in the region. the understanding of the mechanism of rna replication has been aided by the use of in uitro-transcribed di rna generated from cloned cdna. when di rna was transfected into virus-infected cells, the leader rna was rapidly replaced by that of the helper virus (makino and lai, 1989b; . this leader exchange is dependent on the presence of the nine-nucleotide sequence (uuuauaaac) in the di rna (makino and lai, 1989b) , consistent with the finding that this sequence serves as an mrna start signal for discontinuous transcription . the use of the cloned di rna also allowed the determination of the cis-acting signals for rna replication (kim et al., 199313; lin and lai, 1993) . it was shown that more than 400 nucleotides at both the 5' and 3' ends of the di rna are required for rna replication, and that some mhv di rnas also required a stretch (130 nt) of internal sequence in the gene 1 region for rna replication; however, the requirement for the internal sequence was not observed in other mhv or bcv di rna constructs (chang et al., 1994; luytjes et al., 1996) . thus, this internal sequence probably plays a role in maintaining the overall rna conformation for some di rnas (y. n. . again, the requirement of a 3' end sequence (436 nt) that is longer than that required for negative-strand rna synthesis (55 nt) is a surprise. these 3' end sequences are probably required for positive-strand rna synthesis during rna replication. this finding is reminiscent of the sequence requirement for rna transcription discussed above and suggests that there is a direct or indirect rna-rna interaction between the 5' and 3' ends during rna replication. these di rna studies also showed that replication of di rna is inhibited when an mrna is transcribed from an ig site within the same di rna, and that the mechanism of inhibition is due not to competition for the same transcription machinery (jeong and makino, 19921 , but most likely to the overlap of the cis-acting signals for these two different processes. however, the sequence requirements for replication and transcription are different, indicating that these two processes are distinguishable. the mrna transcription and genomic rna replication may be regulated by the same mechanism throughout most of the viral replication cycle. however, the ratio between the genomic rna and subgenomic rnas, as detected by radioactive uridine incorporation, increases during the late stages of the bcv replication cycle (keck et al., 1988a) , suggesting a possible switching mechanism from transcription to replication. it has been shown that genomic rna replication is coupled to encapsidation, since no free genomic rna is found . since the encapsidation of rna requires the n protein, this protein may participate in the regulation of switching between transcription and replication. the sequences of coronavirus mrnas usually start from a site immediately upstream of a gene. these mrnas, except for the smallest mrna, are structurally polycistronic, containing multiple orfs. only the 5' most orf in the mrnas is translatable; the remaining orfs are usually functionally silent. thus, most of these mrnas are functionally monocistronic (see fig. 7) . the s, he, m, and n proteins, and in most coronaviruses the e protein, are translated from separate mrnas by this mechanism; initiation of their translation is unremarkable, utilizing a cap-dependent translation mechanism. many ns proteins, however, are translated from truly polycistronic mrnas, i.e., two or three proteins are translated from the same mrna. for these mrnas, the first orf, e.g., 3a of ibv or 5a of mhv, is probably also translated by the same mechanism as the structural protein genes. for internal orfs, e.g., e protein of ibv and mhv, an alternative mechanism must be employed to initiate translation internally. one characteristic of coronavirus mrnas is the presence of the leader rna sequence at the 5' end, which not only participates in rna transcription, but also regulates the efficiency of translation. it has been shown that the presence of the mhv leader sequence on a heterologous mrna in a chimeric rna construct can enhance its translation in virus-infected cell lysates but not in uninfected cell lysates (tahara et al., 1994) . this effect conceivably will enable the efficient translation of viral mrnas in the face of shutoff of translation of cellular mrnas in the infected cells (siddell et al., 1980; hilton et al., 1986) . the mechanism of translational enhancement by the leader rna has not been determined. it has been shown that during persistent infection of bcv, the leader rna sequence underwent frequent mutations (hofmann et al., 1993b) . one of these mutants had an intraleader short orf and a lower translation efficiency, indicating that the leader sequence in-deed can modulate translation. another region which can potentially regulate the translation of coronavirus mrnas is the 5' utr (other than the leader sequence) of mrnas. the genomic rna (mrna 1) has a particularly long 5' . an mhv with a specific point mutation within the 5' utr was selected during persistent infection in uitro (chen and baric, 1995) . this mutant had a significantly higher translation efficiency than the wild-type virus. different subgenomic mrnas had 5' utr of various lengths, which may also affect their translation. for the translation of internal orfs, several different mechanisms are used by coronaviruses: a. ribosomal frameshifing within the polymerase gene. all of the coronavirus genes 1 (polymerase) sequenced so far contain two overlapping orfs. several features of the ibv polymerase gene sequence , coupled with the absence of a distinct mrna for orf lb, suggested that translation of orf l b involved ribosomal frameshifting from orf la, thus synthesizing a large polyprotein containing both l a and l b sequences. subsequently, a highly efficient (30% frequency) -1 frameshift was demonstrated experimentally in uitro (brierley et al., 1987; somogyi et al., 1993) and in uiuo (brierly et al., 1990) . this mechanism has been shown to operate in gene 1 of mhv, hcv-229e, and tgev as well (bredenbeek et al., 1990; lee et al., 1991; eleouet et al., 1995) . in all cases, the mechanism involves two essential elements: a slippery site followed by an rna pseudoknot (brierley et al., 1989) . the site at which the ribosome slips backward has the sequence uuuaaac. the pseudoknots of ibv and mhv are similar, comprising two base-paired regions stacked coaxially in a quasi-continuous manner and connected by two singlestrand loop regions. the hcv-229e pseudoknot is more complex . it is the overall shape and stability of the pseudoknot that are important, not the nucleotide sequence per se. two reasons have been put forward to explain why coronaviruses should employ ribosomal frameshifting to translate orf l b (brown and brierly, 1995) . one reason is that this is done primarily to control the relative amounts of the l a and l b products. that could be achieved in other ways, of course, e.g., by translating orf l b from a separate mrna, this will require that the transcription of l a and l b mrnas is tightly regulated. the other reason may be to avoid making a l b mrna. such an mrna might be packaged into virions in competition with genomic rna, as the rna region corresponding to the l b orf of mhv contains a sequence that is essential for packaging into virions (fosmire et al., 1992 ) (see section vi,e,l). mrna. the e proteins of ibv and mhv are encoded by the third and second orf, respectively, of the corresponding genes 3 and 5 (fig, 5) . cells infected with ibv contain the products of all three of the gene 3 orfs (liu et al., 1991) . both of the mhv gene 5 orfs are translated in vitro (budzilowicz and weiss, 19871 , but only the 5b orf product has been detected in virus-infected cells . experiments have shown that the e protein orf of both ibv and mhv mrnas is translated by a cap-independent, internal ribosomal entry mechanism (liu and inglis, 1992b; thiel and siddell, 1994) . furthermore, if the 3a and 3b orfs were eliminated from the ibv mrna, translation of the 3c (e) orf did not occur (liu and inglis, 1992b) . this suggested that the 3d3b region contains an internal ribosome entry site (ires) for the e protein orf. le and colleagues have predicted the existence of secondary structures in the 3d3b region of ibv which resemble the ires elements of picornaviruses (le et al., 1994) . they predicted a 265-nucleotide sequence in 3d3b which would fold into five stem-loops, forming a compact structure by the interaction of two pseudoknots. c. translation of nonstructural proteins. in addition to the ns proteins encoded from the 5'-most orfs of mrnas, several other ns proteins are encoded from an internal orf of some viral mrnas, e.g., 3b of ibv and hcv-229e, 4b of bcv, and 7b of fcv (fig. 5) . most of these products have been detected in virus-infected cells; however, the mechanism of the internal initiation of translation has not been elucidated. bcv and mhv rna contains an additional internal orf within the n protein gene. this orf (termed i) is in a different reading frame from that of n protein and encodes a hydrophobic protein (senanayake et al., 1992; fischer et al., 1997) . this protein is translated in virusinfected cells by a leaky ribosomal scanning mechanism from the bicistronic mrna of n gene (senanayake et al., 1992) . it is a nonessential gene. the mechanism of its regulation is not yet clear. the gene 1 product is predicted to be nearly 700-800 kda. it is probably processed into multiple proteins posttranslationally by its own proteases. the processing pathway has just begun to be explored. remarkably, the protease domains and potential cleavage sites predicted by computer analysis (gorbalenya et al., 198913; lee et al., 1991) have largely been confirmed by experimental data. initially, in vitro translation of virion rna of mhv revealed several polypeptides of more than 200 kda (leibowitz et al., 1982b; denison and perlman, 1986) . in addition, a 28-kda product was detected and shown to have originated by cleavage from the n terminus of a precursor (denison and perlman 1986; soe et al., 1987) , now known to be the beginning of the orf l a polyprotein (fig. 6) . the cleavage which generates p28 is carried out by plp 1 (fig. 6) . it cleaves between residues gly-247 and val-248, mutation of either residue resulting in almost no cleavage (dong and baker, 1994; hughes et al., 1995) . in addition to p28, the mhv orf l a encodes a protein of more than 400 kda, which is cleaved to a 290-kda product, which, in turn, is cleaved to produce a 50-kda and a 240-kda product (denison et al., 1992) (fig. 6) . another protein of 65 kda is derived from sequence immediately downstream of the p28-encoding region, thus representing the n-terminal part of the large polyprotein initially found in in uitro translation (probably more than 400 kda) (fig. 6) . the cleavage of p65 from the polyprotein was also carried out by plpl (bonilla et al., ,1997 . inhibition of the c-terminal cleavage of p65 by e64d, an irreversible inhibitor of cysteine (thio) proteinases, inhibited mhv replication . in addition, the 3clp domain is cleaved from the polyprotein by the autocatalytic cleavage activity of 3clp itself to generate a 27-34 kda protein, which contains both the trans-and cis-acting proteolytic activities (lu et al., , 1996 liu and brown, 1994; ziebuhr et al., 1995) . e64d also inhibited the 3clp protease activity. the processing pathway of the l b protein sequence is less clear. there is experimental evidence with ibv and hcv-229e that the l b polyprotein is cleaved in trans by the 3clp encoded by orf l a (liu et al., 1994; ziebuhr et al., 1995; grotzinger et al., 1996) . a polypeptide of approximately 100 kda, representing the extreme c terminus of orf l a and the n terminus of the frame shifted orf lb, was immunoprecipitated from ibv-infected cells. the cleavage sites of the 100-kda protein appear to be at the q/s sites, as predicted from the computer analysis and consistent with the known substrate specificity of the picornavirus 3c protease. a similar observation was recently made with hcv-229e (grotzinger et al., 1996) . this 100-kda protein contains the putative rna polymerase motif and thus may represent the coronavirus polymerase. the coding region for this protein belongs to complementation group d, which has been shown to effect mrna transcription (fig. 6) . b. processing of the structural proteins the s protein is co-translationally glycosylated with nlinked glycans. conversion of the high mannose (simple) glycans of the s protein to complex ones is a slow process, the half-life being one to several hours (vennema et al., 1990a) . the s protein undergoes multiple disulfide linkages to fold into a more complex structure (opstelten et al., 1993) and oligomerize into a trimer in the golgi complex (delmas and laude, 1990) . the s prepropolypeptide is converted to a propolypeptide by removal of the n-terminal signal peptide. whether the propolypeptide is cleaved to generate s1 and s2 depends on the virus species and strain and, to some extent, on the cell type in which the virus is grown (frana et al., 1985) . the sl-s2 cleavage site in ibv and mhv is adjacent to several basic residues (cavanagh et al., 1986a; luytjes et al., 1987) . those coronaviruses whose s protein is not cleaved, e.g., fcv, tgev, and ccv, have no such pairs of basic residues. cleavage of the mhv s protein occurred after conversion of the glycans from simple to complex forms (vennema et al., 1990a) . after cleavage, the s1 and s2 subunits are held together by noncovalent linkages (cavanagh et al., 1986b; sturman et al., 1990) . the s2 protein of mhv is acylated, possibly involving some of the many cysteine residues in the c-terminal, hydrophilic tail of s (schmidt, 1982; sturman et al., 1985; van berlo et al., 1987) . the processing of s proteins is reviewed in greater detail by cavanagh . modification of the m protein depends greatly on the virus species. the major modification is glycosylation. the oligosaccharides of ibv and the tgev group are of the co-translationally added n-linked glycans (stern and sefton, 1982b) . the conversion of the high mannose to complex glycans is not very efficient. in contrast, viruses of the mhv group have o-linked glycans which are added posttranslationally (holmes et al., 1981; niemann et al., 1982; 1984; tooze et al., 1988; locker et al., 1992a; krijnse-locker et al., 1994) . the m protein of tgev is also sulfated (garwes et al., 19761 , but whether this is linked directly to the polypeptide or to glycans is unknown. unlike the m proteins of ibv and the mhv group, which have an internal membrane insertion sequence, those of the tgev group have an n-terminal membrane insertion sequence that is absent from the mature m protein (laude et al., 1987) . this signal sequence, however, is not an essential requirement for the membrane insertion of the m protein (kapke et al., 1988; vennema et al., 1991) . 3. he protein. the he glycoprotein has n-linked glycans which are converted to complex ones in the golgi complex. the n-terminal signal sequence is cleaved from the mature protein, which then forms dimers by disulfide bonds (king et al., 1985; hogue et al., 1989; kienzle et al., 1990; yo0 et al., 1992) . of mhv e protein (yu et al., 1994) . however, this was not observed for the e protein of tgev when expressed in insect cells (godet et al., 1992) . 5. nprotein. the n protein is phosphorylated, the phosphate linkage being exclusively to serine residues (stohlman and lai, 1979) . the role of phosphorylation is unknown. in virus-infected cells, the assembly of virus particles presumably starts with the formation of rnp, which interacts with the components of viral envelope proteins to form enveloped virus particles and bud into the endoplasmic reticulum (er) and golgi complex. several recent advances shed light on this process: early studies have shown that the s proteins are not necessary for virus particle formation; thus, denuded virus particles without spikes can be formed in the virus-infected cells treated with tunicamycin, which inhibits n-glycosylation and transport of the s and he proteins (holmes et al., 1981) . further, recent studies have shown that the minimum requirement for the formation of viruslike particles (vlp), i.e., empty virus particles, is the m and e proteins (bos et al., 1996; vennema et al., 1996) ; 2. the sites of virus budding are in the er and golgi, near the sites of accumulation of the m protein (dubois-dalcq et al., 1982; tooze et al., 1984; tooze and tooze, 1985; klumperman et al., 1994) ; thus, the interaction between the m and e proteins appears to be the key event for virus particle assembly. the incorporation of the nucleocapsids and s and he proteins into virus particles may involve subsequent interactions of these components with the m-e complex. the virus assembly and release process has been studied in most detail for mhv (j. tooze et al., 1984 tooze et al., , 1987 tooze and tooze, 1985; s. a. tooze et al., 1988; krijnse-locker et al., 1994) , and the gross features have recently been confirmed for ibv, tgev, and fipv . recently, an ultrastructural study of the replication of ibv in renal ductotubular epithelial cells of infected chicks has also been very informative (chen and itakura, 1996) . the first virions form in the perinuclear region, in small, smooth vesicles/ tubules between the rough er and the cis side of the golgi stack. later, the rough er becomes the major site of virion assembly, extending beyond the perinuclear region. virions then proceed through the golgi complex, at the trans side of which they are collected into vesicles of the constitutive exocytic pathway and subsequently released from the cell. the major determining factor for the site of virus assembly appears to be the site of localization of the m protein, which is in the golgi complex. there are some points of difference among the coronaviruses. when the m protein of mhv was expressed, it accumulated in the trans-golgi membranes, consistent with its o-linked glycosylation, which occurs efficiently (locker et al., 1992a; klumperman et al., 1994) . in contrast, expression of the ibv m protein from cdna resulted in its accumulation in cis-golgi membranes; consequently the highmannose n-linked glycans of the m protein were not efficiently converted to complex ones (machamer et al., 1990; klumperman et al., 1994) , in agreement with the properties of the m protein in the ibv virions. glycosylation of the coronavirus m protein is not essential for its translocation or for virus particle formation. the m protein exists as monomers in the er, but it oligomerizes to form variously sized complexes during transport through the golgi and trans-golgi network (locker et al., 1995) . it is likely that the m molecules in the virus particles are in complexed form. the sequence requirements for insertion of the nascent m polypeptide into the rough er have not been precisely defined. with the exception of the tgev group, the coronavirus m proteins do not have an amino-terminal signal peptide. even in the case of the tgev group, the signal peptide is not essential for membrane insertion of the m protein (kapke et al., 1988; vennema et al., 1991) . rather, one of the three transmembrane sequences of the m protein is responsible for the insertion of m into the er and its final localization in the golgi complex (machamer and rose, 1987; mayer et al., 1988; armstrong et al., 1990; locker et al., 199213) . different domains of the m protein of ibv and mhv have been identified as the sequences responsible for the final localization of the protein. the first membrane-spanning domain of the ibv m protein performs this function, the m protein being concentrated in the cis-golgi membranes (machamer and rose, 1987; machamer et al., 1990 swift and machamer, 1991) . in contrast, the carboxyterminal domain of the mhv m protein, probably in combination with a middle domain, directs the protein to the trans-golgi (armstrong and patel, 1991; weisz et al., 1993; krijnse-locker et al., 1994) . it should be borne in mind, however, that the major site ofvirus particle formation is proximal to either of the golgi compartments, namely, in an intermediate compartment between the er and the golgi complex . thus, it is proximal to the major site of m accumulation. what is responsible for that? the answer would appear to be that the s glycoprotein and the nucleocapsid interact with the m protein molecules before the m proteins have migrated to the golgi, precipitating virus particle formation. it has been shown that the coronavirus m protein can interact with nucleocapsids (sturman et al., 1980) . this interaction requires the presence of viral rna, since the n protein alone cannot be incorporated into the vlps (bos et al., 1996; vennema et al., 19961 , suggesting either that m interacts with viral rna, or that rna-n protein binding induces a conformational change in the n protein, enabling it to interact with m. interaction between the m and s proteins has also been demonstrated. the m and s proteins co-sediment under certain ionic conditions after dissolution of virions with mild detergents (cavanagh, 1983b) , and cell-associated complexes containing m and s have been detected (opsteltenet al., 1995) . the s protein undergoes certain conformational changes induced by disulfide linkage before it is able to interact with m (opstelten et al., 1993 . inhibition of correct oligomerization of s by dithiothreitol prevented interaction of s with m and, as a result, the rate of transport of the m protein to the trans-golgi increased (opstelten et al., 1993) . this result suggests that s-m interaction can retard the transport of the m protein. the ability of the s or he protein to interact with the m protein appears to be a prerequisite for their incorporation into virus particles. in this regard, it is interesting to note that mhv ts mutants with a deletion in the ectodomain of the s protein or those with defects in oligomerization of the s protein do not incorporate the s protein (ricard et al., 1995; luytjes et al., 1997) . also, partial deletions in the ectodomain of the he protein prevent its incorporation into virus particles . these results suggest that the interaction of s or he with m occurs through the ectodomain or requires the correct protein conformation in the ectodomain. the formation of the s-m complex occurs in the pre-golgi complex, whereas the s-m complex progresses until the golgi complex, indicating that this interaction is not sufficient to localize it in the pre-golgi complex, the ultimate site of virion budding . thus, m-nucleocapsid interaction may also contribute to the determination of the site of virus assembly. in this regard, it is important to note that the recent discovery that m is present in the viral rnp core, as well as in the envelope (risco et al., 1996) may further indicate the crucial role of the m protein in the virus assembly process. only the m and e proteins are required for the production of vlps (bos et al., 1996; vennema et al., 1996) . these particles were formed when the m and e proteins were expressed from transfected plasmids. s protein was incorporated into the vlps if expressed. in the absence of viral rna, the n protein also was not incorporated. when all the structural proteins were expressed from plasmids in the presence of an mhv di rna, which contains a packaging signal, and in the absence of helper virus, the vlps contained the di rna (bos et al., 1996) . moreover, these vlp were "infectious," i.e., on transfer of the released vlps to a new cell culture, they were able to infect the cells, as revealed by the rescue of the di rna by helper virus. these results show that n is dispensable for the formation of vlps but the packaging of rna into virion requires an interaction between m and the n-containing ribonucleoprotein, as previously demonstrated (sturman et al., 1980) . the expression of the m protein alone in the cells did not lead to vlp formation or induction of curvature in the m-containing intracellular membranes. the presence of the e protein together with the m protein triggered both events, but the ratio of m:e in virions was as high as 1oo:l (vennema et al., 1996) . this has led to the suggestion that e does not have frequent, regular positions in the lattice formed by m but rather occupies strategic positions within the lattice to cause membrane curvature. alternatively, its role may be to close the neck of the virus particle as it pinches off from the membrane in the final stage of budding. what determines the site of virion budding? it is possible that the e protein dictates the site of budding, since this protein is also localized in the perinuclear region and associated with membrane (godet et al., 1992; yu et al., 1994) . alternatively, it may be the interaction of the rnp-nucleocapsid with the s-m complexes which halts the migration of the latter and promotes budding. relevant to this notion is the observation that the nucleocapsids and free n protein have affinity for membranes (anderson and wong, 1993) . it should be remembered, however, that in the absence of s, he, and nucleocapsids, the e and m proteins alone can induce budding to form vlps (bos et al., 1996; vennema et al., 1996) . it is not yet clear whether the budding site of vlp containing only m and e is the same as that for the complete virion. empty virus particles have previously been isolated from ibv, which were grown in embryonated fowl eggs (macnaughton and davies, 1980) . this supports the view that even during natural infection, virus budding can be induced without involvement of the viral nucleocapsid. parallels have been drawn between the e protein of coronaviruses, the m2 protein of orthomyxoviruses, and the 6k protein of alphaviruses. all are minor envelope proteins that play a role in virus assembly. once the virus particles bud into the pre-golgi compartment, they are transported through the golgi complex. whether the golgiassociated posttranslational modifications occur before or after incorporation of the proteins into virus particles is not known. retrograde transport of the proteins may be required for some steps of the virus assembly process. finally, the release of virus particles from the cells appears to be restricted to certain areas of cells. tgev grown in polarized llc-pk1 cells both enter and exit by the apical surface (rossen et al., 1994) , whereas mhv-a59 enters polarized murine kidney cells (mtal) by the apical surface but is released via the basolateral surface (rossen et al., 1995a) . however, the site of virus release varies with different cell lines (rossen et al., 1997) . the factors governing this process are not known (rossen et al., 1995b) . probably because of the large size of their rna genomes, coronaviruses have developed a variety of genetic mechanisms, among which are rna recombination and generation of di rna, to maintain their genetic stability and, as a side product, generate diversity. coronaviruses also readily undergo genetic mutation, a characteristic common to all rna viruses. thus, they evolve rapidly and are heterogeneous. these genetic phenomena provide virologists with useful tools for understanding coronavirus biology, particularly because reverse genetics studies for coronaviruses are not yet feasible. using a variety of chemical mutagens, several laboratories have isolated mhv temperature-sensitive (ts) mutants which cannot produce infectious virus particles or cause different plaque morphology at the nonpermissive temperature (haspel et al., 1978; robb et al., 1979; wege et al., 1981; koolen et al., 1983; schaad et al., 1990) . some of these mutants have been characterized with respect to their ability to synthesize rna and have been grouped into at least seven complementation groups (leibowitz et al., 1982a1 , five of which have the rna (-1 phenotype (i.e., cannot synthesize rna at the nonpermissive temperature) (leibowitz et al., 1982a; schaad et al., 1990 ) (see fig. 6 ). with the use of recombination analysis (see below), the possible genetic defects of the mutants were mapped on the rna genome fu and baric, 1994) . it appears that all of the rna (-) mutants have genetic defects within gene 1, suggesting that gene 1 encodes rna polymerase and other proteins involved directly or indirectly in viral rna synthesis. the genetic defects of some of these mutants have been confirmed by rna sequence analysis of the mutants and their revertants . these five different complementation groups have been demonstrated to affect different steps of rna synthesis, including the synthesis of leader rna, negative-strand rna, and positive-strand rna (fig. 6) , suggesting that different steps of rna synthesis require different viral proteins . it is still not possible, however, to correlate the genetic defects definitively with the known processed products of the gene 1 polyprotein. among the rna (+) mutants, two complementation groups have been assigned to the gene encoding the s protein fu and baric, 19941 , but the phenotype of these mutants has not been well characterized. another rna (+) mutant, alb 18, has a single amino acid substitution in the n-terminal domain of s protein and cannot incorporate s protein into the virus particles (ricard et al., 1995) . still another group of rna (+) mutants have a defective n protein (koetzner et al., 1992; masters et al., 1994; peng et al., 1995a) and produce smaller plaques at the nonpermissive temperature; several of these mutants have a deletion in the n gene (masters, 1992) and are defective in rna-binding activity (peng et al., 1995a) . most wildtype revertants have a second-site mutation in the n protein and restored rna-binding activity (peng et al., 1995a) . another class of viral mutants was obtained by a specific selection scheme, e.g., by treating viruses with neutralizing mab and selecting mutant viruses resistant to neutralization. since neutralizing antibodies are usually directed against the s protein, all of the neutralizationescape mutants were presumed to have defects in the s gene. this was indeed the case (reviewed by . depending on the neutralizing mab used for selection, the mutants obtained had either deletions or point mutations in the neutralization epitopes of the s protein (gallagher et al., 1990; wang et al., 1992) . these mutants generally retain growth properties very similar to those of the parental virus but often have significantly different pathogenic properties with altered tissue tropism (dalziel et al., 1986; fleming et al., 1986 fleming et al., , 1987 wege et al., 1988) . during serial virus passages in tissue culture or in animals, coronaviruses often undergo various deletions or substitutions even in the absence of experimentally applied selection pressure. these genetic changes probably provide the emerging virus variants with evolutionary advantages under experimental conditions or in natural infection. the deletions occur most frequently within the s gene, particularly within a hypervariable region encoding the s1 subunit (s. e. wang et al., 1992) . in fact, some natural isolates of mhv have a deletion of 150-460 nucleotides in this region (fig. 4) . similar deletions have been detected in virus variants during central nervous system (cns) infections of rats . in persistent infections of cultured cells of cns origin, viruses with point mutations or deletions in the gene encoding s protein are frequently selected (gallagher et al., 1991; gombold et al., 1993; rowe et al., 1997) . these viruses often have altered cell fusion and pathogenic properties. the most striking effect of deletions during natural virus infection is illustrated by the emergence of prcv from tgev. tgev causes epizootic enteric infection in pigs, resulting in a very high mortality rate in newborn pigs. an attenuated virus strain that is related t o tgev but infects only respiratory tissues was isolated in western europe in the early 1980s (pensaertet al., 1986 ). an independent isolate of prcv was subsequently obtained in the united states (wesley et al., 1990) . both of these prcv isolates have similar extents of deletion in the n terminus of the s1 protein, in addition to smaller deletions in gene 3, which eliminates its expression (rasschaert et al., 1990; wesley et al., 1991; laude, 1993) . although it is not yet possible to link the changes in viral pathogenicity to the deletions in the s gene or gene 3, the tgev-prcv evolution illustrates the power of deletions in coronavirus evolution. different ts mutants with defects in different coronaviral genes have been demonstrated to complement each other. the available ts mutants of mhv have been divided into at least seven complementation groups, five of which have an rna (-phenotype (leibowitz et al., 1982a) (fig. 6) . it is worth noting that these five rna (-) complementation groups have been mapped in gene 1 , which is translated into a polyprotein. the existence of five complementation groups within this gene indicates that this polyprotein is processed into at least five different proteins that function independently. it is not possible, however, to complement the genetic defects of a virus by expressing a wildtype viral protein from an exogenous vector. mixed infection with mhv and murine leukemia virus in tissue culture cells yielded a pseudotype mhv which contained a murine leukemia virus envelope protein and was neutralized by antibodies against both murine leukemia virus and mhv (yoshikura and taguchi, 1978) . this phenotypic mixing of viral proteins suggests the lack of a stringent requirement for a virus-specific spike protein for the formation of coronavirus particles. pseudotype formation of virus particles has also been achieved by expressing a viral protein, e.g., he protein, from a di rna vector (see section vi, e), which was incorporated into virus particles . one unique genetic feature of coronaviruses is their ability to undergo rna recombination at a very high frequency; this is particularly true of mhv, in which recombinant viruses containing parts of the genomic sequences of both parental viruses could be isolated at high frequency when two strains of mhv with defined genetic markers were co-infected into culture cells or animals. this genetic phenomenon was first discovered using two ts mutants of mhv . subsequently, many different recombinant mhvs were isolated (keck et al., , 1988b makino et al., 1987) using a combination of selection markers, such as ts markers, resistance to neutralizing antibodies, and cytopathic effects (the ability of the virus to cause fusion). based on the distribution of the crossover sites on the viral rna genome, it appears that recombination can occur practically anywhere on the viral genome, although some combinations of virus strains favor selection of viruses with certain recombination sites (lai, 1992) . for example, between the mhv a59 and jhm strains, recombination occurs mostly at the 5' end of the genome and rarely at the 3' end. in contrast, recombination between the mhv-2 and jhm strains occurs readily a t the 3' end . the most surprising finding with regard to mhv recombination is the extremely high frequency of recombination, which has been calculated to be nearly 25% for the entire mhv genome . this high frequency of recombination is reminiscent of the reassortment of segmented rna genomes in viruses such as influenza virus and reovirus. the recombination map for mhv is nearly linear, suggesting the random occurrence of recombination ; however, more careful analysis of the recombination frequency showed that there is an increasing gradient of recombination frequency (in the direction of 5'+3') across the genome, suggesting that subgenomic mrnas, which represent preferentially the 3 ' end sequences, may participate in rna recombination baric, 1992, 1994) . recombination has now been demonstrated experimentally for ibv (kotier et al., 1995) and tgev (ballesteros et al., 1997) in embryonated eggs or tissue culture; however, the recombination frequency for these viruses has not been determined. recombination can provide a powerful tool for virus evolution. for example, in a study in which ts mutants of the a59 strain of mhv were co-infected with a wild-type jhm strain, the majority of the progeny viruses after a single passage were recombinants which contained the 5' end of the a59 genome (makino et al., 1986a) , suggesting that this recombinant virus has evolutionary advantages. recombination has also been demonstrated during virus infection in animals (keck et al., 198813) . similar to the situation in other rna viruses, coronavirus recombination probably occurs by a copy-choice mechanism (lai, 1992) . it has been shown that mhv rna synthesis normally pauses at certain sites on the rna genome (baric et al., 1987) . the nascent, incomplete rna transcripts may dissociate from the template rna and then rebind to the template to resume rna synthesis. when the nascent rna binds to a different template, the resumed rna synthesis will result in a recombinant rna. whether coronavirus recombination occurs more frequently at certain rna sites with more complex secondary structure is not yet known. when rna recombination was examined under nonselective conditions (by reverse transcription-polymerase chain reaction detection of the intracellular rna from virus-infected cells), recombination sites appeared to be random; only after serial passages did "hot spots" of rna recombination become apparent . this finding indicates that the recombination hot spots may be the result of selection. recombination has been detected during natural infections of coronaviruses, most notably ibv. sequence analysis of natural ibv strains has provided convincing evidence that some ibv strains are recombinants between different ibv strains; recombination sites have been detected so far in the 5' half of the s gene and at the 3' end of viral rna (kusters et al., 1989; cavanagh and davis, 1992; wang et al., 1993 wang et al., , 1994 jia et al., 1995) . thus, recombination is a natural evolutionary strategy for coronaviruses. rna recombination may also explain the difference in genome structure among different coronaviruses. for example, ibv contains an additional gene, gene 5 (a nonstructural protein gene) inserted between gene m and gene n (fig. 5) . this insertion could be the result of a recombination mechanism involving the consensus ig sequence, which provides a favored recombination site. since all of the coronavirus genes are flanked by consensus ig sequences, each gene can be considered a gene "cassette," which can be rearranged by homologous recombination involving the consensus ig sequence. a nonhomologous recombination event between coronavirus rnas and other virus or cellular rnas may also explain the gene insertions in some coronaviruses. for example, mhv and bcv contain an additional gene, he, which is similar in sequence to the he gene of influenza c virus (luytjes et al., 1988) . this gene may have been derived by recombination between a coronavirus and influenza c virus. comparison between genome structures of coronavirus and torovirus also suggests that several recombination events may have been involved in rearranging the order of several genes during the evolution of these viruses (snijder et al., 1991) . recombination has been demonstrated to occur between viral rna and a transfected rna fragment derived from the viral genome (koetzner et al., 1992; liao and lai, 1992) . since transfection of both the positive-and negative-strand rna fragments led to recombination, these results suggested that recombination can occur during both positive-and negative-strand rna synthesis (liao and lai, 1992) . recombination can also take place between di rnas and viral rna reciprocally, i.e., the viral rna sequence can be incorporated into di rna, and vice versa, during viral rna replication. the incorporation of a helper viral rna sequence into di rna accounts at least partially for the continuous evolution of mhv di rna species during serial passages in cultured cells (see the next section). this phenomenon also explains why some genetic markers in the di rna were rapidly replaced by the helper viral rna sequences during di rna replication kim et al., 1993a) . on the other hand, the incorporation of di rna sequences into viral rna by recombination provides an important tool to introduce desired sequences into the viral genome. for example, when an mrna 7 or di rna containing the n gene of mhv was transfected into cells infected with an mhv ts mutant containing a defective n protein, recombination occurred between the di rna and the wild-type viral rna, resulting in recombinant viruses which had a wild-type rna sequence derived from the transfected rna in place of the defective n gene (koetzner et al., 1992; van der most et al., 1992; masters et al., 1994; peng et al., 1995a ). an mhv recombinant containing a chimeric n protein of bcv and mhv has also been derived by this rna recombination strategy (peng et al., 199513) . this targeted rna recombination promises to be a powerful tool. recombination is thus one of the most unique aspects of coronavirus biology. it can potentially provide a genetic mechanism by which coronaviruses maintain their sequence integrity. in view of the large size of the coronavirus rna, it is predictable that most of the viral rna molecules would contain mutations due to the high error frequencies of rna polymerases; recombination may provide a repair mechanism for the virus (lai, 1992 ). similar to most rna viruses, coronaviruses can readily generate di particles when viruses are passaged in tissue culture at a high multiplicity of infection. this has been demonstrated for mhv, ibv, and tgev. when mhv was serially passaged, different types of di rna appeared at different passage levels, suggesting that di rnas continue to evolve and that new dis have a selective advantage under the evolving cellular conditions . however, the ibv and tgev dis appear to be more stable (penzes et al., 1994; mendez et al., 1996) . the generation of di rnas is probably caused by polymerase jumping during rna replication or nonhomologous rna recombination. although no sequence homology exists at the fusion sites of different rna regions within the di rna, a high degree of potential secondary structure does exist at some of its rna fusion sites (makino et al., 1988b) , which may have facilitated the pausing and template switching of rna polymerase during synthesis. if nonhomologous recombination is involved in generating di rna, it probably occurs between two different rna molecules because di rnas are generated only at high multiplicity of infection. recombination between an existing di rna and helper virus rna has been shown to contribute to the evolution of mhv di rnas during virus passages . the coronavirus di rnas can be grouped into three types. the first type is of nearly genomic size and is typified by dissa rna of mhv . this di rna is efficiently packaged into virus particles and contains several deletions in the viral genome, but it contains a functional gene 1, which encodes rna polymerase, and a functional gene 7, which encodes n protein. these two functional gene products are sufficient to support di rna replication (k. h. ; thus, this type of di rna can replicate without a helper virus (makino et al., 1988a; k. h. kim and makino, 1995) . by definition, it is not a di rna, inasmuch as it is not defective in replication; however, because it is smaller than the genomic rna and is produced at a high multiplicity of infection, it is classified as a di rna. this type of di is unique to coronavirus. a 22-kb di rna has been described for tgev (mendez et al., 19961 , but whether it can replicate in the absence of a helper virus has not been examined. the second type di rna is typified by disse of mhv (makino et al., 198813) . this di rna is truly defective and can replicate only in the presence of helper viruses. it replicates very efficiently, but is poorly packaged into virus particles because it lacks a specific rna-packaging signal. this type of di rna typically contains both the 5' and 3' ends of the wild-type viral rna and one or several discontiguous regions of the wild-type rnas. because of the high efficiency of replication, this type of di can still be serially passaged in tissue culture for at least several passages, probably because a small amount of di rna can be nonspecifically packaged into the virion. the third type of di rna is represented by dissf of mhv-jhm (makino et al., 1990) and di-a of mhv a59 (van der most et al., 1991). it is similar to the second type but contains an rna-packaging signal and is thus packaged efficiently into virus particles. this type of di rna has been detected in ibv (penzes et al., 1994) and tgev (mendez et al., 1996) . a small di rna (2.2 kb) of bcv may also belong to this type , but whether this di rna can be specifically packaged into virion is not certain. all three types of di rnas contain an orf, which encodes a protein fused from two different viral proteins. this orf is not required for the replication of mhv di rna (liao and lai, 19'95) ; nevertheless, mhv di rnas with a functional orf usually have an evolutionary advantage over those without one or with a smaller orf kim et al., 1993a) . therefore, a di rna containing a short orf was often rapidly replaced by di rnas containing a longer orf that had been generated by recombination or mutation kim et al., 1993a) . the translatability of the orf may be more important than the nature of the actual protein translated from this orf (van der most et al., 1995) , suggesting that translation of rna may facilitate rna replication. reduction of the orf of a n ibv di rna to just 20 amino acids did not diminish its capacity to be replicated or packaged (penzes et al., 1996) . however, it has been shown for a bcv di rna that a bcv-specific n protein translated from the di orf (a cis-acting protein) is required for efficient di rna replication . the variation in the sequence requirement for rna replication of these di rnas may be related to their overall rna conformation. the significance of di rna in the biology and natural evolution of coronaviruses is not known. di rnas provide useful tools for studying the sequence and structural requirements for various functions of viral genomic rna. as they contain cis-acting signals for rna replication, they are mini-versions of the viral genomic rna. however, it should be cautioned again that because of the small size of the di rna compared to the genomic rna, the structural requirements for various rna functions, as determined from the use of di rna constructs, may be different from those of the whole viral genome. the following cis-acting signals for various rna functions have been determined using various di rnas: 1. rna-packaging signal. in a comparison of mhv di rnas that are efficiently and inefficiently packaged, it was determined that the packaging signal for mhv di rna is localized near the 3' end of gene 1 (in the l b region, approximately 20 kb from the 5' end) (makino et al., 1990; van der most et al., 1991; fosmire et al., 1992) . this packaging signal forms a stem-loop structure which may be required for the rna-packaging activity (fosmire et al., 1992) . it is necessary and sufficient for the packaging of di rna or a heterologous rna into the virions (woo et al., 1997) . the fact that this packaging signal is localized in gene 1, which is present in genomic but not subgenomic rnas, is consistent with the packaging of genomic but not subgenomic rnas in virus particles. the packaging signal for di rnas of other coronaviruses has not been determined. however, some coronaviruses have been shown to package subgenomic mrnas at low efficiency (sethna et al., 1989; hofmann et al., 1990; zhao et al., 1993) . these are probably packaged nonspecifically; however, the possibility that these viruses may have a different rna packaging signal cannot yet be ruled out. similarly, di rnas that do not contain this packaging signal, such as disse rna of mhv (makino et al., 198813) and di rna of bcv chang et al., 19961, can be packaged at low efficiency, thereby maintaining themselves for at least several passages in tissue culture. for mhv di rna, it has been shown that only 55 nucleotides at the 3' end plus a stretch of poly(a) sequence are required for negative-strand rna synthesis ; no specific upstream rna sequences are required. however, when an mrna is transcribed from an ig site in the same di rna, the negative-strand rna synthesis from this di rna is inhibited, suggesting a common element involved in mrna transcription and negative-strand rna synthesis . one unanswered question is whether or not the sequence requirements for the synthesis of genomic and subgenomic negative-strand rna are identical. 3. replication signal. sequential deletion analysis has shown that the replication (i.e., complete cycles of negative-and positive-strand rna synthesis) of mhv disse or dissf rnas requires approximately 400-800 nucleotides from both the 5' and 3' ends. the minimum sequence requirement for rna replication may vary with different di rnas. these issues have been discussed in section v,f. 4. transcriptional signal. di rnas normally do not transcribe subgenomic mrnas because they do not have ig sequences. thus, natural di rnas can synthesize only the full-sized di rna. however, by introduction of the consensus ig sequences into di rna (makino et al., 19911, it has been possible to use di rna as a vector for determining the sequence requirement for subgenomic rna transcription. the cis-and trans-acting signals for transcription have been described in section v,e. 5 . recombination. di rnas of mhv have been demonstrated to undergo a high frequency of recombination with helper virus rna. as discussed above, this accounts for the evolution of mhv di rna species during serial passages of viruses . furthermore, mhv di rnas with a smaller orf are frequently replaced by a di rna with a larger orf by recombination with the helper virus rna (de groot et al., 1992; kim et al., 1993a) , suggesting that recombination between di rnas and helper virus rnas occurs readily. the reciprocal recombination between di rna and helper virus rna, i.e., the transfer of di rna sequences to the helper virus rna, also has been observed. as a result, the genetic markers on the di rna can be incorporated into the helper virus rna (koetzner et al., 1992) . recombination between two di rnas, however, has not been described. sequence requirements for rna recombination also have not been studied. bcv di rnas also undergo frequent recombination . however, di rnas of ibv and tgev appear to be more stable. coronavirus research has made tremendous progress in the last decade. the virus family has grown in size, and many of the features thought to be unique to coronaviruses have now been found to be shared by some other viruses. since the last time this serial publication published the first comprehensive review of the molecular biology of coronaviruses (sturman and holmes, 1983) , the literature on this virus has grown to exceed anyone's ability to do a comprehensive review of every topic relating to coronaviruses. in this review, we have concentrated on areas which have shown the most progress and which present the most challenges. our choice of literature was meant to be representative but is by no means comprehensive. notably missing from this review are the molecular studies related to viral pathogenesis and the interactions between the virus and cells. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. despite two decades of studies on the molecular biology of this virus, there are still many problems to be solved: 1. with regard to the mechanism of rna transcription, many conflicting data remain. coronavirus undoubtedly utilizes a unique, discontinuous transcription mechanism, but how it acts is a subject of debate. an in uitro rna transcription system, so necessary for an understanding of rna synthesis, is still in its infancy. related to this question is the nature of rna polymerase. the sheer size of the polymerase gene presents a daunting task. the availability of the cdna clones and expression vectors for this gene has just begun to allow this black box to be cracked open. this will undoubtedly be a fruitful area of future research. 2. the last two years have seen the unraveling of the mechanism of coronavirus assembly, which, as it turns out, involves a littlecharacterized e protein. how the various viral structural proteins interact with each other in the various subcellular compartments to form a complete virus particle is an exciting frontier. 3. after more than 30 years since the first coronavirus was seen under electron microscope, an unexpected new feature of the virus, namely, an icosahedral core with a helical nucleoprotein, was recently uncovered. this structure places coronavirus in a unique position among rna viruses because it takes on the characteristics of positive-, negative-and double-strand rna viruses in morphology. this recent finding challenges us to reevaluate the structure of coronaviruses. 4. the ability to perform reverse genetic studies of coronavirus is still very limited. expression of individual viral genes and targeted recombination of very limited rna regions are the only available genetic means for examining the structure and function of the coronavirus genome. perhaps it is an unrealistic dream, but progress in polymerase chain reaction technology may one day allow an infectious cdna for coronavirus rna to be made. the early events of viral replication have so far been largely ignored. identification of the cellular receptors for the viruses may finally provide penetrating molecular tools to allow these issues to be examined. it will not be a surprise to discover that virus penetration and uncoating play defining roles in the cellular tropism of viruses. 6. are nonstructural protein genes really unnecessary? even if they are auxiliary genes, they may prove to play significant roles in the 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cord cultures genetically-determined sensitivity to mhv3 infections is expressed in vitro in lymphoid cells and macrophages cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a59 mouse hepatitis virus strain a59 and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor mouse hepatitis virus receptor activities of an mhvwmph chimera and mhvr mutants lacking n-linked glycosylation of the n-terminal domain identification of the coronavirus mhv-jhm mrna 4 product complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus molecular basis of transmissible gastroenteritis virus epidemiology the coronavirus avian infectious bronchitis virus requires the cell nucleus and host transcriptional factors the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavirus jhm (mhv-4): molecular identification of a viral determinant of paralytic disease replication of murine coronaviruses in somatic cell hybrids between murine fibroblasts and rat schwannoma cells identification and characterization of a coronavirus packaging signal proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion evidence for variable rates of recombination in the mhv genome map locations of mouse hepatitis virus temperaturesensitive mutants: confirmation of variable rates of recombination three different cellular proteins bind to the complementary sites on the 5'-end positive-and 3'-end negative-strands of mouse hepatitis virus rna natural evolution of coronavirus defective-interfering rna involves rna recombination interaction of mouse hepatitis virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with a n mhv strain that expresses the hemagglutinin-esterase glycoprotein murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the aminoterminal half of the spike glycoprotein alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus the polypeptide structure of transmissible gastroenteritis virus isolation of subviral components from transmissible gastroenteritis virus the polypeptide of m, 14,000 of porcine transmissible gastroenteritis virus: gene assignment and intracellular location tgev coronavirus orf4 encodes a membrane protein that is incorporated into virions major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus fusion-defective mutants of mouse hepatitis virus a59 contain a mutation in the spike protein cleavage signal an ntpbinding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rnaviral replication coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis putative papain-related thiol proteases of positive-strand rna viruses single amino acid changes in the s2 subunit of the mhv surface glycoprotein confer resistance to neutralization by s1 subunit-specific monoclonal antibody characterization of a 106-kda polypeptide encoded in gene 1 of the human coronavirus hcv 2293 temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv 2293 polymerase mrna nucleotide sequence of the human coronavirus 2293 rna polymerase locus translational control in murine hepatitis virus infection quantification of individual subgenomic mrna species during replication of the coronavirus transmissible gastroenteritis virus investigation of the control of coronavirus subgenomic mrna transcription by using t7-generated negative-sense rna transcripts anew superfamily ofreplicative proteins the 5'-end of coronavirus minus-strand rnas contains a short poly(u) tract bovine coronavirus mrna replication continues throughout persistent infection in cell culture leader-mrna junction sequences are unique for each subgenomic mrna species in the bovine coronavirus and remain so throughout persistent infection a translation-attenuating intraleader open reading frame is selected on coronavirus mrnas during persistent infection structural proteins of human respiratory coronavirus oc43 synthesis and processing of the bovine enteric coronavirus hemagglutinin protein coronaviridae and their replication tunicamycin-resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein analysis of a 9.6 kb sequence from the 3' end of canine coronavirus genomic rna identification ofthe murine coronavirus p28 cleavage site the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens synthesis of subgenomic mrna's of mouse hepatitis virus is initiated independently: evidence from uv transcription mapping mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic rna transcription on rna replication evidence for coronavirus discontinuous transcription coronavirus transcription mediated by sequences flanking the transcription consensus sequence a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains mutagenic analysis of the coronavirus intergenic consensus sequence the effect of two closely inserted transcription consensus sequences on coronavirus transcription the amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation multiple recombination sites at the 5'-end of murine coronavirus rna temporal regulation of bovine coronavirus rna synthesis in vivo rna-rna recombination of coronavirus in mouse brain rna recombination of murine coronaviruses: recombination between fusion-positive mhv-a59 and fusion-negative mhv-2 inhibition of coronavirus 2293 replication by actinomycin d structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e64d two murine coronavirus genes suffice for viral rna synthesis characterization of a murine coronavirus defective interfering rna internal cis-acting replication signal generation and selection of coronavirus defective interfering rna with large open reading frame by rna recombination and possible editing analysis of cis-actingsequences essential for coronavirus defective interfering rna replication bovine coronavirus hemagglutinin protein infectious bronchitis coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding host genetic control of mouse hepatitis virus type-4 (jhm strain) replication. 11. the gene locus for susceptibility is linked to the svp-2 locus on mouse chromosome 7 repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination differential premature termination of transcription as a proposed mechanism for the regulation of coronavirus gene expression early events of importance in determining host cell permissiveness to mouse hepatitis virus infection differentiation of acid-ph-dependent and -nondependent entry pathways for mouse hepatitis virus temperature-sensitive mutants of mouse hepatitis virus strain a59: isolation, characterization and neuropathogenic properties toroviruses of animals and humans: a review experimental evidence of recombination in coronavirus infectious bronchitis virus characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step tandem placement of a coronavirus promoter results in enhanced mrna synthesis from the downstream-most initiation site early interaction between mouse hepatitis virus 3 and cells entry of mouse hepatitis virus 3 into cells localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein structural and fimctional analysis of the surface protein of human coronavirus oc43 phylogeny of antigenic variants of avian coronavirus ibv. virology sequence and expression of the ns2 coronavirus leader rna-primed transcription: an alternative coronavirus: organization, replication and expression of genome rna recombination in animal and plant viruses mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv 3 the presence of leader sequences in the mrna of mouse hepatitis virus characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus-a cytoplasmic rna virus recombination between nonsegmented rna genome8 of murine coronaviruses coronavirus: a jumping rna transcription coronavirus: how a large rna viral genome is replicated and transcribed localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus jhm coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes porcine respiratory coronavirus: molecular features and virus-host interactions the coronavirus nucleocapsid protein single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed distinct structural elements and internal entry of ribosomes in mrna3 encoded by infectious bronchitis virus the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase synthesis of virus-specific rna in permeabilized murine coronavirus-infected cells thevirus specific intracellular rna species of two murine coronaviruses: mhv a59 and mhv-jhm genetic analysis of murine hepatitis virus strain jhm cell-free translation of murine coronavirus rna detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d coronavirus ibv-induced membrane fusion occurs at near-neutral ph rnarecombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mrna transcription a cis-acting viral protein is not required for the replication of a coronavirus defective-interfering rna coronavirus defective-interfering rna as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase deletion mapping of a mouse hepatitis virus defectiveinterfering rna reveals the requirement of an internal and discontiguous sequence for replication identification of the cis-acting signal for minus-strand rna synthesis of a murine coronavirus: implications for the role of minus-strand rna in rna replication and transcription the 3' untranslated region of the coronaviral rna is required for subgenomic mrna transcription from a defectiveinterfering rna characterisation and mutational analysis of an orf la-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus l d l b polyprotein identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus a polycistronic mrna specified by the coronavirus infectious bronchitis virus a 100-kilodalton polypeptide encoded by open reading frame (orf) l b of the coronavirus infectious bronchitis virus is processed by orf la products 0-glycosylation of the coronavirus m protein. differential localization of sialyltransferases in n-and 0-linked glycosylation membrane assembly of the triple-spanning coronavirus m protein. individual transmembrane domains show preferred orientation the cytoplasmic tail of mouse hepatitis virus m protein is essential but not sufficient for its retention in the golgi complex oligomerization of a trans-golgvtrans-golgi network retained protein occurs in the golgi complex and may be part of its retention intracellular and in vitro-translated 27-kda proteins contain the 3c-like proteinase activity of the coronavirus mhv-ass identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 primary structure of the glycoprotein e2 of coronavirus mhv-a59 and identification of the trypsin cleavage site. virology sequence of mouse hepatitis virus a59 mrna 2: indications for rna-recombination between coronavirus and influenza c virus amino acid sequence of a conserved neutralizing epitope of murine coronaviruses replication of synthetic defective interfering rnas derived from coronavirus mouse hepatitis virus-a59 characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a59 with maturation defects in the spike protein a specific transmembrane domain of a coronavirus e l glycoprotein is required for its retention in the golgi region the e l glycoprotein of an avian coronavirus is targeted to the cis golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain two particle types of avian infectious bronchitis virus ribonucleoprotein-like structures from coronavirus particles rna-dependent rna polymerase activity in murine coronavirus-infected cells effect ofintergenic consensus sequence flanking sequences on coronavirus transcription evolution of the 5'-end of genomic rna of murine coronaviruses during passages in vitro high-frequency leader sequence switching during coronavirus defective interfering rna replication structure of the intracellular defective viral rnas of defective interfering particles of mouse hepatitis virus high-frequency rna recombination of murine coronaviruses leader sequences of murine coronavirus mrnas can be freely reassorted: evidence for the role of free leader rna in transcription rna recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers defective interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas primary structure and translation of a defective-interfering rna of murine coronavirus discontinuous transcription generates heterogeneity a t the leader fusion sites of coronavirus mrnas analysis of efficiently packaged defective-interfering rnas of murine coronavirus: localization of a possible rnapackaging signal a system for study of coronavirus mrna synthesis: a regulated, expressed subgenomic defective-interfering rna results from intergenic site insertion localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus membrane integration and intracellular transport of the coronavirus glycoprotein e l , a class i11 membrane glycoprotein molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity attenuation ofmurine coronavirus infection by ammonium chloride endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein human coronavirus oc43 rna 4 lacks two open reading frames located downstream of the s gene of bovine coronavirus coronavirus infects and causes demyelination in primate central nervous system influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis virus localization of the rna-binding domain of mhv nucleocapsid protein post-translational glycosylation of coronavirus glycoprotein el: inhibition by monensin the carbohydrates of mouse hepatitis virus (mhv) a59: structures of the 0-glycosidically linked oligosaccharides of glycoprotein el the encephalomyocarditis virus 3c protease is rapidly degraded by an atp-dependent proteolytic system in reticulocyte lysate immunoglobulin fc binding activity is associated with the mouse hepatitis virus e2 peplomer protein mhv s peplomer protein expressed by a recombinant vaccinia virus vector exhibits igg fc-receptor activity molecular mimicry between fc receptor and s peplomer protein of mouse hepatitis virus, bovine coronavirus, and transmissible gastroenteritis virus monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly cloning and in vitro expression of the gene for the e3 haemagglutinin glycoprotein of bovine coronavirus expression and secretion of the bovine coronavirus hemagglutinin-esterase glycoprotein by insect cells infected with recombinant baculoviruses sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein sequence analysis reveals extensive polymorphism and evidence of deletions within the e2 glycoprotein gene of several strains of murine hepatitis virus analysis of cell fusion induced by bovine coronavirus infection analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted rnarecombination construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis characterization of a replicating and packaged defective rna of avian coronavirus infectious bronchitis virus replication and packaging of coronavirus infectious bronchitis virus defective rnas lacking a long open reading frame mhvnucleocapsid synthesis in the presence of cyclohexamide and accumulation of negative-strand mhv rna functional analysis ofthe coronavirus mhv-jhm surface glycoproteins in vaccinia virus recombinants lactate dehydrogenase-elevating virus, equine arteritis virus and simian haemorrhagic fever virus, a new group of positive strand rna viruses porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions a conditionallethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins pathogenic murine coronaviruses. 111. biological and biochemical characterization of temperature-sensitive mutants of jhmv rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells mhv-a59 enters polarized murine epithelial cells through the apical surface but is released basolaterally coronavirus infection of polarised epithelial cells mouse hepatitis virus strain a59 is released from opposite sides of different epithelial cell types assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the e l glycoprotein of coronavirus mouse hepatitis virus a59 the coronavirus membrane glycoprotein analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis persistent infection of cultured cells with mouse hepatitis virus (mhv) results from epigenetic expression of the mhv receptor evidence for new transcriptional units encoded at the 3' end of the mouse hepatitis virus genome genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand rna synthesis nucleotide sequence ofthe gene encoding the surface projection glycoprotein of coronavirus mhv-jhm acylation of viral spike glycoproteins: a feature of enveloped rna viruses bovine coronavirus uses n-acetyl-9-0-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells hemagglutinating encephalomyelitis virus attaches to n-acetyl-9-0-acetylneuraminic acidcontaining receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus the s protein of bovine coronavirus is a hemagglutinin recognizing 9-0-acetylated sialic acid as a receptor determinant isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptordestroying and receptor-binding activity neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells the nucleocapsid protein gene of bovine coronavirus is bicistronic coronavirus subgenomic minusstrand rnas and the potential for mrna replicons minus-strand copies of replicating coronavirus mrnas contain antileaders expression and characterization of a recombinant murine coronavirus 3c-like proteinase identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome in vitro interaction of mouse hepatitis virus and macrophages from gneetically resistant mice. i. adsorption of virus and growth curves the coronaviridae the coronaviridae: an introduction the small membrane protein coronavirus jhm: intracellular protein synthesis monoclonal antibody to the receptor for murine coronavirus mhv-a59 inhibits viral replication in vivo identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily the molecular biology of toroviruses the coronavirus superfamily comparison of the genome organization of toro-and coronaviruses: evidence for two nonhomologous rna recombination events during berne virus evolution sequence and translation of the murine coronavirus 5'-end genomic rna reveals the n-terminal structure of the putative rna polymerase ribosomal pausing during translation of an rna pseudoknot isolation and identification of virus-specific mrna in cells infected with mouth hepatitis virus (mhv-ass) coronavirus mrna synthesis involves fusion of non-contiguous sequences proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity coronavirus multiplication strategy identification and characterization of virus-specified rna coronavirus multiplication strategy. 11. mapping the avian infectious bronchitis virus intracellular rna species to the genome synthesis of coronavirus mrnas: kinetics of inactivation of infectious bronchitis virus rna synthesis by uv light coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins phosphoproteins of murine hepatitis viruses specific interaction between coronavirus leader rna and nucleocapsid protein monoclonal antibodies differentiate between the haemagglutinating and the receptordestroying activities of bovine coronavirus the molecular biology of coronaviruses isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell fusing activity of virions by trypsin and separation of two different 90k cleavage fragments conformational change of the coronavirus peplomer glycoprotein at ph 8.0 and 37 degrees c correlates with virus aggregation and virus-induced cell fusion hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice analysis ofreceptor-binding site ofmurine coronavirus spike protein a golgi retention signal in a membranespanning domain of coronavirus e l protein fusion formation by the uncleaved spike protein of murine coronavirus jhmv variant cl-2 the s2 subunit of the murine coronavirus spike protein is not involved in receptor binding coronavirus translational regulation: leader affects mrna efficiency myelin basic protein and human coronavirus 2293 cross-reactive t cells in multiple sclerosis internal ribosome entry in the coding region of murine hepatitis virus mrna 5 a region of the coronavirus infectious bronchitis virus l a polyprotein encoding the 3c-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus infection of att20 murine pituitary tumour cells by mouse hepatitis virus strain a59: virus budding is restricted to the golgi region replication of coronavirus mhv-a59 in sac-cells: determination of the first site of budding of progeny virions sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att20 cells site of addition of n-acetyl-galactosamine to the e l glycoprotein of mouse hepatitis virus-a59 induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea the 9-kda hydrophobic protein encoded at the 3'-end ofthe porcine transmissible gastroenteritis coronavirus genome is membrane-associated a domain a t the 3'-end of the polymerase gene is essential for encapsidation of coronavirus defective interfering rnas homologous rna recombination allows efficient introduction of site-specific mutations into the genome of coronavirus mhv-a59 via synthetic co-replicating rnas subgenomic rna synthesis directed by a synthetic defective interfering rna of mouse hepatitis virus: a study of coronavirus transcription initiation translation but not the encoded sequence is essential for the efficient propagation of the defective interfering rnas of the coronavirus mouse hepatitis virus rat glial c6 cells are defective in murine coronavirus internalization regulation of coronavirus mrna transcription sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the e3 protein of bovine coronavirus is a receptor-destroying enzyme with acetyltransferase activity human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity evidence of natural recombination within the s1 gene of the infectious bronchitis virus evolutionary implications of genetic variations in the s1 gene of infectious bronchitis virus genetic variation of neurotropic and non-neurotropic murine coronaviruses the peplomer protein e2 of coronavirus jhm as a determinant of neurovirulence: definition of critical epitopes by variant analysis evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse 16 hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions the proposed family toroviridae: agents of enteric infections. brief review pathogenesis of feline infectious peritonitis: nature and development of viremia oligomerization of a membrane protein correlates with its retention in the golgi complex natural cytotoxicity against mouse hepatitis virus-infected cells. 11. a cytotoxic effector cell with a b lymphocyte phenotype evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus the replication of murine coronaviruses in enucleated cells evidence for a pseudoknot in the 3'-untranslated region of the bovine coronavirus genome receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins in vivo and in vitro models of demyelinating disease: efficiency of virus spread and formation of infectious centers among glial cells is genetically determined by the murine host in vivo and in vitro models of demyelinating disease. possible relationship between induction of regulatory subunit from camp dependent protein kinases and inhibition of jhmv replication in cultured oligodendrocytes murine coronavirus packaging signal confers packaging to nonviral rna human aminopeptidase n is a receptor for human coronavirus 2293 mouse hepatitis virus s rna sequence reveals that nonstructural proteins ns 4 and ns 5a are not essential for murine coronavirus replication mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors the receptor for mouse hepatitis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus heterogeneity of gene expression of hemagglutinin-esterase (he) protein of murine coronaviruses hemagglutininesterase (he)-specific monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus coronavirus mrna transcription: uv light transcriptional mapping studies suggest an early requirement for a genomiclength template a spike proteindependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the e3 region of adenovirus the 92 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells mouse hepatitis virus strain mhv-s: formation of pseudotypes with a murine leukemia virus envelope a conserved motif a t the 3' end of mouse hepatitis virus genomic rna required for host protein binding and viral rna replication specific binding of host cellular proteins to multiple sites within the 3'-end of mouse hepatitis virus genomic rna mouse hepatitis virus gene 5b protein is a new virion envelope protein unusual heterogeneity of leader-mrna fusion in a murine coronavirus: implications for the mechanism of rna transcription and recombination interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus r n a correlation with the amounts of subgenomic mrna transcribed a 5'-proximal rna sequence of murine coronavirus as a potential initiation site forgenomic-lengthmrna transcription the hemagglutinidesterase glycoprotein of bovine coronaviruses: sequence and functional comparisons between virulent and avirulent strains biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child coronavirus leader rna regulates and initiates subgenomic mrna transcription, both in trans and in cis presence of subgenomic mrnas in virions of coronavirus ibv the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 3' terminus of the genome characterization of a human coronavirus (strain 2293) 3c-like proteinase activity mouse hepatitis virus orf 2a is expressed in the cytosol of infected mouse fibroblasts we would like to thank the following individuals for making data available prior to publication: luis enjuanes, hubert laude, peter rottier, and pierre talbot. we would also like to thank daphne shimoda for her tremendous help with editorial revisions, preparation of the figures, and the typing of the manuscript. m.m.c.l. is investigator of the howard hughes medical institute. key: cord-338307-vfutmwxq authors: sturman, lawrence s.; holmes, kathryn v. title: the molecular biology of coronaviruses date: 1983-12-31 journal: advances in virus research doi: 10.1016/s0065-3527(08)60721-6 sha: doc_id: 338307 cord_uid: vfutmwxq publisher summary coronaviruses have recently emerged as an important group of animal and human pathogens that share a distinctive replicative cycle. some of the unique characteristics in the replication of coronaviruses include generation of a 3' coterminal-nested set of five or six subgenomic mrnas, each of which appears to direct the synthesis of one protein. two virus-specific rna polymerase activities have been identified. many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. the intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the e1 glycoprotein, which acts as a matrix-like transmembrane glycoprotein. e1 also exhibits distinctive behavior by self-aggregating on heating at 100°c in sodium dodecyl sulfate (sds) and by its interaction with rna in the viral nucleocapsid. the e1 of mouse hepatitis virus (mhv) is an o-linked glycoprotein, unlike most other viral glycoproteins. thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of o-linked cellular glycoproteins. this contribution is dedicated to the memory of our colleague and friend, frederik bang, who uncovered some of the most intriguing questions about the biology of coronaviruses. beginning with the discovery that mouse hepatitis virus (mhv) could grow in and destroy macrophages in culture (bang and warwick, 1959) and that macrophage susceptibility in uitro reflected a genetic determinant for susceptibility in the mouse, bang and warwick, (1960; kantoch et al., 1963 ) constructed a simple model to explain genetic resistance and susceptibility to this virus. for more than two decades, bang (1978 bang ( , 1981 and his colleagues explored genetic and environmental aspects of host resistance and susceptibility to mhv, and the effects of mhv on macrophages. at the wurzburg symposium on the biochemistry and biology of coronaviruses in 1980, dr. bang (1981) recounted the changes in his thinking about mechanisms of host resistance, as many of the conclusions drawn from earlier experiments were reinterpreted in the light of subsequent findings. as always, he spoke with humility and humor, and displayed his distinctive interest in host-parasite interrelations. unfortunately, that was his last opportunity to address his colleagues in this rapidly expanding field. we shall miss him. coronaviruses have been known, although not by that name, for almost five decades. this previously unrecognized group emerged during the 1960s in the aftermath of the discovery of several new human respiratory pathogens. avian infectious bronchitis virus (ibv), mouse hepatitis virus, and some newly described human respiratory viruses were noted t o have a similar appearance (almeida and tyrrell, 1967; mcintosh et al., 196713; becker et al., 1967) . in contrast to myxoviruses, with which they had been previously compared (berry et al., 1964; mallucci, 1965) , these viruses displayed a characteristic fringe of large, distinctive, petal-shaped peplomers or spikes which resembled a crown, like the corona spinarum in religious art; hence the name coronaviruses ( fig. 1a ; tyrrell et al., 1968) . in addition to their morphological similarities, some of the human coronaviruses (hcv) were noted to be antigenically related to mhv (tyrrell et al., 1968; mcintosh et al., 1969; bradburne, 1970) . between 1968 and 1974 , research on coronaviruses emphasized morphologic and immunologic relationships and comparative biology. several new viruses were added t o the coronavirus group: porcine transmissible gastroenteritis virus (tgev), porcine hemagglutinating encephalomyelitis virus (hew, rat coronavirus (rcv), sialodacryoadenitis virus of rats (sdav), turkey bluecomb disease virus (tcv), and neonatal bovine diarrhea coronavirus (bcv). a comprehensive review by mcintosh (1974) provides an excellent overview of early coronavirus research. in 1975, the international committee on the taxonomy of viruses approved the creation of a new family, coronaviridae, with one genus, coronavirus . additional species were added later including canine coronavirus (ccv), feline infectious peritonitis virus (fipv), and human enteric coronaviruses (hecv) (tyrrell et al., 1978) , rabbit coronaviruses (small et al., 1979; lapierre et al., 1980) , and several others (reviewed by wege et al., 1982) . table i shows the coronaviruses with their natural hosts and the major diseases which they induce. clearly the coronaviruses are important pathogens of man and domestic animals. timely reviews of several aspects of the biology and pathogenesis of coronaviruses have been published by virelizier (19811, macnaughton and davies (1981) , . during the past several years, substantial progress has been made in understanding the structure and replication of coronaviruses. the symposium on the biochemistry and biology of coronaviruses held in wurzburg in october, 1980 dramatized the emergence of exciting frontiers in coronavirus research . siddell et al. (1982) have written an excellent review of the structure and replicathe purpose of this article is to describe the development of our present understanding of the molecular biology of coronaviruses. we have taken an historical approach to the subject, using figures from some of the key papers to illustrate the development of our present concepts. we are grateful to our colleagues for making these figures and their unpublished data available to us. we hope that this contribution will convey the sense of excitement and cooperation which has characterized this period in coronavirus research. negative stains of coronaviruses from eggs, clinical specimens or media over tissue, or organ cultures infected with coronaviruses revealed the characteristic viral structure shown in fig. 1a (berry et al., 1964; tyrrell and almeida, 1967; mcintosh et al., 1967a; almeida and tyrrell, 1967; apostolov et al., 1970; oshiro et al., 1971; johnson-lussenberg, 1975-1976) . the virions were spherical, enveloped particles ranging from 80 nm to 160 nm in diameter. they showed some pleiomorphism, and frequently had a shallow central hollow containing some negative stain. indeed, on the basis of negatively stained images of ibv, bingham and almeida (1977) suggested that the morphology of coronavirus virions might resemble a punchedin sphere. whether this represents the true morphology of the virions or is a deformation resulting from drying and negative staining remains to be determined. coronavirus morphology has been reviewed by mcintosh (1974) , oshiro (19731, pensaert and callebaut (1978) , and robb and bond (1979a) . most coronaviruses appear to have only one morphologic type of surface projection or peplorner. the peplomers of coronaviruses are large and roughly club shaped. for mhv, each peplomer is about 20 nm long by 7 nm wide a t the tip . there are approximately 200 peplomers per virion. it is not known how many glycoprotein molecules form each peplomer. the peplomers of different coronaviruses have somewhat different appearances in negatively stained preparations (caul and egglestone, 1977; davies and macnaughton, 1979) . for bcv, bridger et al. (1978) have suggested that there may be two morphologically distinct types of peplomers. analysis of the structural proteins of bcv (section iic) also suggested that there might be an additional species of glycoprotein in the virion (king and brian, 1982) . possibly this additional glycoprotein forms the second type of peplomer. two types of peplomers have been observed rarely on hev and mhv virions (greig et al., 1971; sugiyama and amano, 1981) . it is not clear to what extent the morphological differences between peplomers of coronaviruses reflect differences in amino acid sequence, glycosylation, proteolytic cleavage, or reduction of disulfide bonds (see sections ii,c and iv,a). many preparations of cornaviruses contain some virus particles which partially or completely lack peplomers. storage of virions may lead to detachment of peplomers, and under some conditions virions lacking peplomers can be formed in infected cells (holmes et (1980) . the e l glycoprotein forms aggregates of irregular sizes x 100.000. (d) the peplomeric glycoprotein e2 of mhv purified by the same technique forms rosettes or single peplomers. arrows indicate rosettes. x 100,000. (c and d from k. v. holmes.) 1981a,b; see section 111,d). isolated noninfectious, empty ibv particles. such particles have not yet been identified for other coronaviruses. the virions of coronaviruses are rather fragile and tend t o disrupt upon storage and/or during the negative-staining procedure (apos-tolov et al., 1970) . this can be prevented by fixation of virions with glutaraldehyde prior to negative staining. in spontaneously disrupted preparations of coronaviruses, fragments of viral envelopes are frequently seen. however, the internal component of coronaviruses has been more difficult to visualize in negatively stained preparations. thin sections of infected cells or virions demonstrated a flexible, cylindrical nucleocapsid which was probably helical (see section 111,f). johnson-lussenberg ( 1975-1976) showed that threadlike nucleocapsids, 8-9 nm in diameter, were released from disrupted hcv-229e virions. these were very pleiomorphic and may represent strands which had uncoiled from helical nucleocapsids. the most tightly coiled helical nucleocapsids were obtained from virions which were spontaneously disrupted by storage at 25°c overnight. found hollow, helical nucleocapsids, 14-16 nm in diameter and up to 0.32 pm long, released from purified preparations of hcv-229e and mhv-3 virions. the unit length of coronavirus nucleocapsids has not yet been determined, although nucleocapsids up to 6 pm long have been observed . caul et al. (1979) purified helical nucleocapsids from hcv-229e (fig. 1b) . the nucleocapsid was about 9-11 nm in diameter. coronavirus nucleocapsids appeared to be more flexible and easier to uncoil than paramyxovirus nucleocapsids. recent studies of helical nucleocapsids of negative-stranded rna viruses (heggeness et al., 1980 (heggeness et al., , 1982 showed that these properties depended upon the ionic strength and cation composition of the buffers. it appears likely that the appropriate conditions for further characterization of the structure of coronavirus nucleocapsids will be identified in the near future. in addition t o permitting analysis of the viral nucleocapsid, disruption of coronaviruses with nonionic detergents such as np-40 or triton x-100 has permitted the isolation of the envelope glycoproteins of coronaviruses (section 11,g). the morphology of the two envelope glycoproteins of mhv isolated from detergent-disrupted virions on sucrose density gradients is shown in fig. 1c and 1d . early studies of coronaviruses were hampered by limited virus growth and difficulties with virus purification. coronaviruses exhibited restricted host ranges in cell culture, low virus yields were usually obtained, and the viruses were highly labile. these difficulties have now been overcome for many coronaviruses . suitable permissive cell types and virus strains have been identified and conditions have been described which result in greater virus stability. many human coronaviruses were first identified by their growth and cytopathogenicity in human embryonic tracheal and nasal organ cultures (tyrrell and bynoe, 1965; almeida and tyrrell, 1967; mcintosh et al., 1967a) . some of these viruses have not yet been grown in continuous cell lines, and great difficulties are still encountered with primary isolation of hcvs. although permissive cell hosts have been described for hcv-229e (hamre and procknow, 1966; hamre et al., 1967) as well as for tissue culture-"adapted" hcv-oc43 (bradburne and tyrrell, 1969; kapikian et al., 1969; bruckova et al., 1970; gerna et al., 1979; , virus yields of <lo8 pfu per milliliter are usually obtained. however, 0. w. obtained high yields (up to lo9 pfu per milliliter) of tissue cultureadapted strains of hcv-oc43 and -2293 in a human rhabdomyosarcoma cell line. good growth of some human enteric coronaviruses has also been reported in a human rectal carcinoma cell line (laporte and bobulesco, 1981) . early studies of mhv revealed that productive infection could be obtained in nctc1469 and l929 mouse cell lines (manaker et al., 1961; tanaka et al., 1962; hartley and rowe, 1963; mosley, 1961) . however, both of these cell lines contained endogenous retroviruses (david-ferreira and manaker, 1965; dales and howatson, 19611 , thus limiting their usefulness for propagation and biochemical studies of mhv. in 1972, sturman and takemoto reported that the growth of mhv was enhanced in al/n and balb/c 3t3 mouse cells which were transformed by papovaviruses or retroviruses. a "spontaneously" transformed balb/c 3t3 cell line, designated 17 c1 1, was also shown to be a highly permissive host for mhv, yielding >lo8 pfu per milliliter. two additional transformed cell lines have also been found to produce high yields of mhv: dbt, a schmidt-ruppin rous sarcoma virus-induced mouse tumor cell line (hirano et al., 1974 (hirano et al., , 1976 (hirano et al., , 1978 , and sac (-1, a maloney sarcoma virus-transformed mouse cell line which is defective in retrovirus production . in contrast to hcv and mhv, no continuous cell line is available which produces large amounts of ibv. limited growth of a few strains of ibv has been obtained in vero and bhk-21 cells (cunningham et al., 1972; coria and ritchie, 1973; otsuki et al., 1979) . however, high yields of some strains of ibv have been obtained in primary chick embryo kidney cells (otsuki et al., 1979; stern and kennedy, 1980a) . another development which facilitated progress in this field was the recognition that coronaviruses are most stable between ph 6.0 and 6.5 alexander and collins, 1975; sturman, 1981) (fig. 2) . at ph 6.0, the half-life of mhv infectivity at 37°c in the presence of 10% fetal bovine serum was 24 hours, whereas at ph 8.0, a 50% loss in virus infectivity occurred in less than 1 hour. the rapid loss stability of viral infectivity is shown as the ratio of viral titer at 24 hoursititer at 0 time, x100. at 4°c the virus is quite stable from ph 4 to 10. however, at 37°c the virus exhibits marked thermolahility at phs c4.5 and >6.5. (reproduced from sturman, 1981, with permission.) of infectivity at ph 8.0 was associated with aggregation of the peplomeric glycoprotein (sturman, 1981) . several unique features of the coronavirus glycoproteins, as well as the lack of suitable permissive cell types for some coronaviruses and residual host-cell contamination of other coronaviruses, delayed recognition that coronaviruses all possess a similar pattern of structural proteins. recently, the general organization of coronavirus structural proteins has become apparent. figure 3 illustrates a model of the structure of mhv-a59. we will use this model and the nomenclature developed during our studies of the structural proteins of mhv for the following discussion of coronavirus structural proteins and their organization. the envelope of the intact virions contains two envelope glycoproteins, e l and e2, in a lipid bilayer. the helical nucleocapsid is composed of a single long strand of message sense, genomic rna with the nucleocapsid protein, n. glycosaminoglycan is associated with the viral envelope. (b) treatment of the virions with pronase or bromelain removed the bulk of e2, a 5k glycosylated portion of e 1, and the glycosaminoglycan, but leaves the nucleocapsid intact. (c) virions released from cells treated with tunicamycin lack e2 but contain normal amounts of glycosylated e l and nucleocapsid. in 1975, garwes and pocock characterized the structural polypeptides of tgev, a porcine coronavirus. although analysis of coronavirus polypeptides had been attempted earlier, those studies were less definitive since they utilized virus produced in animals or eggs which may have been contaminated with some host-cell components (hierholzer et al., 1972; bingham, 1975) . working with radiolabeled tgev produced in cell culture, garwes and pocock identified four major polypeptide peaks on sodium dodecyl sulfate (sds)-polyacryl-amide gels. treatment of virions with bromelain removed the peplomers and the largest glycoprotein (200k1, which is analogous to the one which we have called e2 in fig. 3 . a single, nonglycosylated, arginine-rich, 50k species similar to n, and two smaller (28 and 30k) glycoproteins, analogous to e l in fig. 3 , were also identified and partially characterized (garwes et al., 1976) . using double-labeled virus grown in tissue culture, the structural polypeptides of the a59 strain of mhv were described next (sturman, 1977; sturman and holmes, 1977) . this virus was shown to contain a nonglycosylated basic polypeptide, n (50k) and five glycoprotein peaks which were separated into two families based on the ratios of incorporation of different radiolabeled precursors. the e2 glycoproand 1%-amino acids ( 0 -4 ) were solubilized in sds and analyzed by page. two classes of glycoproteins, e l and e2, are identified by the ratios of labels, and a nonglycosylated nucleocapsid protein, n, is seen at 50k. designation and molecular weights of the viral structural proteins are indicated above the arrows. ( b ) virions labeled with 14c glucosamine (0-0) and sh fucose (o---o) showed two classes of glycoproteins. the e l glycoproteins were labeled with glucosamine but not with fucose, whereas the e2 glycoproteins were labeled with both. (adapted from sturman and holmes, 1977, with permission.) teins (gp180 and gp90) were labeled with both fucose and glucosamine, whereas the e l glycoproteins (gp23, gp38, and gp60) were labeled with glucosamine but not fucose (fig. 4b) . a surprising finding was the demonstration that e l (gp23) aggregated when heated to 100°c in the presence of sds and mercaptoethanol, generating several forms of higher apparent molecular weights (sturman, 1977; fig. 4b) . when virions were solublized in sds at 37"c, only a single broad peak at 23k was observed (fig. 5a) . the 180k form of e2 could be converted quantitatively to 90k by treatment of intact virions with trypsin, which did not remove the peplomers ( fig. 6 ; sturman and holmes, 1977) . when the 180 and 90k e2 glycoproteins were extracted from polyacrylamide gels and further digested with trypsin, virtually identical tryptic peptide patterns resulted. these results suggested either that proteolytic were prepared for page by incubating at 37°c for 30 minutes instead of boiling. the e l glycoprotein did not aggregate into dimers and trimers as shown in fig. 4 , but migrated as a broad peak of 23k. designations and molecular weights of the viral structural proteins are indicated above the arrows. (b) when virions similarly labeled were incubated with the proteolytic enzyme bromelain, the e2 glycoprotein and the 5k glycosylated portion of e l were removed, leaving an 18k portion protected within the viral envelope with the nucleocapsid protein n (vp 50k). (adapted from sturman and holmes, 1977 , with permission.) virions with 10 pgiml trypsin, the 180k form of e2 was quantiatively converted into the 90k formb), while the other two structural proteins were unchanged. (adapted from sturman and holmes, 1977, with permission.) cleavage of the 180k form of e2 yielded two different 90k forms which comigrated, or that the 180k e2 might be a covalently linked dimer of a single 90k species. thus, although the mhv virion was composed of only three major structural proteins, multiple forms of e l and e2 were generated by aggregation and proteolysis. similar observations have been made with some other coronaviruses (see below). the structures and functions of these two glycoproteins will be considered in detail in section iv. the study of mhv-a59 also provided information about the relative ratios of the structural proteins and their orientation in the virion. on the basis of incorporation of radioisotopic labels, we estimated that the proteins occur in virions in a ratio of 8 n : 16 el:l e2 . as with tgev, treatment of virions with bromelain or pronase resulted in the loss of e2 (fig. 5b ) and removal of the peplomers or spikes on the virion (sturman and holmes, 1977) . pronase also removed a 5k glycosylated portion of e l , which suggested that a terminal glycosylated region of e l was exposed on the outer surface of the viral envelope, while a larger (18k) nonglycosylated region was protected within the envelope. since pronase treatment of intact virions did not affect n (50k1, n was thought to be an internal protein. the structural relationships of these three viral polypeptides are summarized in the model in fig. 3 . wege et al. (1979) , investigating the structural proteins of the jhm strain of mhv, detected two species of e l on sds-polyacrylamide gel electrophoresis (sds-page) which appeared to correspond to nonglycosylated and glycosylated forms of e l . in addition, they showed that the e2 (90k) of mhv-jhm could be resolved into two distinct bands. in contrast to e2 from mhv-a59, the e2 of jhm became aggregated with e l when heated to 100°c and remained near the origin of the resolving gel (wege et al., 1979; siddell et al., 1981b) . comparison of a variety of strains of mhv revealed that some of the homologous polypeptides from different strains were distinguishable by page (stohlman and lai, 1979; anderson et al., 1979; bond et al., 1979; cheley et al., 1981b) . this may be useful in genetic and complementation studies. stohlman and lai (1979) demonstrated that the n polypeptide of mhv was phosphorylated on serine residues (fig. 7) . subsequently, '*i siddell et al. (1981a) identified a cyclic amp-independent protein kinase activity which copurified with the virion (fig. 8) . the functional significance of phosphorylation for transcription, translation, or maturation of the viral nucleocapsid is not yet known. the nucleotide sequence of the rna encoding the nucleocapsid protein of mhv-a59 was recently determined by armstrong et al. (1983) . this sequence contained a single long open reading frame encoding a protein of molecular weight 49,660 which was enriched in basic residues. there was also a second short, open reading frame in this sequence predicting a polypeptide of 90 amino acids. however, no such product has yet been identified. the pattern of three major structural proteins and their organization in the virion as shown for mhv-a59 in fig. 3 is generally applicable to most other species of coronaviruses (reviewed in detail by siddell et al., 1982) . although the proteins of different coronaviruses have different molecular weights, similar polypeptide patterns have been obtained with the porcine coronaviruses, transmissible gastroenteritis virus, and hemagglutination encephalomyelitis virus pocock and garwes, 1977; callebaut and pensaert, 1980; k. moreau and d. a. brian, personal communication) , and with canine coronavirus i71 (garwes and reynolds, 1981; carmichael and binn, 1981 ; see the review by garwes, 1980) . the structural polypeptides of several of the mammalian coronaviruses, including those from rats, cats, and rabbits, have not yet been investigated. macnaughton (1980) and schmidt and kenny (1982) reexamined the polypeptide composition of the human coronaviruses 2293 and oc43 and obtained results similar to mhv. schmidt and kenny demonstrated that the e l of oc43 aggregated upon heating in sds under reducing conditions, whereas e l from 2293 did not. several coronaviruses may have an additional envelope glycoprotein. in some bovine and porcine coronaviruses, three or four large glycoprotein peaks associated with the virus peplomers have been identified by sds-page (king and brian, 1982; callebaut and pensaert, 19801 , and more than one morphologically distinguishable spike has been detected by electron microscopy (bridger et al., 1978) . it has been suggested that these viruses possess several different types of peplomers. however, the relationship between the components detected on sds gels and the morphologic subunits of these viruses has not yet been elucidated. for some time it appeared that the structural polypeptides of an avian coronavirus, ibv, were more complex than those of mammalian fig. 8 . demonstration of virion-associated protein kinase activity. virion-associated protein kinase activity is demonstrated in detergent-disrupted mhv virions by the incorporation of 32p from orthophosphate into protein. the page pattern on the right indicates that the only viral structural protein phosphorylated during this reaction is n. lanes indicate times after initiation of the reaction; molecular weight standards are shown on the right. (reproduced from siddell et al., 1981a, with permission.) coronaviruses (bingham, 1975; collins et al., 1976; alexander and collins, 1977) . however, macnaughton and madge (1977a) demonstrated that harsh conditions of sample treatment generated spurious additional bands on sds gels. these studies were extended by collins and alexander (1980a,b) and lancer and howard (1980) . by 1981, it was apparent from the work of several investigators (cavanagh, 1981; wadey and westaway, 1981; lomniczi and morser, 1981; macnaughton, 1981; that there were three major classes of ibv structural polypeptides. contamination by host polypeptides seems to have caused many of the problems associated with studies using virus grown in embryonated egge (wadey and westaway, 1981; cavanagh, 1981) . lomniczi and morser (1981) showed that the n polypeptide of ibv was phosphorylated like that of mhv (stohlman and lai, 1979) . they found that actin was bound to the surface of purified ibv virions. in contrast, actin has not been detected in purified mammalian coronaviruses. the polypeptide composition of another avian coronavirus, turkey bluecomb disease virus, has not yet been reported. differences in the carbohydrate compositions of e l and e2 for mhv were first indicated by results obtained from metabolic labeling of virions with radioisotopic precursors. e2 was labeled with both fucose and glucosamine, whereas e l was labeled with glucosamine but not with fucose (sturman and holmes, 1977) . the carbohydrate compositions of e l and e2 were analyzed by niemann and klenk (1981a,b) . their results are shown in table 11 . as coronaviruses were often grouped with myxoviruses in early classification schemes, it is interesting to note that both e l and e2 contained sialic acid, unlike the myxovirus glycoproteins. e2 also contained substantial amounts of mannose and galactose plus fucose, glucose, and n-acetylglucosamine. these sugars are all found in high mannose and complex oligosaccharides which are derived from a mannose-trisaccharide core nglycosidically linked to asparagine residues in the protein. the oligosaccharide side chains of e l were strikingly different from those of e2, in that they lacked fucose and contained a high proportion of nacetylgalactosamine, which was absent from e2. recent evidence indicates that in mhv-infected cells labeled with glucosamine, the glucosamine is converted to n-acetylgalactosamine prior to incorporation into the e l glycoprotein (h. niemann, personal communication). the carbohydrate composition of the e l glycoprotein suggested that e l oligosaccharides might possess 0-glycosidic linkages to serine or threonine residues in the protein. the same conclusion was reached by others (holmes et al., 1981a,b; siddell et al., 1981c; rottier et al., 1981b) based on the resistance of the glycosylation of e l to tunicamycin, an inhibitor specific for n-glycosylation. such 0-linked glycoproteins had not been found previously in viruses. the glycopeptides of e l and e2 were readily distinguishable by electrophoresis on borate-polyacrylamide gels at high ph (sturman, 1981; holmes et al., 1981a) and by column chromatography on biogel p6 (niemann and klenk, 1981a) . niemann and klenk (1981b) demonstrated conclusively that the oligosaccharide moieties on e l were attachd by 0glycosidic linkages since they could be released by p-elimination with sodium borohydride. recently, h. niemann et al. (personal communication) have used high-performance liquid chromatography (hplc) to characterize two species of oligosaccharide chains released from e 1 by p-elimination. these are shown in fig. 9 . it is highly probable that the virus makes use of host-cell enzymes for 0-linked glycosylation as other viruses do for n-linked glycosylation. thus, the e l glycoprotein of mhv is of considerable interest as a model for studying the synthesis and glycosylation of 0-linked cellular glycoproteins (see sections ii1, d and iv, b) . there appears to be considerable variation in the glycosylation of the e l glycoproteins of different coronaviruses. the e l of some mhv strains could not be labeled with glucosamine (anderson et al., 1979) and the e l of tgev was labeled with both fucose and glucosamine ; also see review by games, 1980) . recent neunac 2 1 6 la neunacz~3cal1---3galnac-oh evidence from endoglycosidase h sensitivity and tunicamycin inhibition studies indicated that the e l glycoprotein of ibv is n-linked, rather than o-linked . the biological signficance of n-versus o-linkage of the oligosaccharides is not known. other constituents of e l and e2 have been identified. e l and e2 could both be labeled with 35s sulfate (garwes et al., 1976; l. s. sturman, unpublished data). by analogy with glycoproteins of orthomyxoparamyxo-, and rhabdoviruses (nakamura and compans, 1977; prehm et al., 1979; hsu and kingsbury, 1982) , these sulfate moieties may be ester linked to glucosamine or galactosamine residues of oligosaccharide side chains and may contribute to the charge heterogeneity of the viral glycoproteins. protein sulfation on tyrosine resides may also occur (huttner, 1982) , but this has not yet been reported for viral proteins. the significance of sulfate groups for antigenicity or other biological functions of viral glycoproteins is not known. m. f. g. schmidt and his co-workers (1979; schlesinger, 1979, 1980; schmidt, 1982a,b) demonstrated that palmitic acid was covalently attached to certain glycoproteins from a variety of enveloped viruses. niemann and klenk 11981a) showed that palmitic acid was present on the e2 glycoprotein of mhv, but not on e l or n. schmidt reported similar results with bcv (1982) . recently, we separated two e2 (90k) species from trypsin-treated mhv-a59 by sds-hydroxyapatite chromatography (l. s. sturman and k. v. holmes, unpublished data) . only one of these 90k species contained palmitic acid. this palmitic acid may be useful as a marker to identify one 90k subunit of the e2 molecule. minor protein species associated with some coronaviruses have also been identified, such as gp65 in mhv-jhm (wege et al., 1979; siddell et al., 1981b) . the reiationship of such minor components to the other structural proteins of the virion is not yet known. these may represent virus-specific polypeptides which are found in infected cells (see section 111,d). as with the structural proteins, the earliest data on the coronavirus genome were misleading. initial observations suggested that coronaviruses contained segmented or multimeric genomes (tannock, 1973; . there were also indications of possible rna polymerase activity associated with the virion . these findings were in accord with the belief then current that coronaviruses were probably similar to myxoviruses. then watkins et al. (1975) demonstrated that high-molecular-weight rna could be extracted from ibv virions. the large, single-stranded, linear rna molecules obtained from ibv were shown to be polyadenylated and infectious (schochetman et al., 1977; lomniczi, 1977; lomniczi and kennedy, 1977 ; macnaughton and madge, 197713) . lomniczi and kennedy (1977) characterized the ibv genome by electrophoresis on methyl mercury gels and by t1 oligonucleotide fingerprinting. this approach also proved to be very useful in the comparative analysis of coronavirus genomes and subgenomic virus-specific mrnas (see section iid). extracted high-molecular-weight (60-70 s) rna from tgev and hev. however, they observed that the rna was dissociable above 60°c into 35 and 4 s species. tannock and hierholzer (1977) also reported releasing 4 s rna from hcv-oc43 rna and fragmentation of the 70 s rna after heating at 60°c. lai and stohlman (1978) found a variable amount of 4 s rna in mhv preparations, in addition to 60 s rna. garwes et al. (19751, pocock and garwes (19751, tannock and hierholzer (19771, and lai and stohlman (1978) demonstrated that the size and heterogeneity of the virion rna which was isolated was affected by the time of virus harvest, the ph of the culture medium, the method of virus purification and duration of virus storage, and the method of rna extraction. greater fragmentation of coronavirus rna was observed if virions were harvested at 24 hours postinfection versus 16 hours, if cultures were kept at ph 7.2 or 8.0 and not at ph 6.5, if virus was purified on potassium tartrate equilibrium gradients instead of sucrose gradients, if virions were stored for 24 hours at 4°c before rna extraction, and if the rna was extracted in the presence of phenol. fragmentation was readily detected after heating the rna at 60°c or higher, and by centrifugation through dimethyl sulfoxide gradients. this behavior of coronavirus rna, as well as other aspects of the structure and physiocochemical properties of coronaviruses, is discussed in the excellent review by garwes (1980) . it now appears that coronavirus genomes are 5.4-6.9 x lo6 daltons in size, which corresponds to about 16,000-21,000 nucleotides. rna genomes in this size range have been detected in virions of ibv (schochetman et al., 1977; macnaughton and madge, 197713; macnaughton, 1978; stern and kennedy, 1980a) , mhv (yogo et al., 1977; lai and stohlman, 1978; wege et al., 1981b; macnaughton, 1978; spaan et al., 1981; weiss and leibowitz, 19811, hcv (tannock and hierholzer, 1977; macnaughton and madge, 1978; macnaughton, 1978) , tgev and hev brian et al., 19801, and bcv (brian et al., 1980) . the genomic rna is infectious (lomniczi, 1977; schochetman et al., 1977; wege et al., 1978; brian et al., 19801, capped (lai and stohlman, 1981; lai et al., 1982a) , and polyadenylated (schochetman et al., 1977; lomniczi, 1977; yogo et al., 1977; madge, 1977b, 1978; wege et al., 1978; lai and stohlman, 1978; guy and brian, 1979; . for further details, see the reviews by garwes (1980) and siddell et al. (1982) . in 1980, stern and kennedy (1980b) mapped the location of the t1 oligonucleotides of the genome in a partial 3' to 5' order. large fragments of rna were produced by partial alkali fragmentation of the genome. these fragments were fractionated into different size classes by sedimentation on sucrose density gradients, and polyadenylated rnas were selected for t1 oligonucleotide analysis. the same approach was applied to mhv by lai et al. (1980) . these studies disclosed that there was no significant reiteration of oligonucleotides within the genome. analysis of the genetic complexity of the virion rna indicated that it was all of the same sense. since genomic rna was infectious, the virion rna must be of positive or message sense. t1 oligonucleotide analysis of coronavirus genomes has been employed for comparisons of virus isolates and strains, for characterization of mutants, and for epidemiological investigations. the t1 oligonucleotide fingerprints of the two isolates of the beaudette strain of ibv which were studied by lomniczi and kennedy (1977) and stern and kennedy (1980a) were found to be quite different. subsequently, clewley et al. (1981) distinguished 11 oligonucleotide fingerprint patterns from 13 isolates of ibv, revealing differences between ibv serotypes, and also between different preparations of a single serotype. this suggested that considerable variation in the genome may be characteristic of ibv, and possibly of other coronaviruses as well. it is characteristic of rna viruses that the frequency of spontaneous mutations is high (holland et al., 1982) , and coronaviruses appear t o be no exception to this rule. analyses of murine coronaviruses have also indicated that there is considerable diversity between different strains. wege et al. (1981a) and found that the oligonucleotide fingerprint pattern of the -jhm strain differed significantly from those of mhv-1, -2, -3, -s, and -a59, and that mhv-3 and -a59 were closest in oligonucleotide patterns. have attempted to correlate strain-specific oligonucleotide differences with hepatotropism and neurotropism of mhv-3 and -a59. stohlman et al. (1982b) have characterized oligonucleotide patterns of plaque morphology variants of j h m that differ in neuropathogenicity. the genetic relatedness of coronaviruses has also been analyzed by nucleic acid hybridization. using a cdna probe representative of the entire genome of mhv-a59, weiss and leibowitz (1981) found that mhv-3 and -a59 were more closely related to one another than either was to -jhm. hcv-229e appeared to be quite unrelated to mhv by this technique. cheley et al. (1981b) , using a cdna probe prepared against the mrna of mhv-a59 which coded for the nucleocapsid protein, obtained evidence of 7 0 4 0 % homology by analysis of hybridization kinetics of viral rna from cells infected with mhv-1, -3, -s, and -jhm. the lipid composition of coronavirus virions has not been analyzed in detail. it appears likely that the lipids of the viral envelope will reflect the lipid composition of the intracellular membranes from which virus budding occurs (see section 111,f). indeed, pike and garwes (1977) observed that tgev contained less cholesterol and fatty acid than was found in the plasma membrane of the cells in which the virus was grown. when the virus was grown in different cell types, the viral lipids reflected the overall lipid composition of the cells in which it was grown. these studies need t o be extended using various cell-membrane fractions. the role of the lipid composition of cellular membranes in coronavirus maturation remains to be elucidated. it would be interesting to know whether e l and the viral nucleocapsid preferentially associate with membranes of a certain lipid composition. boundary lipids associated with the hydrophobic portions of e l and e2 have not been identified, although palmitic acid has been shown to be covalently attached to e2 (niemann and klenk, 1981a,b; schmidt, 1982a ,b) (see section 11,c). the derivation of coronavirus envelopes from intracellular membranes may render the viral envelope less susceptible to solubilization by bile salts and other detergents than enveloped viruses which bud from the plasma membrane. greater stability of the viral envelope to solubilization by bile salts would be consistent with the survival and replication of these viruses in the enteric tract. comprehensive studies on the relative susceptibility to solubilization of coronavirus envelopes and other viral envelopes have not yet been carried out. several components of normal host cells or tissues have been found to copurify with coronaviruses. the difficulty of obtaining ibv free from host-cell contamination and the association of actin with ibv have already been mentioned (section ii,b and c). in early studies of coronavirus antigens, host antigens were often detected in association with the purified virions. some components of fetal bovine serum adsorb to and copurify with coronaviruses (kraaijeveld et al., 1980) . it is not yet clear whether the protein kinase associated with coronavirus particles is a host contaminant or a product of the viral genome. another class of host molecules which copurified with several different coronaviruses was glycosaminoglycans (gag) (garwes et al., 1976; sturman, 1980) . these are polyanionic, linear polysaccharides such as hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, and heparin, which are secreted by cells and which may remain associated with their external surfaces (roden, 1980; oldberg et al., 1977; prinz et al., 1980) . they tend to aggregate spontaneously with like molecules to form large complexes. multiple chains of gag molecules which are linked to core proteins are called proteoglycans. garwes et al. (1976) showed that some sulfated gag was associated with tgev. glycosaminoglycan associated with virions of mhv-a59 was partially characterized by enzymatic and chemical degradation (sturman, 1980) . the gag associated with mhv-a59 resembled the heparan sulfate species produced by uninfected transformed mouse cells, which contained a reduced level of 6-0 sulfated glucosamine residues (sturman, 1980; keller et al., 1980; winterbourne and mora, 1981) . the virion-associated gag illustrated in fig. 3 is known to be on the external surface of the viral envelope since it can be removed by protease treatment of intact virions. the number of gag molecules associated with each virion is not yet known. cellular gags have been found in association with many types of enveloped viruses pinter and compans, 1975; lindenmann, 1977; kemp et al., 1982) , but the biological significance of this association is not known. in cells, heparan sulfate may play an important role in cell-cell and cell-substrate adhesion, growth control, and masking of cell surface receptors (vannucchi and chiarugi, 1977; rollins and culp, 1979; oldberg et al., 1979; kraemer and smith, 1974) . one could speculate that virion-associated gags or proteoglycans could alter viral functions by modifying viral structure, antigenicity, or susceptibility to proteolytic enzymes. coronavirus particles may spontaneously disrupt to yield membrane fragments and threadlike or helical nucleocapsids (see section 11,a). viral subunits have also been generated for structural and immunologic analysis by solubilization of the envelope with nonionic detergents such as np-40 or triton x-100 in low salt concentrations. after detergent disruption of the viral envelope, solubilized glycoproteins could be separated from the nucleocapsid by sedimentation in sucrose density gradients. garwes et al. (1976) were the first to report isolation of the surface projections and nucleocapsid of a coronavirus, tgev, by this technique. immunization with np-40-solubilized, gradient-purified tgev suface projections (e2) induced neutralizing antibodies (see section iv,a). johnson-lussenberg (1975-1976 ) isolated a nucleoprotein from 2293 by a similar method. similar studies were carried out with hev and mhv-jhm (pocock and garwes, 1977; wege et al., 1979) . in these studies, the nucleocapsid sedimented at a density of 1.24-1.29 gm/ml and was found to consist of rna in association with both n and e l structural proteins. electron microscopy revealed spherical "cores" 60 to 70 nm in diameter, sometimes with a strand approximately 9 nm in diameter inside. helical nucleocapsids (fig. 1b) were not observed in these studies. in similar studies on np-40-disrupted mhv-a59 (sturman et al., 19801 , we found that the temperature of solubilization determined whether e l would be isolated separately or in association with the nucleocapsid. solubilization a t 4°c yielded separate peaks of e l , e2, and nucleocapsid (p = 1.28 gm/ml; fig. loa) , whereas incubation of the viral extract at 37°c for 30 minutes resulted in quantitative binding of e l t o the nucleocapsid forming an e1-n-rna complex (p = 1.22 gm/ml; fig. 10b ). in negatively stained preparations, purified e l formed irregular aggregates of varied size, whereas purified e2 was in the form of single peplomers or rosette-like aggregates of about 12 peplomers ( fig. 1c and d). purified e l and e2 were used to raise monospecific antisera for the analysis of the functions of the glycoproteins (holmes et al., 1981b; section iv) . the reasons for the difficulty in isolating helical nucleocapsids from coronaviruses are not yet clear. e. 0. caul (personal communication) has suggested that the helical nucleocapsid in the virion may be in the sturman et al., 1980, with permission.) form of a labile supercoiled structure which may be identical to the 60to 70-nm spherical forms described above. alternatively, the 60to 70nm particles could represent incompletely solubilized nucleocapsids within a membrane-like structure containing e l . in some intracellular inclusions of nucleocapsids, such a structure has been visualized by transmission electron microscopy (dubois-dalcq et al., 1982; section 111,f) . holmes and behnke (1981) observed that mhv-a59 virions changed from spherical to flattened, disk-shaped particles during migration from the rough endoplasmic reticulum to the golgi (section 111,f). such a change may be correlated with supercoiling of the nucleocapsid strands. several investigators have studied the susceptiblity of isolated coronavirus nucleocapsids to digestion with proteases or rnase. using an electron microscopic assay, davies et al. (1981) showed that the nucleocapsid of ibv was destroyed by trypsin or pronase and was partially susceptible to degradation by pancreatic ribonuclease. during the past few years, a comprehensive understanding of coronavirus replication has begun to emerge. our current concept of coronavirus replication is shown in fig. 11 . although coronaviruses have structural similarities to the large, enveloped, negative-stranded orthomyxo-and paramyxoviruses, the coronaviruses demonstrate several unique features in their replicative cycle. a nested set of five or six subgenomic mrnas are elaborated, each of which codes for a single protein. there is some evidence which suggests that rna fusion may play a role in coronavirus replication. in some cases an o-linked envelope glycoprotein is formed which exhibits restricted intracellular transport. the cellular secretory apparatus may be used for release of virions. while much clearly remains to be learned about the replication of coronaviruses, it is already apparent that they utilize many novel ways of solving the problems of virus replication. relatively little is known about the earliest stages in coronavirus replication. although the marked host and tissue tropisms of coronaviruses have frequently been ascribed to possible host-cell receptor specificities, studies on early virus-cell interactions have been limited. in studies analogous to those done on myxoviruses, early studies of the interaction of coronaviruses with membranes used hemagglutination as a model for binding to the cell surface receptor. several coronaviruses, including hev (greig et al., 19621, ibv (corbo and cunthe entire replicative cycle occurs in the cytoplasm. the genomic rna acts as mrna to direct the synthesis of viral rna-dependent rna polymerase. this enzyme copies the genomic rna to form full-length, negative-stranded templates. from these templates, using the viral rna polymerase, a series of subgenomic mrnas are synthesized. these mrnas form a nested set with common 3' ends. all are capped and polyadenylated. each of the mrnas apparently codes for a single gene product. the functions of the nonstructural gene products ns 14k and ns 35k are not known. rna7 codes for the synthesis of n protein on free ribosomes. the n protein associates with newly formed genomic rna to form the viral nucleocapsid. rna3 and -6 are translated on membranebound ribosomes t o yield e2 and e l , respectively. the peplomeric glycoprotein e2 is cotranslationally glycosylated at asparagine residues and the core oligosaccharides are trimmed as the glycoprotein migrates through the golgi apparatus. the membrane glycoprotein, e 1, migrates to the golgi apparatus where oligosaccharides are added to the serine residues posttranslationally. virions are formed by budding in the rough endoplasmic reticulum and golgi apparatus, but not at the plasma membrane. e2 migrates readily to the plasma membrane, but intracellular transport of e l is limited to the golgi apparatus. virions are released from intact cells by fusion of post-golgi vesicles with the plasma membrane, possibly using the cellular secretory apparatus. numerous virions adsorb to the plasma membrane of infected cells. coronavirus-induced cytopathic effects include cell fusion and rounding of infected cells. ningham, 1959; bingham et al., 19751, bcv (sharpee et al., 1976) , rabbit enteric coronavirus (lapierre et al., 19801, hcv-oc38/43 (kaye and dowdle, 19691, and mhv (sugiyama and amano, 1980; walker and clantor, 19801 , can cause hemagglutination (see section iv,a). mengeling et al. (1972) showed that hev bound to avian erythrocytes by the tips of the viral peplomers. binding of ibv to erythrocytes was studied by bingham et al. (19751, who found that the binding was inhibited by protease and neuraminidase treatment, and enhanced by phospholipase c treatment of erythrocytes. shif and bang (1970) suggested that mhv-2 bound equally well to macrophages from mice genetically susceptible or resistant to mhv-2. they demonstrated equivalent amounts of infectious virus remaining in the supernatant medium over macrophage cultures after virus adsorption. binding of mhv-a59 to l2 cells in spinner culture was studied by richter (19761, who found that the cell surface receptor activity was removed by protease treatment of the cells. this suggested that the receptor was a protein. attachment occurred at 4 c and more rapidly at 37°c. saturation of virus receptor sites was achieved with only about 700 virus particles per cell. additional studies showed that at 4°c mhv-a59 bound to splenic lymphocytes from susceptible and resistant mice, but not to thymocytes. the interaction of mhv-3 with l cells and cells from susceptible and resistant strains of mice was studied by krystyniak and dupuy (1981) . at 37"c, radioactive virions bound to macrophages, spleen cells, t lymphocytes, and thymocytes. the virus bound equally well to macrophages from genetically susceptible and resistant mice. this supports the conclusion of shif and bang (1970) that genetic susceptibility and resistance are not determined at the level of virus receptors. the binding of coronaviruses to cells appeared to be via the peplomeric glycoprotein since antibody to the peplomeric glycoprotein e2 inhibited virus infectivity (garwes et al., 1978-19791 , and isolated e2 competed with intact virions for the same cell surface receptor (k. v. holmes, unpublished observation; see section iv,b). scanning electron microscopic analysis of the binding of hcv-229e to cultures of mrc human diploid cells showed that the virions bound randomly to the cell surface at 4°c (patterson and macnaughton, 1981) . warming the cultures to 37°c resulted in the loss of virions from the cell periphery, apparently by an energy-dependent capping mechanism. the penetration and uncoating of enveloped rna viruses has been studied extensively in recent years using biochemical and electron microscopic techniques. semliki forest virus, influenza virus, and vesicular stomatitis virus (vsv) virions bind to cell surface receptors and are internalized via coated pits (helenius et al., 1980a; marsh and hel-enius, 1980; matlin et al., 1981 matlin et al., , 1982 . the vesicles containing virions, which may be similar to the receptosomes of willingham and pastan (1980) , then appear to fuse with endosomes. at the low ph within the endosomes, the viral envelopes fuse with the endosomal membranes releasing nucleocapsids into the cytoplasmic sap. proteolytic cleavage of the peplomeric ha glycoprotein is required for fusion of influenza virus envelopes (huang et al., 1981; white et al., 1981) . although detailed studies of coronavirus penetration and uncoating have not yet been performed, there are some indications that a similar pathway of virus uptake may occur. electron microscopic studies of the uptake of mhv and ibv suggested that viral entry was by means of viropexis or endocytosis (tanaka et al., 1962; david-ferreira and manaker, 1965; sabesin, 1971; patterson and bingham, 1976) . however, other investigators suggested that coronaviruses entered the cell by fusion with the cell membrane (doughri et al., 1976) . the possibility that mhv-3 may enter the cell by fusion with the plasma membrane was supported by the observation of krystyniak and dupuy ( 1981) that mhv-3 could infect cells treated with cytochalasin b to prevent phagocytosis. adsorbed virions rapidly became associated with lysosomes (david-ferreira and manaker, 1965; sebesin, 1971) . virus uptake via coated pits was also observed (chasey and alexander, 1976; arnheiter et al., 1982) . chloroquine, a lysosomotropic drug that elevates the ph in lysosomes and prevents penetration of semliki forest virus and influenza virus through the endosomal membrane (helenius et al., 1980b, 19821 , was found to inhibit the replication of mhv by affecting a stage subsequent to virus adsorption (mallucci, 1966) . in 1979, in the first report on virus-specific rnas in coronavirusinfected cells, robb and bond (1979b) identified multiple size classes of virus-specific rnas in mhv-jhm-and -a59-infected cells by fractionation of mrnas on sucrose density gradients. soon thereafter, siddell et al. t 1980) demonstrated that different size classes of poly(a)containing intracellular mhv-jhm rnas fractionated on sucroseformamide gradients directed the synthesis of different structural proteins in cell-free translation systems. the n protein was translated from the smallest (17 s) fraction, whereas e l was translated from a larger (19 s) class of rna. at almost the same time, stern and kennedy (1980a,b) showed, in a classic study of ibv rna, that coronavirus infection resulted in the production of a nested set of subgenomic messenger rnas with common 3' ends. in ibv-infected cells, six virus-specific rna species were in this diagram each oligonucleotide in the rnase t1 fingerprint is labeled according to the smallest subgenomic mrna species in which it appears. the unlabeled nucleotides were not included in subgenomic rna e and hence, presumably, represent sequences between the 5' end of the genome and rna e. (adapted from stern and kennedy, 1980a,b, with permission.) identified by electrophoresis on agarose-glyoxal gels. all of these rnas were polyadenylated and therefore likely to be of message sense. together, the five subgenomic species greatly exceeded the total size of the genomic rna, suggesting that the subgenomic rnas shared some sequences. comparison of t1 oligonucleotide digests of these intracellular rnas, as shown in figs. 12 and 13, revealed that they formed a "nested' set of sequences. the oligonucleotides of the smallest mrna were contained within the next larger mrna, and so on. these data also showed that the genomic rna was of the same sense as the mrna since they shared the same oligonucleotides. the possible origin of several unique oligonucleotides found in rnas a and c will be discussed later. by ordering the t1 oligonucleotides of the genomic rna through analyses of poly(a)-containing fragments produced by limited alkaline hydrolysis (see section ii,d), stern and kennedy 11980b) showed that all of the subgenomic mrnas shared common sequences extending from the 3' terminus of the genome. a similar structure for the intracellular rnas of mhv was also demonstrated. spaan et al. (1981) isolated six subgenomic, virus-specific rnas from polysomes of mhv-a59 infected cells, identified six intracellular rna species in cells infected with mhv-jhm. weiss and leibowitz (1981) and cheley et al. (1981a) showed, by hybridization with a cdna probe against the 3' end of the smallest message, that all of the subgenomic rnas of mhv-a59 contained sequences common to the 3' end. t1 oligonucleotide fingerprints of subgonomic rnas of mhv were analyed by leibowitz et al. (19811, lai et al. (19811, and spaan et al. (1982) . the studies showed that, like avian coronaviruses, for the murine coronavirus mhv-a59, the oligonucleotides of each of the subgenomic rnas were included within the next larger species, starting from the 3' end of the genome. a tentative map of the mhv-a59 genome based on these data is shown in fig. 14. recently, a similar pattern of overlapping mrnas has been demonstrated by heilman et al. (1982, and personal communication) for the early and late regions of the dna-containing bovine papilloma virus. using a cdna probe for the 3' end of mhv rna, cheley et al. (1981b) found differences in the elctrophoretic mobility of several subgenomic rnas of mhv-s in comparison with homologous rnas of mhv-1, -3, -a59, and -jhm. found that the intracellular rnas appeared to be present in different ratios in cells infected with different strains of mhv (table 111 ). the ratios of mrnas to each other did not change significantly during the course of viral infection (stern and kennedy, 1980a; wege et al., 1981b; . messenger rna coding assignments for the structural proteins of mhv have been established by eell-free translation experiments by siddell et al. , 1981~1, leibowitz and weiss (1981 , and leibowitz et al. (1982b) , and from translation experiments in xenopus laeuis oocytes by rottier et al. (1981a) (summarized in fig. 11 , and see section 111,d). the intracellular rnas of mhv have been designated 1 through 7, beginning with genomic rna1, and proceeding to the smallest mrna, 7. each virus-specific primary translation product has been shown to be associated primarily with one mrna (fig. 11 ). fig. 14. tentative map of the genomic rna of mhv. based on the in uztro translation of isolated mrnas for several mhv strains by siddell et al. (1980 siddell et al. ( ,1981c , leibowitz et al. (1982b31, and rottier et al. (1981a1, and the oligonucleotide mapping of lai et al. (1981) , we have drawn a tentative map of the mhv genome. although additional details will become available, it seems clear that the genes which have been identified to date are arranged in this order. comparison of each rna with the size of its translation products suggested that only the gene proximal to the 5' end of each mrna was translated. as shown in fig. 15 from the study by rottier et al. (1981a1, the product of rna7 was protein n (see section iii,c,l), that of rnag was e l , and of rna3, e2. the coding assignments for several nonstructural proteins were deduced by siddell et al. (1980 , 1981~1, leibowitz and weiss (1981 ,and leibowitz et al. (198213) . rnab directed the synthesis of a 30-35k nonstructural (ns) protein; rna4 or -5, a 14k ns protein; and rna1, several related ns proteins which were >200k. these data have been incorporated into the tentative map for the genomic rna of mhv-a59 shown in fig. 14 . thus, the current dogma of coronavirus replication is that each mrna directs the synthesis of a single protein using only the gene at the 5' end of the rna. however, some caution must be exercised in accepting this as proven. in all of the in uitro translation studies, rnag has been shown to direct the synthesis of e l , plus a fairly large amount of n (siddell, 1980 (siddell, , 1981c rottier et al., 1981a; leibowitz and weiss, 1981; fig. 15 ). this has been ascribed to contamination of rnag with rna7 and a higher efficiency of translation of rna7. it is possible that rnag alone might direct the synthesis of several gene products, both e l and n. in semliki forest virus, an enveloped, positive-stranded rna containing alphavirus, a large polycistronic mrna is translated to yield both glycosylated envelope glycoprotein and nonglycosylated capsid protein (schlesinger and kaariainen, 1980) . the translation of rna4 and -5 of mhv has been difficult to study because these rna species are present in such small amounts. so far only the 14k ns protein has been identified as a product for these two mrnas. in vitro translation studies using microsomal membrane fractions have not yet been done with coronavirus mrnas. such studies would permit the analysis of glycosylation, acylation, and processing of the glycoproteins. initial studies of intracellular virus-specific proteins anderson et al., 1979) identified the three major structural proteins, e l , e2, and n, and several nonstructural proteins. no high-molecular-weight polyproteins were detected in infected cell . concurrent studies of the structural proteins of the virion and analyses of the mrnas and their translation products set limits on the probable number of virus-specific polypeptides, and established that the structural proteins were not derived from a large polyprotein precursor. the intracellular proteins of mhv and ibv were identified in many laboratories at almost the same time. much of this information was presented at the 1980 coronavirus symposium and was summarized in the review by siddell et al. (1982) . the intracellular proteins identified included n, e l , and e2, and three nonstructural species: 30-35k and 14k nonglycosylated polypeptides, and a 65k glycoprotein. the 30-35k species has not been detected in virions. however, minor proteins of approximately 14k and 65k have been found in some coronaviruses (wege et al., 1979; siddell et al., 1981a; rottier et al., 1981b , though not in others (e.g., sturman, 1977; macnaughton, 1981; rottier et al., 1981b; lomniczi and morser, 1981; niemann and klenk, 1981a; bond et al., 1981; see siddell et al., 1982 , for further references). the significance of these findings is not known. the 65k species has not yet been associated with a virus-specific mrna. much of our understanding about the synthesis and processing of coronavirus proteins in uiuo has been obtained from pulse-labeling and pulse-chase experiments with mhv. several different virus-cell systems have been studied ranging from highly lytic ones involving l, dbt, and sac(-) cells, in which virus infection induced syncytia formation and destroyed the cells within 10-12 hours (anderson et al., 1979; cheley et al., 1981b; siddell et al., 1980; spaan et al., 1981) , to a more moderate infection in 17 c1 1 cells, in which the infected cells could survive for more than 24 hours without significant cytopathic effects (cpe) (robb and bond, 1979b; sturman et al., 1980; holmes et al., 1981b) . not surprisingly, differences were detected in the rates of shutdown of cellular protein synthesis in these different systems. other differences between lytic and moderate coronavirus-cell interactions were also noted. synthesis of mhv proteins in lytic infections appeared to be coordinated throughout the replication cycle rottier et al., 1981b) , whereas in cells showing minimal cpe, the synthesis of n was detected much earlier than that of e l and e2 (j. behnke, personal communication). pulse-labeling experiments at different times after infection and at different temperatures revealed that the synthesis of e l and e2 in these cells was coordinated, but different from the synthesis of n. the transit time for the synthesis of the viral glycoproteins, their incorporation into virions, and release of the virions from the infected cells also differed in moderate and lytic infections. in moderate infecfig. 16. synthesis and processing of mhv structural polypeptides (cont, control; inf, infected). at 6 hours after infection with mhv-a59, cells were pulse labeled for 15 minutes with [3h]-leucine and then incubated with excess cold leucine. there were no polyproteins observed. e2 is synthesized as the high-molecular-weight form, and no 90k e2 was observed in these cells. the n protein was synthesized in large amounts and appeared to be processed into two faster migrating species. the e l was synthesized as a nonglycosylated species which was posttranslationally glycosylated. both the glycoproteins were chased out of the cell into mature virions within 2-3 hours after labeling. only a small fraction of the n protein was chased into mature virions. (adapted from holmes et al., 1981b, with permission.) tion, e l and e2 were quantitatively chased out of cells and into virions within about 2 hours (holmes et al., 1981b; fig. 16 ). however, in lytic infection of sac(-) cells, e l chased into virions within 2-2.5 hours after labeling, while much of the e2 remained cell associated for up to 3 hours . another difference between lytic and moderate infections related to the appearance of e l and e2 on the surface of infected cells. both e l and e2 antigens of mhvjhm were detected on the surface of lytically infected l cells prior to the release of progeny virions (collins et al., 1982) . in contrast, e2 was the predominant viral glycoprotein on the surface of 17 c1 1 cells during the early stages of a moderate infection with mhv-a59 (doller and holmes, 1980 ; section iii,d,2). to date, cleavage of the e2 glycoprotein has been detected only in lytically infected cells. indeed, cleavage of e2 may be responsible for the extensive cell fusion seen in lytically infected cultures (l. s. sturman and k. v. holmes, unpublished observation) . these host-dependent differences may reflect differences in host-dependent processes required for maturation of coronaviruses. the features of the synthesis, processing, and transport of coronavirus structural proteins which are consistent in all cell systems are described in the following paragraphs. although the time of appearance of labeled n paralleled the time of appearance of the labeled glycoproteins in virions, no significant decrease in the amount of n in the infected cell was detected during a 90to 120-minute chase period (anderson et al., 1979; bond et al., 1981; holmes et al., 1981b; rottier et al., 1981b; siddell et al., 1981b) . this suggested that there was a large intracellular pool of n, most of which did not chase into virions, but was incorporated into nucleocapsids or replicative intermediates which never left the cell. recently, it was shown that n was synthesized on free ribosomes (niemann et al., 1982) . intracellular n was phosphorylated at serine residues, as was n in the virion (siddell et al., 1981a,b) . during a 2-hour chase, an intracellular form of n with slightly greater electrophoretic mobility was detected within infected l cells (anderson et al., 1979) . this process was believed to be due to proteolytic processing of n (cheley and anderson, 1981) . the proportion of the smaller species increased following immunoprecipitation (siddell et al., 1980; rottier et al., 1981a) , which led to the suggestion that these additional species represented molecules of n partially degraded by serum or cellular proteases. in pulse-labeling studies, these forms of n have been detected in cells without immunoprecipitation late in the infectious cycle of mhv-a59 (j. n. behnke and k. v. holmes, unpublished observations) . analysis of peptide maps of these different forms of n will be required to determine their relationships. e l appeared to be synthesized as a nonglycosylated 20k apoprotein which was posttranslationally glycosylated (holmes et al., 1981b; niemann and klenk, 1981a; rottier et al., 1981b; siddell et al., 1981~) . the addition of sugars to this o-linked glycoprotein began 15 to 30 minutes after completion of the apoprotein, and continued for 1-2 hours (holmes et al., 1981b; rottier et al., 1981b) with production of two or three discrete glycosylated species. however, not all of the e l which entered the virion was glycosylated (rottier et al., 1981b) . the oligosaccharide side chains of the e l glycoprotein are shown in fig. 9 . niemann et al. (submitted for publication) have estimated that there are three oligosaccharide chains per e l molecule. glycosylation of e l is resistant to tunicamycin (holmes et al., 1981a,b; niemann and klenk, 1981a; rottier et al., 1981b; siddell et al., 1981~) . recent cell fractionation studies (niemann et al., 1982; m. frana, unpublished observation) showed that e l was translated on membrane-bound polysomes. monensin inhibited glycosylation of e l (niemann et al., 1982) . the intracellular transport of the e l glycoprotein is markedly different from that of other viral glycoproteins. immunofluorescent staining with monospecific anti-el antibody showed that early in the infectious cycle e l was restricted to the perinuclear area of infected cells and accumulated in the golgi apparatus (doller et al., 1982; fig. 17 ). in contrast, e2 rapidly dispersed throughout the cell membranes and appeared on the plasma membrane. the mechanisms and signals for intracellular transport of e l are not yet understood. possibly e l is transported by cellular mechanisms which transport cellular glycoproteins destined for the golgi apparatus. since e l is a transmembrane protein and has been shown to interact with viral nucleocapsid in uitro, it appears likely that the localization of e l on the rough endoplasmic reticulum and golgi membranes determines the site of virus budding. e2, the peplomeric glycoprotein, was recently shown to be synthesized on membrane-bound ribosomes (niemann et al., 1982) . pulselabeling studies showed that a large, 150-180k glycoprotein was the first form detected in infected cells (holmes et al., 1981a; rottier et al., 1981b; siddell et al., 1981~) . as noted below, there is considerable controversy over the relationship between the 180 and 90k forms of e2. this controversy may relate to differences in processing of e2 in different cell types. a 110-120k apoprotein was detected in mhv-a59 infected sac(-) and 17 c1 1 cells treated with tunicamycin rottier et al., 1981b; niemann and klenk, 1981a) . this is similar to the in uitro translation product of rna3 in l-cell or reticulocyte lysates in which glycosylation does not occur (rottier et al., 1981a; siddell et al., l980,1981c) , and probably represents the protein moiety of the e2 glycoprotein. translation of mrna3 inxenopus laeuis oocytes permitted glycosylation and yielded a 150k e2 glycoprotein (rottier et al., 1981a; fig. 15 ). pulse-labeling experiments with mhv-a59 and -jhm in sac(-) cells showed that the first form of e2 detected fig. 17 . localization of e l in the golgi apparatus. (a) monospecific anti-el antiserum was used to stain e l antigens within the cytoplasm of infected cells. the e l accumulated in a sharply demarcated region near the nucleus and did not migrate to the plasma membrane as readily as did e2. (b) the same cells were labeled by the thiamine pyrophosphatase histochemical reaction which labels the terminal saccules of the golgi apparatus. this demonstrates that the e l accumulates in the golgi apparatus. x960. (from e. w. doller.) in these cells was a 150k species (siddell et al., 1981b,c; rottier et al., 1981b) . similar experiments using mhv-3 and -a59 in l and 17 c1 1 cells, respectively, demonstrated a 180k, rather than a 150k, product (cheley and anderson, 1981; holmes et al., 1981b) . the difference between the 180 and 150k moieties has not been identified, but could relate to additional glycosylation, trimming, or sulfation of e2. the appearance of the 90k species of e2 is different in different cell types. although in moderate infection of mhv-a59 in 17 c1 1 cells, little or no 90k e2 was detected at 8-10 hours after infection (holmes et al., 1981b; fig. 16) , in sac ( -) cells infected with mhv-jhm or -a59, a substantial amount of 90k e2 was detected after 60-to 90-minute chase (siddell et al., 1981c; rottier et al., 1981b) . siddell et a2. (1981~) have suggested that the 150k precursor to e2 may be cleaved to a 90k protein which dimerizes to form the 180k e2. based on our studies with mhv-a59, we believe that two different 90k forms of e2 result from proteolytic cleavage of the 180k e2 which occurs as a late step in the intracellular transport or processing of e2 and the maturation of the virions. recently, stern and sefton (198213) showed by tryptic peptide mapping that the two large virion glycoproteins of ibv, gp90, and gp84, were produced by cleavage of a 155k glycoprotein precursor (gp155). although the cellular location at which the e2 glycoprotein undergoes acylation has not been identified, acylation of the vsv glycoprotein has been shown to occur near the golgi complex (schmidt, 198213) . the intracellular transport of e2 appears to be similar to that of nlinked glycoproteins of other viruses such as orthomyxo-, paramyxo-, rhabdo-, and alphaviruses. immunofluorescent staining of e2 with monospecific antiserum stained the cytoplasm diffusely, e2 appeared on the plasma membrane at a time when intracellular transport of e l was directed to the golgi apparatus (holmes et al., 1981b; doller et al., 1983) . treatment of mhv-infected cells with tunicamycin resulted in a marked inhibition in the synthesis of e2 (holmes et al., 1981b) , although late in infection some nonglycosylated e2 could be detected (niemann and klenk, 1981a) . virions isolated from tunicamycintreated cells contained no e2 (sturman, 1981) and no peplomers (holmes et al., 1981a,b) . this suggested that e2 was not required for virus budding or for the release of virions from infected cells. brian (1981, 1982) and b. w. j. mahy et al. (1983) first detected rna-dependent rna polymerase activity in cells infected with tgev and mhv. this activity was insensitive to antinomycin d and was associated with cytoplasmic membrane fractions like that of alphaviruses. brayton et al. (1982) and lai et czl. (1982b) have characterized the viral rna polymerase activity in mhv-a59-infected cells. by pretreatment of cells with actinomycin d for 1 hour before infection and synchronization of infection by adsorbing virus at 0-4"c, brayton et al. (1982) detected virus-specific rna synthesis in mhv-a59-infected cells as early as 1 hour after infection. two peaks of virusspecific rna synthesis were demonstrated, one early (2 hours) and the other late (6 hours) after infection (fig. 18 ). corresponding to these, two different virus-specific rna polymerase activities were detected, early and late in infection. these two polymerases were distinguished by different responses to potassium and different ph optima. the rna product of the early polymerase was of negative-strand polarity, complementary to the genomic rna, whereas the products of the late polymerase activity were predominantly of positive polarity (p. r. brayton, personal communication) . only a single virus-specific, negative-stranded rna species was detected. this was the size of the complete viral genome. a double-stranded rna form was isolated which contained rna of genomic size. when this double-stranded rna was isolated without rnase treatment and heated, subgenomic mrnas were released. this suggested that the virus-specific mrnas were incorporation of radiolabel into virus-specific rna in cells infected with mhv-a59 occurs in two peaks which show different ionic requirements. the early peak is believed to represent synthesis of full-length, negative-stranded rna templates, and the later peak is believed to represent synthesis of mrnas and genomic rna. (reproduced from brayton et al., 1982, with permission.) transcribed from a negative-strand rna template of genomic size . the mechanism which regulates the frequency of transcription of each mrna species from the negative-stranded rna template remains t o be elucidated. the relative rates of synthesis of the mrnas appear to be constant throughout infection (stern and kennedy, 1980a; wege et al., 1981b; spaan et al., 1981; . the smaller mrnas (numbers 7 and 61, which represent the 3' end of the genome, are far more abundant than the sum of all the others (jacobs et az., 1981; table 111 ). in different virus strains, the relative abundance of the different viral mrna species varies considerably. have shown ( table 111 ) that the relative molar amount of rna5 synthesized in mhv-jhm-infected cells is about one tenth of that found in cells infected with mhv-a59. a central question in the molecular biology of coronaviruses is how the subgenomic mrnas and genomic rna are transcribed from the negative-strand template. ultraviolet transcription mapping was done to identify the size of the template(s) for the synthesis of the mrnas. jacobs et al., (1981 ; table iv ) and stern and sefton (1982a) found that b k x t was calculated from the relationship in (n,no) = k x t x t, where n t represents the incorporation of ["iuridine into rna after t seconds of uv irradiation, n o is the rna synthesis in the unirradiated culture, t is the target size, and k is a constant. the calculation was made from the data points by using linear regression analysis. the value ofk was calculated as 6.38 x 10 -9s -1 by substituting a value of 5.6 x 106 for the target size of r n a l (experiment l), or as 10.3 x by inserting a value of 1.4 x 106 for rna4 (experiment 2). c by using the two values for k described in footnote b, the target sizes for the other rnas were calculated. d the molecular weights of the denatured, virus-specific rnas were determined by agarose gel electrophoresis. the uv target sizes of the templates for the mrnas of mhv-a59 and ibv were the same as the sizes of the respective mrnas. since the uv mapping studies were done at 5.5 and 6.0 hours postinfection, when synthesis of the negatively stranded template may have been completed, it appears likely that only the synthesis of the mrna species was being inactivated by uv. comparison of the oligonucleotide fingerprints of subgenomic mrnas and virion rna of mhv suggested that some rna splicing mechanism or other modification of the rna may take place during mrna synthesis. stern and kennedy (1980a,b) found several t1 oligonucleotides in mrna species which were not present in larger mrnas. , , and spaan et al. (1982) also identified several t1 oligonucleotides in subgenomic mrnas which were not present in the viral genome of mhv, or were not found in the same region in the genome. preliminary evidence indicated that some of these oligonucleotides from several subgenomic rnas had similar sequences . it has been suggested that these may represent junction sequences from the splicing of two unlinked stretches of rna . since coronaviruses can replicate in enucleated cells brayton et al., 1981) , it appears unlikely that this splicing is done by cellular mechanisms located in the cell nucleus. lai et al. (1982a) found that the nucleotides adjacent t o the cap structures of each of the subgenomic mrnas contained the same sequence, 5'-cap-n-uaag. it is not known how many additional nucleotides are shared at the 5' end of these mrnas. it is not clear how the cap with its adjacent nucleotide sequences is added to each mrna. one possibility would involve rna splicing. a leader sequence which originates by splicing from large precursor molecules would appear to be contraindicated by the results of uv transcriptional mapping. however, a leader sequence with or without an attached cap may be derived from a small rna of viral or cellular origin. since viral rna synthesis was not inhibited by antinomycin d, this leader rna may be virus specific or perhaps derived from a stable, small, cellular rna. the sequence -uaag which was found adjacent to the cap structure at the 5' end of viral subgenomic mrnas and genomic rna (lai et al., 1982a) is also present in some small host coded cytoplasmic rnas, including 5s ribosomal rna (delihas and andersen, 1982) and 7s rna (busch et al., 1982) , and u2 and u3 small nuclear rnas (busch et al., 1982) . furthermore, the same order is contained in the consensus sequence a t the 5' exon-intron boundary of many splice junctions (lerner et al., 1980) . the complementary sequence which is present in u1 snrna is thought to assist in the proper orientation of exons for splicing of rna. several models have been postulated for the utilization of a leader sequence in coronavirus transcription (lai et al., 1982a; spaan et al., 1982) . 1. such a leader could act as a primer for the initiation of rna transcription along the full-length, negative-strand template. 2. alternatively, a leader sequence could be fused onto the 5' end of newly synthesized viral transcripts. this sequence could come from viral or cellular rna. 3. an rna polymerase jumping mechanism could explain the common nucleotide sequences at the 5' end of each viral mrna and genomic rna. thus, a short segment of the 5' region would be transcribed from the 3' end of the negative-strand template, and then the polymerase would translocate through intervening sequences on the negative-strand template and resume transcription at the beginning of a particular cistron. the uv transcription data do not contradict any of these models, since the leader sequence may be too small a target to have been detected. some of the early studies on coronavirus replication emphasized ultrastructural changes in infected cells (svoboda et al., 1962; tanaka et al., 1962; david-ferreira and manaker, 1965; hamre et al., 1967; becker et al., 1967) . all of these studies showed that the entire replicative cycle of coronaviruses occurred in the cytoplasm. indeed, mhv has been shown to replicate in enucleated cells wilhelmsen et al., 1981) , although replication of ibv in enucleated bhk-21 cells was significantly reduced (evans and simpson, 1980) . ultrastructural studies on the binding and penetration of coronaviruses have been described (section ii1,b.i. the early morphological events associated with coronavirus infection were rather nonspecific ones, such as increase in cytoplasmic membranes or in the size of polysomes (david-ferreira and manaker, 1965; sebesin, 197 1) . toward the end of the viral latent period, about 6-7 hours postinfection, spherical virions approximately 60-100 nm in diameter were observed in the lumens of the rough endoplasmic reticulum, golgi apparatus, and smooth-walled vesicles (david-ferreira and manaker, 1965; massalski et al., 1981; ducatelle et al., 1981; dubois-dalcq et al., 1982; fig. 19a-c) . in most instances these virions had electron-lucent centers with electron-dense granular or tubular nucleocapsids associated with the inner surface of the viral envelope. tubular nucleocapsids were also observed under the membranes of the rough endoplasmic reticulum or golgi apparatus (oshiro, 1973; holmes et al., 1981a; massalski et al., 1981 massalski et al., , 1982 dubois-dalcq et al., 1982; fig. 19b and c) . chasey and alexander (1976) showed that the envelopes of budding virions were covered with peplomers. during the early stages of viral infection, virions appeared singly or in small clusters, whereas later in infection, scores of virions were found within large vesicles and occasionally within the lumen of the nuclear membrane (fig. 20) . budding of virions from the plasma membrane was almost never visualized. an exception was a single particle visulaized several days after virus infection of a neural cell culture (dubois-dalcq et al., 1982) . although sugiyama and amano (1981) reported that the virions were budding from the plasma membrane, the scanning electron microscopic (semi images were not of sufficient resolution to demonstrate budding. in some studies, although numerous intraluminal virions were observed, no budding virions were detected. detection of budding images apparently depended on the viral strain, the host cell type, and the time after virus inoculation (watanabe, 1969) . budding virions were most likely to be detected late in the infectious cycle. this suggests that these images represent arrested buds, and that during the early stages of infection coronavirus budding may be a very rapid process. several investigators have suggested that coronaviruses were released by lysis of the infected cells (hamre et al., 1967; oshiro et al., 1971; takeuchi et al., 1976; chasey and alexander, 1976) . release by fusion of virus-filled, smooth-walled vesicles with the plasma membrane has also been observed (doughri et al., 1976) . when the kinetics of release of infectious virus was correlated with cell lysis by microcinematography, however, it was apparent that the infectious virions were released from intact cells (k. v. holmes, unpublished observations) . indeed, it appears that release of virus depends upon the good condition of the host cells. the generally accepted mechanism of coronavirus release from infected cells is via fusion of virus-filled vesicles with the plasma membrane (doughri et al., 1976) . thus, the coronaviruses may be released from cells by utilizing a cellular transport mechanism developed for secretion or exocytosis of the contents of secretory vesicles. i n uiuo, the target cells for replication of many coronaviruses are epithelial cells with tight junctions in the respiratory or gastrointestinal tract. in polarized cells such as these, influenza virus and vsv have been shown to bud specifically from the apical or basilar plasma membranes (rodriguez-boulan and sabatini, 1978) . there is as yet little evidence to show whether the fusion with the plasma membrane of vesicles filled with coronaviruses exhibits specificity for basal or apical membranes. secretory cells do demonstrate strong polarity in the direction of secretion of cellular secretory products such as enzymes from pancreatic acinar cells. it is possible that some polarity of coronavirus release may be identified. to date, in the only study which addresses this point, doughri and storz (1977) observed that porcine coronavirus could be seen on both apical and basal cell surfaces of intestinal epithelial cells. fig. 20 . coronavirus virions in the rough endoplasmic reticulum and nuclear envelope. late in the infectious cycle, numerous spherical virions accumulate within the rough endoplasmic reticulum and the nuclear envelope (n) (arrows). ~3 6 , 0 0 0 . (from j. n. behnke.) late in the infectious cycle, it was common to find very large numbers of virions adsorbed to the surface of infected cells (oshiro et al., 1971; oshiro, 1973; doughri and storz, 1977) . scanning electron microscopy showed these most effectively (sugiyama and amano, 1981 it is not clear what function, if any, may be served by this adsorption to infected cells. most of the virions appeared to remain on the surface without being internalized, although some were found in lysosomes (sabesin, 1971) . characteristic features of coronavirus infection were vacuolization of cells and virus-induced cell fusion (oshiro, 1973; mcintosh, 1974) . the time of appearance of these depended on the virus and the host cell type. it is noteworthy that large intracytoplasmic inclusions of nuholmes.) cleocapsids were not observed in most early studies of coronavirusinfected cells. caul and egglestone (1977) did observe such inclusions in cells infected with hecv, and others have seen them also (watanabe, 1969) . massalski et al. (1982) suggested that nucleocapsid inclusions accumulate after the cessation of virus budding. dubois-dalcq et al. (1982) showed that several types of inclusions that could be produced by the intracytoplasmic nucleocapsids of different strains of mhv in differentiated cultures of central nervous system (cns) cells ( fig. 19d and e) . they also found an increase in "myelin figures" in the cytoplasm of mhv-infected cells. additional features of coronavirus cpe included several types of intracytoplasmic inclusions whose origins and functions remain unclear. david-ferreira and manaker (1965) found "reticular inclusions" in mhv-infected cells. these consisted of masses of interconnected tubules of smooth membranes in continuity with the rough endoplasmic reticulum (fig. 21a) . they have only occasionally been observed with other coronaviruses. a second type of inclusion was observed near the reticular inclusions. this consisted of vacuoles about 200 nm in diameter containing flexible coiled filaments about 30 nm in diameter. these filamentous structures were surrounded by a double membrane ( fig. 21b ; david-ferreira and manaker, 1965; takeuchi et al., 1976) . third, "tubular inclusions" consisting of interconnected tubules 16 to 25 nm in diameter were observed near the reticular inclusions (david-ferriera and manaker, 1965; watanabe, 1969) . inclusions consisting of interconnected virions within the lumen of smoothwalled vesicles have also been observed (oshiro et al., 1971) . within the lumen of the rough endoplasmic reticulum and smooth-walled vesicles, long, rigid, cylindrical structures about 50 nm in diameter have occasionally been detected (fig. 21c; dubois-dalcq et al., 1982) . these may represent an excess of the e l glycoprotein since they occur frequently in tunicamycin-treated infected cells, where synthesis of e2 is greatly reduced (holmes et al., 1981a) . it is evident from the preceding descriptions that coronaviruses produce a wide variety of effects in different cell types. further studies are needed to determine the composition of these different virus-associated structures. in other virus systems, conditional lethal mutants have been important in elucidating many steps in virus replication. with coronaviruses, this effort is still at an early stage. although many variants have been isolated from natural infections. there are few well-charac-terized, chemically induced mutants of coronaviruses. recently, several groups have obtained and partially characterized temperature-sensitive (ts) mutants of mhv. almost all of the mhv mutants have been selected for failure to induce syncytium formation. unfortunately, most of these mutants grow rather poorly, even under permissive conditions. there are few, if any, chemically induced mutants of other coronaviruses. clearly, other phenotypes and additional mutants are needed. the first collections of chemically induced mutants of mhv-jhm were made by haspel et al. (1978) with 5-azacytidine or 5-fluorouracil, and by robb et al. (1979) using n-methyl-n'-nitrosoguanidine and 5fluorouracil. temperature-sensitive mutants were selected for failure to induce fusion of susceptible cells at the nonpermissive temperature (39.5" or 385°c). the majority of the 34 mutants identified by robb et al. were found to be rna negative. only three were rna positive and exhibited synthesis of viral proteins. some of these mutants produced altered neuropathogenesis in mice. leibowitz et al. (1982a) performed complementation analysis of 37 ts mutants of mhv-jhm and identified seven complementation groups. six of these affected virus-specific rna synthesis. the gene product affected by each mutation has not yet been identified. several of the mutants described by haspel and his co-workers, including the ts8 mutant, are of particular interest because they induce demyelination with a much higher frequency than the wild type (haspel et al., 1978; knobler et al., 1981c, 19821 , and because they may form aberrant inclusions in cns cells in uitro (dubois-dalcq et al., 1982) . wege et al. (1981c wege et al. ( , 1983 ) also isolated temperature-sensitive mutants of mhv-jhm with 5-fluorouracil and found that these mutants caused higher rates of subacute and chronic neurological diseases than did wild type virus in suckling and weanling rats. twenty chemically induced (with 5-fluorouracil), temperature-sensitive mutants of mhv-a59 have been partially characterized by koolen et al. (1981 koolen et al. ( ,1983 . most of these mutants, selected for their inability to induce syncytium formation at 40"c, were rna negative also. several of the mutants exhibited altered neuropathogenic properties. a variety of coronavirus mutants have been isolated from persistently infected cultures of mhv, and are described in section ii1,h. clearly, the potential contributions of these and other coronavirus mutants to the study of coronavirus replication are enormous. in combination with cloning of the viral mrnas and genomic rna, which is being done in several laboratories, mutants will be invaluable for the analysis of coronavirus genetics, pathogenesis, and replication. coronaviruses are capable of inducing persistent infection in animals (robb and bond, 1979a; wege et al., 1982) and in tissue cultures. many persistent infections were summarized at the wurzburg symposium on coronaviruses siddell et al., 1982) . the salient feature of these infections was that in most cases the majority of cells remained antigen negative, yet all of the cells were resistant to superinfection with wild-type virus. derived cold-sensitive mutants of jhm from persistently infected neuroblastoma cells. these were rescued from latently infected cells by polyethylene glycol-induced fusion to permissive cells. hirano et al. (1981) obtained small plaque mutants of jhm, and holmes and behnke (1981) isolated small plaque and temperature-sensitive mutants of a59 from persistently infected cells. these mutants have not yet been fully characterized. in order to understand the balance between virus and host which permits this persistent infection, it is necessary to identify the host functions which are utilized by the viruses during the replicative cycle and to characterize in detail the controls exerted upon coronavirus transcription and translation. there are important host controls over coronavirus replication at several levels. immune response genes may play an important role in resistance to coronavirus-induced disease . however, genetic factors are also important at the single-cell level. these are the focus of our discussion. bang and warwick (1960) demonstrated that while the pri strain of mhv (mhv-2) caused fatal hepatitis in mice of the pri strain, this virus did not kill c3h mice. this susceptibility of pri mice to death induced by mhv-2 was found t o be inherited as a dominant gene (bang and warwick, 1960; kantoch et al., 1963) . by backcrossing, created a strain of mice congenic t o c3h mice, but bearing the dominant susceptibility to mhv-2 (c3h-ss strain). extensive studies were done comparing the effects of mhv-2 on c3h and c3h-ss mice taylor et al., 1981; bang, 1981) . by various manipulations, bang and his associates were able to modulate the effects of mhv on the susceptible and resistant mice. treatment of resistant animals with cortisone (gallily et al., 1964) or a proteindeficient diet (bang, 1981) rendered them susceptible so that they died from a small dose of mhv-2, and treatment of susceptible animals with concanavalin a rendered them resistant so that they could survive a normally fatal dose of mhv-2 (weiser and bang, 1977) . the susceptibility or resistance of different mouse strains to mhv-2, as measured by survival of animals, was directly correlated with the response of peritoneal macrophages from each strain in culture to this virus (bang and warwick, 1960; bang, 1981) . however, the cell culture conditions greatly affected these results. when fetal bovine serum was substituted for horse serum in the medium, the difference in virus yields between macrophages from resistant and susceptible mice was significantly reduced (lavelle and bang, 1971; bang, 1981) . the mechanism for this cellular restriction of mhv-2 synthesis in cells from resistant animals under defined conditions has not yet been characterized. shif and bang (1970) demonstrated that the restriction was at a stage subsequent to virus adsorption, and suggested that degradation of virions within the resistant cell might be responsible. later, cody showed that the virus grew equally well in resistant and susceptible cells, but was one-twentieth as infective for resistant cells (cody, 1980; bang, 1981) . in his last publications (bang and cody, 1980; bang, 1981) , bang described recent experiments which suggested that macrophage resistance to mhv-2 was also dependent upon associated lymphocyte action. he also suggested that cell-bound interferon might play a role in protecting the genetically resistant cell. the pioneering studies of bang and his co-workers were extended by other investigators using different strains of mhv. these studies demonstrate that mhv-2, mhv-3, and mhv-jhm exhibit different patterns of host susceptibility and resistance. although replication of mhv-2 was restricted in c3h mice, replication of mhv-3 was only partially reduced (leprevost et al., 1975; virelizier and allison, 1976; yamada et al., 1979; taguchi et al., 1981) . furthermore, a/j mice, which were resistant to mhvs, were susceptible to mhv-jhm (knobler et al., 1981b) . resistance to mhv-3 and mhv-jhm correlated with failure of the virus to replicate and with delayed appearance of cpe after low-multiplicity infection of cultures of peritoneal macrophages (virelizier and allison, 1976; krzystyniak and dupuy, 1981; knobler et al., 1981a,b; stohlman and frelinger, 1981; stohlman et al., 1982a1, neuronal cells (knobler et al., 1981a , and hepatocytes (arnheiter and haller, 1981; arnheiter et al., 1982) . resistance was partially overcome by infection at higher multiplicities, resulting in cell destruction, although virus yields remained low (virelizier and allison, 1976; arnheiter et al., 1982; knobler et al., 1981b) . arnheiter et al. (1982) showed by fluorescent antibody staining that in the first cycle of virus replication, resistant cultures of hepatocytes had fewer cells expressing viral antigen. however, after infection at high multiplicty (multiplicity of infection of loo), all cells contained viral antigens, including e2 and e l , but virus production was delayed and virus yields remained low. the characterization of this cellular restriction to mhv replication remains incomplete. much of our present knowledge about the structure and functions of coronavirus glycoproteins comes from studies of mhv. a model for the structure of the e2 glycoprotein of mhv-a59 is shown in fig. 22 . some of the important features of this model include (1) anchoring of one end of the protein in the viral envelope; (2) covalent attachment of palmitic acid; (3) a single trypsin-sensitive site accessible in the native glycoprotein on the virion; (4) the presence of sh groups and disulfide bonds; (5) noncovalent association between the subunits (90a and 90b) of e2; and (6) oligosaccharide side chains on both subunits. e2 is probably anchored to the viral envelope through a short hydrophobic region, as pronase or bromelain quantitatively removed both 180k and 90k species from the intact virions, while the peplomers were removed (sturman and holmes, 1977) . palmitic acid is covalently attached, probably at or near the hydrophobic domain which anchors the peplomer in the viral envelope, since this has been demonstrated for the influenza virus hemagglutinin (schmidt, 1982a) and the g protein of vsv (petri and wagner, 1980. although detergent-sol-fig. 22 . model of the peplomeric glycoprotein e2. this is a provisional model of e2 which shows some of the important structural features of the peplomeric glycoprotein such as the acylation, glycosylation, presence of both sulfhydryl groups and disulfide bonds, and the existence of a trypsin-sensitive cleavage site in the center of the molecule which results in the formation of two species, tentatively called e2a and e2b, which comigrate a t 90k. ubilized e2 could be digested by trypsin into many peptide fragments, there appears to be only a single trypsin-sensitive cleavage site accessible in the native 180k molecule on the mhv-a59 virion (sturman and holmes, 1977. disulfide linkages mask proteolytic cleavage in the hemagglutinin of hev. upon reduction, the 140k hemagglutinin (e2) of strain vw527 disappeared and a new polypeptide species (gp76) appeared, indicating that the 140k e2 was composed of two 76k subunits which were linked through disulfide bonds (callebaut and pensaert, 1980) . the hemagglutinin of hev strain fs255 was also sensitive to treatment with sulfhydryl reagents; exposure to dithiothreitol resulted in the loss of hemagglutinating activity and release of a 1 2 5 from the virion (pocock, 1978) . several investigators have reported that coronavirus infectivity was most stable below ph 7 alexander and collins, 1975; sturman, 1981) . the ph-dependent thermolability of mhv infectivity shown in fig. 2 appeared to be the result of a conformational change in e2 which led to aggregation of the peplomeric glycoprotein (sturman, 1981) . intrachain disulfide and sulfhydryl groups appeared to be important in determining the conformation of e2, as ph and temperature-dependent aggregation of e2 on virions or of isolated, np-40-solubilized e2 were enhanced by reducing agents and sulfhdryl blocking reagents (sturman, 1981) . there is no evidence for interchain disulfide bridges between the 90a and 90b subunits of mhv-a59 e2; they remained associated noncovalently on the virion after trypsin cleavage of the peplomer (sturman, 1977; sturman and holmes, 1977) . during ph 8 inactivation, however, some 90k e2 was released, and incubation with reducing agents caused release of more 90k e2. this was probably associated with a change in the conformation of this molecule following reduction of intramolecular disulfide bonds. the same appears to be true for the liberation of gp125 from hev strain fs255 (pocock, 1978) . present evidence indicates that e2 possesses six or more biological activities (table v): 1. binding of virions to receptors on the plasma membrane of susceptible cells (adsorption and hemagglutination) appears to be mediated by e2. purified, radiolabeled e2 bound to susceptible cells but not to cells lacking in virus receptors, such as erythrocytes (k. v. holmes, unpublished data) . binding of e2 was inhibited by preincubation of cells with excess mhv. virions from which the peplomers had been removed, or particles without peplomers, exhibited markedly reduced capacities for cell attachment and infection (holmes et al., 1981a) . in hcv-oc43, monospecific antibody to e2 inhibited hemagglutination (schmidt and kenney, 1982) . coronaviruses which hemagglutinate include some strains of ibv, hev, hcv-oc38/43, mhv-3, a murine enteric coronavirus, rabbit enteric coronavirus, and bcv (see section 111,b). initially, trypsin treatment of virions appeared to be necessary for activation of ibv hemagglutinin (corbo and cunningham, 1959) . subsequently, however, the ibv hemagglutinin was shown to be inactivated by trypsin . trypsin also destroyed the hemagglutinating activity of oc38143 viruses (kaye and dowdle, 1969) . there appeared to be significant strain differences in ibv hemagglutination , and the response of ibv t o trypsin was also strain dependent. 2. e2 is responsible for the induction of neutralizing antibody. garwes et al. (1976, (1978) (1979) were the first to show that antibody against purified surface projections (of tgev) possessed virus-neutralizing activity in vivo. in accordance with this, found that most of the antibody induced during infection of human volunteers with hcv-229e was directed against the surface projections of the virus. hasony and macnaughton (1981) also showed that immunization of mice with e2 protected them against infection with mhv-3, whereas immunization with e l or n failed to provide protection against virus challenge. holmes et al. (1981b) demonstrated that monospecific antibody against mhv-a59 e2 neutralized infectivity in cell culture, and similarly kenny (1981, 1982) showed that monospecific antibody to e2 of hcv-oc43 neutralized the fig. 23. virus in uitro. monoclonal antibodies against mhv-jhm e2 were shown by collins et al. (1982) to neutralize mhv-jhm virus infectivity in the absence of complement, whereas monoclonal antibody to e l exhibited neutralizing activity only in the presence of complement. 3. e2 on the surface of infected cells renders them susceptible to cytotoxic effects of spleen cells. the cell-mediated cytotoxicity of spleen cells from uninfected mice to mhv-a59-infected cells was also inhibited by antibody to e2 4. the ph-dependent thermolability of coronavirus virions is due to aggregation of e2. this conformational change in e2, which occurs above ph 6.5 a t 37"c, is sensitive to sulfhydryl reagents (sturman, 1981) . 5. e2 is responsible for cell fusion. cell fusion was frequently a prominent feature of coronavirus infection in uiuo and in uitro. the extent of cell fusion depended upon the virus strain, host cell, and the conditions of infection. the role of e2 in the induction of cell fusion is indicated by the observation that coronavirus-induced fusion was inhibited by monospecific and monoclonal antibodies to e2 (holmes, 1981b; collins et al., 1982; fig. 23a and b) , and by the finding that treatment of infected cells with tunicamycin inhibited both the synthesis of e2 and cell fusion (holmes et al., 1981a) . trypsin in the overlay medium enhanced plaque formation of an enteropathogenic bovine coronavirus and several strains of ibv (storz et al., 1981a,b; otsuki and tsubokura, 1981) . in the presence of trypsin, infection with coronaviruses was associated with cell fusion (storz et al., 1981a,b; toth, 1982) . similar findings were obtained with a mutant of mhv-s which did not ordinarily induce cell fusion (yoshikura and tejima, 1981) . trypsin treatment of infected cells also enabled mhv-s to form fusion plaques on otherwise resistant cells, and allowed mhv-2 to form fusion-type plaques. this effect of trypsin on the ability of coronaviruses to induce cell fusion is similar to that however, if monospecific antibody against the e2 glycoprotein is added to the culture from 2 hours after infection, the virus-induced cell fusion is prevented (b ). monospecific antiserum against the e 1 glycoprotein does not prevent coronavirus-induced cell fusion; this suggests that e2 is the fusion factor of mhv(c). ~1 0 0 . (from k. v. holmes.) ( d and e ) fusion of uninfected l2 cells by direct action of concentrated mhv-a59 on the plasma membrane does not occur within 2 hours (d). however, if the virions had been previously treated with trypsin to cleave the e2 180k to e2 90k, then rapid fusion of uninfected cells was observed (e). this and other observations suggest that coronaviruses have a protease-activated cell fusion factor like those found in other enveloped rna viruses. (from l. s. sturman.) observed with the f protein of paramyxoviruses and the ha protein of myxoviruses . sturman and holmes (1977) showed that trypsin treatment of mhv-a59 resulted in cleavage of 180k e2 to 90k. recently, direct evidence has been obtained for the role of proteolytic cleavage of e2 in cell fusion. regardless of the multiplicity of infection, efforts to obtain rapid cell fusion with a coronavirus had been unsuccessful until we employed virus which had been treated with trypsin which caused cleavage of e2 wok) to e2 (90a + 90b). cell fusion occurred rapidly after addition of this virus and in the absence of viral protein synthesis (fig. 23c and d) . 6 . proetolytic cleavage of e2 may be required for viral infectivity. however, this has not yet been proven. many investigators have studied the effects of trypsin on coronavirus infectivity with mixed results. for example, trypsin treatment at low concentrations (10 p/mu enhanced mhv-a59 infectivity two-to threefold, whereas at high concentrations (1 mg/ml), infectivity was reduced by the same relative degree (sturman and holmes, 1977) . since a large proportion of mhvassociated e2 was already in the 90k form, it has not been possible to assess the role of proteolytic cleavage of e2 as was done with the f, glycoprotein of sendai virus and the ha, glycoprotein of influenza virus . when a source of coronavirus with uncleaved e2 is identified, the effect of specific cleavage of e2 on virus infectivity can be demonstrated conclusively. e l is in many ways a unique viral glycoprotein. it performs functions associated with matrix proteins of other viruses, yet it is glycosylated and protrudes from the viral envelope. e l comprises approximately 40% of the protein of the virion and contains 70% of the methionine label. at first, e l was difficult to study because of its unusual tendency to aggregate upon heating at 100°c in sds (figs. 4 and 5; sturman, 1977) . the generation of multimers of e l produced a variety of polypeptide patterns on sds-page depending upon the conditions of sample treatment. aggregation of e l in sds has been described for other strains of mhv (wege et al., 1979) and for other coronaviruses as well (callebaut and pensaert, 1980; schmidt and kenny, 1982) . multiple forms of e l were also distinguished within the 20-25k apparentmolecular-weight range (wege et al., 1979; siddell et al., 1981b; rottier et al., 1981b; holmes, et al., 1981b) . these may reflect differences in the number or heterogeneity of the oligosaccharide chains on e l . fig. 24 . model of the membrane glycoprotein el. this is a provisional model of the e l glycoprotein which is associated with the coronavirus envelope, showing some of the characteristic features of the molecule. these include the amino-terminal end (n) of the protein which protrudes from the envelope and bears the 0-linked carbohydrate side chains, and the extensive domains of the e l within and beneath the membrane where they can interact with other e l molecules, with the nucleocapsid, or with e2. many of the characteristics of coronaviruses, such as their intracellular budding site, may be determined by the properties of the e l glycoprotein. a model for the e l glycoprotein of mhv-a59 is shown in fig. 24 . e l appears to possess three domains: 1. a small 5k hydrophilic region, containing all of the carbohydrate on the molecule, extends outside the viral envelope and can be removed by pronase of bromelain (sturman and holmes, 1977) . have shown that for the e l of ibv, this external domain represents the amino-terminal portion of the molecule. we do not know anything about the functions of this glycosylated portion of e l which may have important cooperative effects with e2. 2. a hydrophobic domain resides within the lipid bilayer. disulfide bonds are illustrated in this region because aggregation of e l in sds at 100°c was markedly enhanced in the presence of reducing agents, which indicated that reduction of disulfide bonds exposed a highly hydrophobic domain (sturman and holmes, 1977) . 3. the third domain of e l resides on the inner surface of the envelope and may be associated with the nucleocapsid. as described earlier, after solubilization of the viral envelope with np-40 at 4"c, the nucleocapsid could be separated from both e l and e2, but at 37"c, e l reassociated with the nucleocapsid see fig. 10b ). e l has been shown to bind to rna in the nucleocapsid. however, this interaction was not specific for mhv rna. nucleocapsid structures of other coronaviruses also interacted with e l (garwes et al., 1976; pocock and garwes, 1977) . e l appears to be the only glycoprotein required for coronavirus budding, as shown by the fact that in mhv-infected, tunicamycin-treated cells, in which e2 was made in markedly reduced amounts and not incorporated into virions, mhv virions were formed and released normally. therefore, we infer that e l is responsible for the formation of the viral envelope. e l of ibv contained n-linked, rather than 0linked, oligosaccharides . glycosylation of the e l of ibv was inhibited by tunicamycin, but virions which lacked e2 and contained nonglycosylated e l were produced. thus, glycosylation of e l was not essential for formation of ibv virions. the intracellular localization of e 1 on intracytoplasmic membranes ( fig. 17 ) may determine the characteristic budding sites of coronaviruses, which are limited to the endoplasmic reticulum and the golgi apparatus (holmes et al., 1981b; doller et al., 1983) . e l may also contain sites for interaction with the viral peplomers. some monoclonal antibodies to e l have been shown to exhibit neutralizing activity, but only in the presence of complement (collins et al., 1982) . the possibility has not been excluded that e l may also have a role in other functions which are presently thought to involve e2, such as the interaction of virions with cell receptors, production of a cell-mediated immune response, and induction of cell fusion. v. antigenic relationships among coronaviruses antigenic relationships among ibv, hcv, and mhv strains were first studied in detail, by immunofluorescence, hemagglutination inhibition, and neutralization, by mcintosh et al. (1969) and bradburne (1970) . these investigators established that mhv and some human coronaviruses were antigenically related, while ibv did not cross-react with hcv or mhv. mcintoshs data indicated that there were at least two subgroups of human coronaviruses. hcv-oc38, and the very similar oc43, showed a close antigenic relationship to mhv, but none of these cross-reacted with hcv-229e. bradburne also found an antigenic relationship between oc43 and mhv; however, he detected some cross-reactivity between hcv-229e and both oc43 and mhv. more recently, schmidt and kenny (19821, using rocket immunoelectrophoresis, found no evidence of cross-reactivity between any of the structural proteins of hcv-229e and oc43, whereas gerdes et al. (1981a,b) and hasony and macnaughton (1982) detected some antigenic cross-reactivity between the n proteins of hcv-229e and mhv by immunoprecipitation and enzyme-linked immunoassay. using immunofluorescence, pedersen et al. (1978) separated eight mammalian coronaviruses into two antigenically distinct groups. one group consisted of mhv, hev, hcv-oc43, and bcv. the second group included tgev, hcv-229e, fipv, and ccv. additional anti-genic cross-reactions between other coronaviruses have been detected: the rat coronaviruses, rcv and sdav, were antigenically related to mhv (parker et al., 1970; bhatt et al., 1972) , and the rabbit coronavirus which produces pleural effusion disease (rbcv) cross-reacted with both hcv-229e and hcv-oc43 (small et al., 1979) . another mammalian coronavirus, the procine enteropathogenic coronavirus designated cv777, did not cross-react with any other coronavirus tested, including tgev, hev, fipv, ccv, bcv, and ibv . these antigenic relationships are summarized in table vi . recent studies of antigenic relationships among coronaviruses have focused on antigens of individual structural proteins and have employed monospecific and monoclonal antibodies. gerdes, burks, and their co-workers showed that two coronaviruses (sd and sk) isolated from fresh autopsy brain tissue from two patients with multiple sclerosis (burks et al., 1980) were serologically related to mhv-a59 and hcv-oc43 (gerdes et al., 1981a,b) . antisera prepared against each of the four viruses, sd, sk, a59, and oc43, precipitated all three structural proteins ( e l , e2 and n) of the other three viruses, demonstrating that the structural proteins of these viruses were antigenically related. gerna et al. (1981) found a high degree of cross-reactivity between e2s of oc43 and bcv. reynolds et al. (1980) showed cross-reactivity between e2s of tgev and ccv, and horzinek et al. 11982) have demonstrated cross-reactivity between analogous e l , e2, and n proteins of tgev, fipv, and ccv using radioimmune precipitation, electroblotting, and enzyme-linked immunosorbent assay. at the present time, coronaviruses can be classified into two major subgroups: avian coronaviruses and mammalian coronaviruses. mammalian coronaviruses can be further subdivided into a t least two subtypes. within each subtype, individual virus species can be readily distinguished. there appears to be a great degree of antigenic diversity within some coronavirus species which have been studied extensively, including ibv and mhv. this suggests that there may be considerable antigenic drift in these coronaviruses (see section 111,g). coronaviruses have recently emerged as an important group of animal and human pathogens which share a distinctive replicative cycle. some of the unique characteristics in the replication of coronaviruses illustrated in fig. 11 include generation of a 3' coterminal-nested set of five or six subgenomic mrnas, each of which appears to direct the synthesis of one protein. two virus-specific rna polymerase activities have been identified. early rna polymerase snythesizes a negative strand of genome size. a double-stranded form has been identified in the infected cell. the subgenomic mrnas are synthesized from a fulllength, negative-stranded template by a second (late) rna polymerase. rna fusion or some other type of rna modification appears to be involved in mrna synthesis. many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. the intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the e l glycoprotein, which acts as a matrix-like transmembrane glycoprotein. e 1 also exhibits distinctive behavior by self-aggregating on heating at 100°c in sds and by its interaction with rna in the viral nucleocapsid. the e l of mhv is an o-linked glycoprotein, unlike most other viral glycoproteins. thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of o-linked cellular glycoproteins. e2 is a large, multifunctional, peplomeric glycoprotein which exhibits unusual ph-and temperature-dependent conformational changes. as for the myxo-and paramyxoviruses, it appears that specific proteolytic cleavage of e2 is necessary for coronavirus-induced cell fusion, and may also be an important determinant of coronavirus pathogenicity. future research must address the difficult problems of determining the functional relationships and their roles in infection and disease. thus, in recent years a large collection of facts about the replication of coronaviruses has been compiled. the 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fusion by semliki forest, influenza, and vesicular stomatitis viruses the replication of murine coronaviruses in enucleated cells the receptosome: an intermediate organelle of receptor-mediated endocytosis in cultured fibroblasts cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation t-lymphocyte dependent difference in susceptibility between ddd and c3h mice to mouse hepatitis virus, mhv-3 polyadenylate in the virion rna of mouse hepatitis virus role of proteins in mhv-induced cell fusion we wish to thank the many investigators who generously shared their knowledge, illustrations, and unpublished data with us for this article. excellent secretarial assistance was provided by kathleen cavanagh, mary tribley, and elinore dunphy. this work was supported by grants numbered a118997 and gm31698 from the national institutes of health, and r07043 from the uniformed services university of the health sciences. the opinions expressed are the private views of the authors and should not be construed as official or necessarily reflecting the views of the uniformed services university school of medicine or the department of defense.